Eicosapentaenoic Acid: Sources, Health Effects and Role in Disease Prevention

B IOC HE.
\1ISTRY R ESEARCH T RENDS
EICOSAPENTAENOIC ACID
SOURCES, HEALTH EFFECTS AND ROLE
IN DISEASE PREVENTION
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B IOC HE.\1ISTRY R ESEARCH T RENDS
EICOSAPENTAENOIC ACID
SOURCES, HEALTH EFFECTS AND ROLE
IN DISEASE PREVENTION
THEODORE
G. BRADLEY
Al'\TD
FRANCISCO P. V ARGAS
EDITORS
New York
Copyright 20 12 by Nova Science Publishers, Inc.

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CONTENT S
vii
Preface
Cha pter I
Omega-3 Po lyunsaturated Fatty Acid T reatment in Park inson's

Disease; Review o f Current Ev idence
Dirk W Luchtman, .leffrey A. Zidichouski and Cai Song
Cha pter 2
Eicosapentaenoic Acid (EPA) and Bone Metabo li sm

Gerard G. Dumancas, Rangika S. Hikkaduwa Koralege,
Patrisha .f. Pham and Elmer-Rico E. Moj ica
47
Cha pter 3
EPA and DHA Antidepressant Eirect : Alone or Togethe r?

Car/os Horacio Laino, Martin Gahriel Codagnone,
Maria Fernanda Podesta and Ana/la Reimis
75
Cha pter 4
Eicosapentaenoic Acid: The Role in lvlalignant Diseases

Vesna VuCic and Danijela Ristic-Medic
99
Cha pter 5
Role o f Eicosapentaeno ic Acid in Menial Health

Kalpana Poudel- Tandukar and Krishna C. Poudel
117
Chapter 6
Combined Supp lementa tion with EPA-Ricb Fi sh Oil

and Phytostero ls Improve Plasma Lipid Pro fil e and C-react ive
Protein (CR P) in Individuals with Hyperlipidem ia
Michelle A. Micallefand Manohar L. Garg
129
Cha pter 7
Physio logical Role o f Eicosapentaenoic Acid (EPA) in Reduc ing

th e Coronary Heart D iseases
Sumudu N. Warnakulasuriy a and H.P. Vasantha Rupasinghe
143
Cha pter 8
Hypocholestero lemic and Hypolipidem ic Effects of Emu lsified

Surimi Feeding in lvl ice
Teruo Mmsushita, Ry usuke Tanaka, Hi.deto Fukushima
and Yutaka Fukuda
155
VI
Contents
Chapter 9
ldenti tication and Characterization of n-6 and n-3 Fatty Acid

Desaturase and E longase from Acanthopagrus Schlegelii
Sun Hee Kim, .long Sug Park, Yo Soon .fang, So Yun Kim,
Jung-Bong Kim, Ky ung Hee Roh, Hy un Uk Kim,
Ky eong -Ry eol Lee and .long Bum Kim
165
Chapter 10
Fractionation and Concentration ofOmega-3 by lvlolecu lar

Distillation
Pablo Rossi, Nelson Ruben Grosso, Maria del Carmen Pramparo
and Valeria Nepote
177
I ndex
205
PREFACE
Omega-3 long chain pol yunsaturated fatty aci ds have been part of the human diet since
the beginn ing of mankind. W ith the recent discovery of the tremendous health benefits
provided by fatty acids, especiall y eicosapentaenoic acid (EPA); a growing progress is
occurr ing in understanding their biological funct ions and mode of action as well as the
introduct ion of EPA-en riched value-added foods and nutraceut icals. Th is book discusses the
sources, health effects and role in disease prevention of EPA. Topics include the
eicosapentaenoic acid role in bone metaboli sm, malignant diseases, mental health, and
reducing coronary heart disease; iden ti fication and character izat ion of n-6 and n-3 fatty acid
desaturase and elongase from acanthopagrus shlegelii; fractionation and concentration of
omega-3 by molecu lar di st illat ion; and combined supplmentation w ith EPA-r ich fish oi l and
phytosterols can improve p lasma lip id profile and c-react ive prote in in hyper lip idem ia.
Parkins on's disease (P D) is a neu rodegenerative disorder cl inicall y characterized by
motor dysfunct ion but also mood disorders and cognit ive impairmeni. PD neuropathology is
characterized hy the selecti ve degeneration of dopaminergic neurons projecting from the
substantia nigra pars compacta (SNpc) in the mesencephalon (middle brain ), leading to
massive dopaminergic denervat ion in the striatum, but oth er neurotransmitter systems, in
part icular the noradrenergic system, and ct -synuclein metabol ism, are also affected. An
indispensable tool for acquiring new insights into the neuropath ology and pathogenesis of
PD, and testing therapeutics, is the 1-methy/-4-pheny/-1, 2, 3, 6, -tetrahydropyridi ne (MPTP)
model of PD. Research with th is model has demonstrated that oxidative stress, inflammat ion
and apoptosis are among the primary pathogen ic factors . The current therapy is li mited to
symptom reduct ion, produces aversive side effects and, in some cases, can be quite invasive.
Increasing evidence suggest that natural, side-effect free omega-3 (n-3) polyunsaturated fa tty
acids (PUFA) may be beneficial to PD, but their mec-hanisms are compli cated and findings
regarding the ir effects heterogeneous. However, neuroprotect ive, ant i-depressant and
cognitive enhancing effects of n-3 PUFA have been w idely reported in the context of other
human neurodegenerat ive and psych iatric diseases and are relevant to the therapeutic
potent ia l of n-3 PUFA in PD.
The first part of Chapter I descr ibes the clinical appearance and the neuropathology of
PD, w ith particu lar focus on the neurochemical changes that occur in the disease as well as
the M PT P mode l. Th is is followed by an overview of the major PD pathogenesis, supported
by findi ngs from the MPT P model. The m iddl e part provides a general accoun t of n-3 PUFA
metabol ism and funct ion in the brain. The final part first focuses on epidemiological studies
T heodore G . Bradley and Francisco P. Vargas

of PUFA intake and PO in the general population, and evaluates up to date evidence for the
effect ives of n-3 PUF A against PO as obtained from clinical and animal studi es . Findings
from our research group concerning the effect of omega-3 P UFA eicosapentaenoic acid
(E PA) in PO and related mode ls are discussed in deta il. The chapter w ill conclude w ith a
summary and propose current limitat ions and future research di rect ions.
This book chapter provides updated and important informat ion for understand ing how to
potent ia lly prevent PO from nutrit ional aspect and for understanding the mechanism of n-3
PUFA in the treatment of PD. The chapter also provides evidence for developing therapy
involving adjuvant n-3 fatty ac ids w ith DA-rep lacement/ agonist drugs. This chapter w ill also
be recogn ized by doctors, graduate students and scient ists for exploring new research
di recti ons in PD.
Polyunsaturated fauy acids (P UFAs) are precursors of a w ide range of metabolites, for
example eicosanoids li ke prostaglandi ns and leukotr ienes, which play crit ical roles in the
regu lation o f a var iety of biological processes, including bone metabolism. PU FAs can be
classified into t0-6 and t0-3 groups. Eicosapentaenoic acid (EPA) is an co-3 PU FA obtained in
the human di et by the consumpt ion o f mari ne products. It is also found in the human breast
mil k. In the ske leton, there is evidence show ing that alteri ng the relative composition of
di etary fatty acids can affect bone cell metabolism. Several studies have shown the benefits of
EPA in bone. An EPA-enriched di et prevented loss of bone weight and strength in
ovariectomized (OVX) rats fed w ith a lower calcium diet. It is also known to counteract
weight lessness-induced bone loss by inhibiti ng N F-KB, a transcr ipt ional activator o f many
genes, including some that lead to muscle atrophy and bone resorpt ion. In another study, EPA
was shown to be efrective for di abetic osteopen ia. Despite these benefits of EPA, a study
indi cated that supp lementat ion of fish oil in large quant ities and the associated increase in
di etary fat content can lead to substant ial degradation of the morphologica l and mechanical
properties of cortical bone. Chapter 2 will di scuss the positive and negat ive effects of EPA
intake in re lat ion to bone metaboli sm as well as the possib le mechanisms that explain such
effects . In addi tion, the effect of co-6/t0-3 rat ios on calc ium balance and bone as well as the
recommen ded dietary intake of EPA will also be discussed.
Even w ith the new generat ion of antidepressants, the treatment of depression st ill shows
mu ltiple needs. Treatment issues usua ll y relate to lack of efl'icacy, prolong onset of
antidepressant act ion and a plethora of side effects (such as sexua l dysfunct ion,
gastroimestinal events, weight gain). Severa l studies have shown that certain biological
constituents, when ingested w ith food or administrated in pure fom1s, can function as
antidepressant agents . Epidemiological findings derived in clinical and prec li nical studies
from which severa l compounds emerged as potentia l new treatments for depression either as
active agents or as adj unct therapy w ith advantages over conventional treatments in tem1s of
side effects and drug interactions. Omega-3 polyunsaturated fatty acids (PU FA) are key
nutr it ional components that exert important physio logical and biochemical etrects on
cardi ovascular, immune and central nervous system (CNS) function. Indeed, di et
supplementat ion with omega-3 PU FA has shown to be benefic ial in the treatment of
depression. However, not all types of omega-3 PUFA are equally efl'icacious. Whil e there is
more infOrmation on docosahexaen oic acid (D HA), li tt le is known about eicosapentaenoic
acid (EPA)' s antidepressant effects and the mechanism of action of these fatty ac ids. Chapter
3 explores clinical and preclin ical evidences related to the antidepressant eftects of D HA,
EPA and fish oil (DHA plus EPA). An overview of DHA and EPA efrects on CNS funct ion
Preface
lX
and ultimately the hypothesis of the central rol e of omega-3 PUFA on depression are also
provided. Undoubted ly, further studi es are needed to shed light on the mechanisms involved
in the potential c li nical benefit of omega-3 supplementat ion for the treatment of depression.
In chapter 4, a number of epidemio logical and exper imental evidences have li nked
polyunsaturated fatty acids (PUFA) of n-3 ser ies to reduced cancer r isk. Particu larly the long
chain n-3 PU FA eicosapentaenoic acid (E PA) an d docosahexaenoic acid (DHA) have been
shown to have potent ant itumor effects. Increased consumpt ion of EPA and DHA, found
naturall y in sea food, may lower th e risk of cancer develo pment. Both EPA and DH A also
exert ant i-angiogen ic effects, inhibit ing product ion of many important angiogen ic mediato rs.
Furth em10re, nutr it ional intervent ion w ith EPA decreases weight loss, promotes weight gain
and increases surviva l t imes in patients affected w ith cancer cachexia.
Nature ofantitumor e !lects of EPA is not clearly understood, but one of the mechanisms
is competitive inhibit ion of th e use of arachidonic acid, an n-6 fatty ac id, for the production of
eicosanoids. Eicosanoids derived from arachidonic acid have been associated with both tumor
promot ion and progression. EPA is also a potent angiogenes is inhibitor, which suppresses
product ion of crucial angiogen ic mediators name ly: Vascu lar Endothelial Growth Factor
(VEG F), Platelet-Derived Growth Factor (PDGF), cyclo-oxygenase 2 (COX-2), Nuclear
Factor Kappa Beta (N FKB) and nitric oxide.
Proport ion ofEPA in plasma or erythrocyte phospholip ids in healthy individuals depends
on the dietary intake and endo genous metabol ism. However, clinical evid ence suggests that
EPA status in new ly diagnosed cancer patients and pat ients undergoing chemotherapy is
usuall y mark edly lower in compar ison w ith healthy population. Low EPA status was found in
patients w ith cancer on di fferent site.s, including pancreat ic, lung, prostate cancer, and nonHodgkin lymphoma. In addition, EPA and the other n-3 PUFA were shown to be particular ly
depleted in advanced cancer patients, dur ing chemotherapy and in cancer pat ients close to
death . Therefore, both the disease itself and therapeut ic treatments may be contr ibut ing
factors in the decli ne of EPA status in patients w ith cancer. The present review w ill focus on
the current know ledge related to the ant icancer effects ofEPA on di fferent stages of disease,
from initiat ion and promot ion, to progression and neop last ic transfom1at ion.
Interest in the role of polyunsaturated fatty acids (PUF As), part icular ly long-chain n-3
PUF As, in mental health is increasing. Ep idem io logic and case-contro l data suggest that
increased dietary intake ofn-3 PU FAs may be of benefit in all eviating di fferent mental hea lth
problems. However, the resul ts of randomized controlled trials are inconsistent and
controversy ex ists as to whether either eicosapentaeno ic acid (E PA) or docosahexaenoic acid
(DHA) or both are responsible for the reported benefits. Evidence is grow ing for the more
infl uential role of EPA in cogn ition, behavior, and mood. T here are many li nes of evidence
indicating why EPA might be more beneficial than DHA in mental health . Chapter 5
discusses briefl y the beneficial ro les of EPA in mental health and, where known, the
underlying mechan isms. This is fo llowed by a review of the emerging literature on the
potentia l therapeutic ut ili ty of pure EPA in di fferent mental disorders or illnesses.
The treatment of hyperlipid emia, particularly reducing LDL-chol esterol and triglycer ide
concentrat ion, is establi shed as an efficacious means of reduc ing both morbidity and mortality
trom CVD. D ietary supplementat ion w ith phytosterols is known to reduce LDL-chol esterol,
wh ilst omega-3 PUFA are hypotriglyceridemic and have immuno-modulatory properties. The
objective of Chapter 6 was to examine the effect of combi ned supplementation w ith EPA-r ich
iish oil and phytosterols on cardiovascu lar r isk factors in individuals with combined
Theodore G. Bradley and Francisco P. Vargas
hyperlipidemia. Fi fty-eight healthy hyperlipidemic adults, were randomly assigned to

consume 3 x I g EPA-r ich (550 mg 20:5n-3 and 120 mg 22:6n-3) fish oil capsules da ily or 3
x I g placebo (sunola oil) capsu les daily, either alone or in combinati on w ith 25 g
phytostero l-enriched spread (2 g phytosterols) da ily fur 3 weeks. Blood pressure, plasma lipid
profile, plasma fatty acids and inflammatory status were analysed. Phytosterol
supp lementat ion sign ifican tly reduced total-cholesterol and LDL-cholesterol (-8.0 % and -7.5
%, P < 0.0 I), wh il st fi sh o il supp lemen tat ion signi ficantly improved triglycerides and H DLcholesterol (-8.5 %, P < 0.05; +6.5 %, P < 0.01). Total-cholesterol, LDL-cholestero l,
triglycerides and H DL-cholesterol were sign ificantly improved ( -9.5 %, P < 0.0 I; -7.5 %, P <
0.01; -22.2 %, P < 0.01 ; +7 .1 %, P < 0.01) foll ow ing the combined dietary supplemen tat ion.
CRP concentration was reduced by 11 % (P = 0.02) in the comb ination group. In conclusion ,
the combined supp lementation of EPA-rich fish oil capsules and a phytosterol-enr iched
spread provide cardiovascu lar health benefits, by way of optim ising plasma lipid profi le and
improving inllammatory status in individuals with comb ined hyperlipidemia.
Omega-3 long chain polyunsaturated Fatty acids (m-3 PU FA) have been a part of human
di et since the beginn ing of mank ind. With the recent revealing of tremen dous health benefits
provided by CD-3 PU FA especially eicosapentaenoic acid (E PA), a growing progress is
occurr ing in understanding the ir biological functions and mode of action as we ll as
introduct ion of EPA-enr iched value-added foods and nutraceuti cals to the consumer. Seafood,
especially fish o il and marine algae as a natural source of EPA, provides prom ising
intervent ion in preventing number of di seases. The role of E PA in human growth and
development and di sease prevention have been characterized by using cell cultures,
experimental an imals and human clinical studi es. The wide range of biol ogical sign ificance of
EPA extends to cardiovascular disease, anti-infl ammatory propert ies, ch ildhood learn ing and
behaviour, adult psych iatric and neurodegenerat ive di sorders and cancer. In vitro studies
indicate th at the role of EPA in biologica l funct ions can be exp lained through many
mechan isms such as its interference in the physical nature of cell membranes, proteinmediated responses, eicosanoid generation, ce ll signali ng and gene expression associated with
many cellular functions. The concurrent administration of EPA w ith drugs has also attracted
attention in research due to the improved physiological effects. The essentiality of EPA for
the human li fe have been clearly shown by animal and epidemiologica l studi es and Chapter 7
is an attempt of review ing the current understandi ng of the role ofE PA in prevent ion of the
coronary heart diseases.
Fish oil , contain ing eicosapentaenoic ac id (EPA) and docosahexaenoic acid (DHA),
shows preventive eil'ects against various diseases. "Surim i" is a th ick paste of fish meat,
wh ich is the cooking material used to make "kamaboko," a processed Japanese seafood
product. However, the main nutr ient of surimi is fish prote in, and the content of lish oil is
very low. We have developed an emulsifi ed surimi in wh ich added fish oil is formed into fine
pan icles and un iformly di stributed th roughout the fish protein. In Chapter 8, we studied the
effects of emulsi fied surim i on hypercholesterolem ia and the absorption of EPA and DH A
through mice intest ines in order to con firm a future role fur emu ls ified surimi in functional
foods. Hypercholesterolemia was induced in mice by the intake of a di et containing
cho lesterol (2%) and choli c acid (0.5%), and the effects of emulsi fled surim i feeding on
hypercholesterol emia in th is di sease mode l were examined. The emulsified surimi was
lyoph ilized, and the powdered emulsified surimi was add ed to the di et for mice in the
experimen t. The mice's serum cholesterol level was clearly increased by Ctlnsuming the
Preface
XI
cholesterol-added di et for 9 days. Furthem10re, the increase of the serum chol esterol level,
induced by the consumpt ion of the cholesterol-added diet, was sign ifi cant ly inhi bited by the
addit ion of a powdered emu lsified surimi to the diet. In addit ion, the EPA and DHA leve ls in
the livers of the powdered emu lsi fied surim i-fed mice were sign ificant ly increased, compared
with the control m ice livers. The resuJts of the present study suggest that intaking emulsified
surimi-used food products might be used to exp loit disease preventing properties .
Polyunsaturated fatty acids (PUFAs}, especially eicosapentaenoic acid (EPA} and
docosahexaenoic acid (DHA) have significantly beneficial etrects on human health . The
pr imary dietary sources of these fatty acids are mar ine fish . T he current high use of fish and fish
oi I leads to research towards the development of a viable alternative sustainable source of
PUFAs. Chapter 9 focuses on genes encoding the pr imary PUFAs biosynthet ic activities and
characterized them. Falty acid delta 6- desaturase (D6DES} and elongase (ELOVL5) are key
enzyn1es in the synthesis of PUFAs. Two plasmids pYES2 and pYES3 were used to
cotransfom1 into INVSc l for the coexpression of D6DES and ELOV L5 in yeast, respectively.
The cotransfom1ed yeast cell, designated as pY2-D6D and pY3-FA E produced y-linolen ic acid
(GLA) and di-homo-y-linolen ic acid (DG LA} from exogenous linoleic acid (LA) and
stearidonic acid (STA) and eicosatetraenoic acid (ETA) from a-linolen ic acid (ALA). In these
resulls, the recombinant A. schlegelii ~DES and ELOV L5 show act ivity on both n-6 and n-3
fatty acid substrates. Thus, these genes may play a cr itical role in the bin-production of both n-6
and n-3 PUFAs and could be good sources for PUF As synthesis in engineered oil seed crops.
As explained in Chapter 10, certain oils are a ri ch source of compounds called omega-3 ,
which are pol yunsaturated fatty ac ids that play an essent ial role in the human di et because of
their ability to prevent disease. These acids parti cipate in the product ion of hom10nes
involved in several physiological systems regulating pain and moisture, while maintaining
adequate blood pressure and o ptimal leve ls of cholesterol and promot ing nerve transmission.
Especiall y two fatly acids, both omega-3 type, the 5,8, 11 , 14, 17-eicosapentaenoic acid (E PA,
20:5ro3) and the 4, 7, I 0, 13, 16, 19-docosahexaenoic acid (D HA, 22:6ro3} are important
funct ional consti tuents of the human body.
The comp lexity in the preparation process of concentrated omega-3 compounds is mainly
based on the sim ilar physicochemical pro pert ies of the falty ac ids. In th is sense, it is di m cult
to separate them from the other fatty ac ids in the oil.
Simple fractionat ion processes do not discriminate between di l:lerent pol yunsaturated
fatty acids present in an oily mixture, so it is necessary to evaluate the di fferent available
al ternatives. Nowadays, at Ctlmmercial product ion scale, mol ecular distillation is the most
widely used technique.
Molecular distillation is a special type of v ery high vacuum di stillat ion, which takes place
in an apparatus constructed so that the distance that the mol ecules must travel between
evaporation and condensation is smaller than their mean free path . In th is way, it is possi ble
10 avoid an excessive exposure of the product 10 deteriorat ing conditions. This feature makes
molecular distillation a good alternative for app lication in the fract ioni ng process o f omega-3
fatly ac ids.
The main obj ect ive of th is chapter is to present a review where it is discussed, in a
theoretical and exper imenta l way, the process of fract ionation and concentration of ethyl
esters o f omega-3, usi ng the fa ll ing fil m mol ecu lar distillat ion.
..
XII
Theodore G. Bradley and Francisco P. Vargas
It is important to highlight that for obtaining a concentrate of omega-3, it is first

necessary to perform a transester ification stage for achieving the separation of the carbon
chain of the glycerol molecule. ln th is react ion, the most favorable condition is achieved by
workjng at high temperatures close to the bo iling point of the eth anol. Moreover, the
conversion of the react ion increases with increasing th e catalyst concentrat ion with in the
studied ranges.
As esters of omega-3 have an intermediate volat ili ty value between those of esters of
light fatty acids and the esters ones, it is requ ired to perfom1 two stages of molecu lar
dist illat ion. Working at low temperatures, both stages result in a low separation efTiciency. On
tbe other hand, working at very high temperatures tbe separation efficiency is not favorable,
but an opt imal po int in the process can be found.
Several works have shown mathematical models which opti mize mo lecular distillat ion
process, aimjng to find condjtions which allow a good recovery of ethyl esters of omega-3
fatty ac ids w ith a high degree of purity. Models also allow analyzing the influence of different
operating variables.
In: Eicosapentaeno ic Acid

Edi tors: T.G. Bradley and F.P. Vargas, pp. 1-46
ISBN: 978- 1-62257-480-3

2012 Nova Science Pub lishers, Inc.
Chapter 1
O MEGA-3 POLY UNSATURATED FATTY A CID

TREATMENT IN PARKINSON'S DISEASE ;
R EVIEW OF C URRENT E VIDENCE
Dirk W. Luchtman 1, .Jeffrey A. Zidichouski1 and Cai Sonl'4'
1
Department of Pharmacology, Univers ity of Alberta, Edmonton, Canada;

1
National Research Institute for Nutrisciences and Heal th, Charlottetown, Canada;
3
Chinese Academy Engineer Institute for the Development of Endangered Medicinal
Resource in Southwest of Cttina, Nanning, China,
4
Department of Psychology and Neurosc iences, Dalhous ie Univers ity, Halifax, Canada
Abstract
Parkinson's disease (PD) is a neurodegenermive disorder clinically characterized by motor
dysfunction but also mood disorders and cognitive impairmem. PD neuropathology is
characterized by the selective degeneration of dopami nerg ic neurons projecting from the
substantia nigra pars compacta (SNpc) in the mesencephalon (middle brain), leading to
massive dopaminergic denerva[ion in rhe srriatwn, bm mher neurmransminer sys[ents, in
panicular the noradrenergic system, and a-synuclein metabolism, are also affected. An
indispensable tool for acquiri ng new ins iglus imo the neuropathology and pathogenesis of PD,
and testing therapemics, is the 1-methyl-4-phenyl-1,2,3,6, -tetrahydropyridine (MPTPj model
of PD. Research with this model has demonstrated that oxidative stress, inflammation and
apoptosis are among the primary pathogenic factors. The currem therapy is limited to
symptom reduction, produces aversive side effects and, in some cases, can be quite invasive.
Increasing evidence suggest that namral, side-effect free omega-3 (n-3) polyunsaturated fauy
acids (PUFA) may be beneficial to PD, bm their mechanisms are complicated and findings
regarding their effects heterogeneous. However, neuroprmective, ami-depressam and
cognitive enhancing effects of n-3 PUFA have been widely reported in the comext of other
human neurodegenerative and psychiatric diseases and are relevam to the therapemic potemial
ofn-3PUFA in PD.
Email address: c.ai.song@dai.ca; Tel: 86~77 12443036; Address: Chines-e Acadenl)' Engine-er Jnstirute for the
Development of Endangere.d Medicinal Resource in Southwest of China, Nanning, China, 4Depanrnent of
Psychology and Neurosd ences, Dalhousie University, Halifax, Canada. (CotTesponding author)
Di rk W. Luchtman, Jeflrey A . Zidichouski and Cai Song

The first pan of this chapter describes the clinical appearance and the neuropathology of
PO, with particular focus on the neurochemical changes that occur in the disease as well as the
MPTP model. This is followed by an overview of the major PO pathogenesis, supported by
findings from the MPTP model. The middle pan provides a general accoum of n-3 PUFA
metabolism and function in the brain. The final pan first focuses on epidemiological studies of
PUFA imake and PO in the general population, and evaluates up to date evidence for the
effectives of n-3 PUPA against PO as obtained from clinical and animal studies. Findings
from our research group concerning the effect of omega-3 PUFA eicosapemaenoic acid (EPA)
in PO and related models are discussed in detail. The chapter will conclude with a summary
and propose currem limitations and future research directions.
This book chapter provides updated and important information for understanding how to
pmemially prevem PO from nmritional aspect and for understanding the mechanism of n3
PUFA in the treatmem of PD. The chapter also provides evidence for developing therapy
involving adj uvam n-3 fatty acids with DA-replacemem/agonist drugs. This chapter will also
be recognized by doctors, graduate studems and sciemists for exploring new research
directions in PD.
1. PD C linical Symptoms
PD is primari ly a motor disorder, but psychiatric symptoms, such as cogmt1ve
impairment and mood disorders are common (reviewed in Lauterbach (2004)). Four cardinal
motor symptoms of PD have been ident ifi ed as crucial for the diagnosis and grouped under
the acronym TRAP (!remo r at rest, rigidity, .!J.k inesi a!bradyk inesia and .eostural instability). In
addjtion, flexed posture and freezing (motor blocks) have been incl uded, among classic
features of park insonism (for a review of motor symptoms, see Jankovic (2008)).
Between 60 and 90% of PD patients report at least one psych iatric symptom, among
wh ich cognitive impaim1ent and depression are the most debili tating (see Farlow and
Cummings (2008); Lauterbach (2004) and Tbanvi et al. (2003) for general reviews of PD
psych iatry). Severe cognitive impairment and dement ia usuall y occurs in about 2044% of
patients, but the prevalence depends o n factors such as age, progression of the disease, male
gender, the presence of Lewy bodies and in some cases, A lzheimer's disease (A D). A PD
pati ent has a 6-fold increased chance of develop ing dement ia compared to heal thy controls.
However, the typical parkjnsonian cognitive impaim1em is more subt le and of a di fferent
nature th an the severe dement ia associated w ith advanced AD, and generall y involves
executive d ysfunct ion (Far low and Cummings, 2008; Thanvi eJ al., 2003).
2. PD Neuropathology
2.1. 1\igrostriatal Dopaminergic 1\europathology
The neuropatho logical hal lmark of PD includes the loss o f nigrostri atal dopaminergic
neurons in the SNpc, co-occuring w ith a depletion of striata l dopamine (DA), w ith relat ive
sparing of adjacent ventral tegmental neurons ( for a detailed review of PD neuropathology,
see Dauer and PrLedborski (2003)).
The nigrostrial dopami nerg ic pathway is one of four major dopami nergic pathways in the
brai n (for a detailed review, see Van den Heuvel and Pasterkamp (2008); Bjl\rkJund and
Dunneu (200 7) and Blandini et al. (2000)), but the nigrostriatal pathway is the one most
Omega-3 Pol yunsaturated Fatty Acid Treatment in Park inson's Disease.. .
affected and also the largest pathway, as it contains 70% of dopaminergic neurons and
const itutes about 80% of brain DA. Th is pathway proj ects from the SNpc mainly to the dorsal
(sen sorimotor) striatum, but proj ects also to the (more li mbic) ventral striatum, hippocampus
and cerebral cortex (Van den Heuvel and Pasierkamp, 2008; Bj ork lund and Dunnett, 2007).
The nigrostriatal dopaminergic pathway is integral in a complex set of interconnected
nucle i called the basal gangli a. PD is essentially a disorder of the basal gangli a, as the DA
dep letion, induced by degeneration of the SNpc, leads to a global dysfunct ion of the basal
ganglia. A complete functional anatomical explanation of basal gangli a dys funct ion in PD is
beyond the scope of this chapter, but f()r detailed reviews, see Galvan and W ichmann (2008);
Groenewegen (2003); Wichmann and Delong (2003); Herrero et al. (2002); Blandini et al.
(2000).
In studi es measuring the effect of candidate therapeutic compounds, such as omega-3
PUF A, on the integrity of nigrostriatal DA, the following components are usually targeted. The
first is obviously nigrostriatal DA itself, which can be depleted up to 85% or more in advanced
stage PD as well as MPTP-treated rodents (Luchtman et al., 2009; Dauer and Przedborski,
2003). DA, similar to other catecholam ines (such as noradrenaline (NA)), is der ived from a
common precursor, the am ino acid tyrosine. Tyrosi ne is converted to 3,4-dihydroxy-Lphenylalanine (L-DOPA) under the action of the enzyme tyrosine hydroxylase (TH).
Once synthesized by TH, L-DOPA is decarboxylated to DA by the act ion of aromat ic Lamino acid decarboxylase (AA DC) (for overview, see Purves et al. (200 1) and Kandel et al.,
2000)). Because TH is the rate-li miting enzyme for the synthes is of both DA and NA, its
presence is a valuable marker for identifying catecholam inergic neurons. With degeneration of
nigra! cell bodies, TH immunohistochemistry is often reduced in the SNpc of MPT P treated
rodents and thus provides a valuable target for assessing the effect of a therapeutic candidate
compound. Another important target is the dopamine transporter ( OAT). The DAT plays an
important role in re-uptaking DA into the pre-synaptic neuron at the (striatal) nerve tern1inals.
In PD and MPT P-treated animals, DAT protein expression is a valid marker for the
presence of dopaminergic nerve tern1inals and is found to be reduced in parall el with DA
dep letion and thus provides another important target. Aside DA T act ion, synapt ic DA can
al so be inact ivated by enzymat ic metaboli sm. DA metaboli sm by monoamine oxidase-B
(MAOB) results in the production of 3,4-dihydroxyphenylacetic acid (DOPAC) and DO PAC
metabolism by catechol-o-methyl transferase (COl'viT) in turn s leads to the product ion of
homovanilic acid (HVA).
A typical findi ng in PD and MPT P treated rodents is that DA is decreased, but its
metabolites DOPAC and HVA are decreased to a lesser extent. Hence, the metabolite/ DA
ratio provid es an index of the rate of DA turnover in the remaining dopaminergic nerve
tem1inals (Rabey and Burns, 2002) and is thus highly valuable in evaluat ing the etTect of a
therapeut ic compound on the functional as well structural integrity of the nigrostriatal
dopaminergic system.
2.2. 1\on-Dopamin ergic Ne uropathology

The major non-dopaminergic neurodegerat ion reported in PD is in the noradrenergic
system. The largest noradrenergic system in the brai n is the one ascendi ng from the locus
coeruleus (LC), which has extensive proj ections into the entire neocortex and the limbic
fore brain (amygdala, septum, hippocampus). Remarkably, both lesions of the LC or chronic
NA deplet ion decreases stri atal DA re lease and resu lt in the compensatory upregu lat ion of
striatal dopamine (D2) receptors (reviewed in Ronm1el fanger and Weinshenker (2007)). The
degree of cell loss in the LC has been est imated to be around 70% in advanced PD, simi lar to
the degree of cell loss in the SNpc. Neurodegenerat ion in the LC may even precede that in the
SNpc (Rommel fanger et al., 2007). The involvement ofNA and a possible protect ive funct ion
has also been indicated in animal models of PD (Rommel funger et al., 2007; Rommel fanger
et al., 2004; Bing er al., 1994; Mavridis et al., 199 1).
Wh ile serotonin (5-HT) has richer innervations of the nigrostriatal system than NA, this
non-catecholaminergic monoamine appears to have less close ties with PD neuropathology
and cli nical symptoms ( Rabey and Burns, 2002). For instance, prior destruction of striatal 5HT neurons did not affect DA dep let ions produced in the striatum by MPT P (Melamed et al.,
1986). However, increased 5-H IAA/5HT rat ios are found in the striatum, frontal cortex and
hippocampus of PD pat ients and MPTP treated m ice, suggesting increased 5-HT turnover in
these brain regions, wh ich cou Id be a compensatory mechan ism (Luchtman et al., 20 12; 2009;
Rozas et al. ( 1998) and reviewed in Rabey and Bums (2002)). Furthermore, compensatory
sprout ing of serotonergic afferents into the striatum after MPT P intoxicat ion was found in
mice (Rozas et al., 1998) and monkeys (Gaspar et al., 1993 ).
2.3. Lewy Bodies

Lewy bodies (LB) are intra-cytoplasmic aggregates masses (plaques) of abnom1all y
fo lded cl-synuclein protein. They appear as rounded eosinophili c neuronal inclusions, and are
composed o f altered neurofi laments. In PD, LBs mainly occur in selected sub-cortical brain
regions, including the SNpc and LC. However, the ro le ofL Bs in PD neuropathology has also
been debated, as they are not unique to PD, and animal models that cause select ive
nigrostriatal neuropathology (such as the lv!PTP model) do generall y not develop LBs (Dauer
and Przedborsk i, 2003), even ! 0 years after MPTP administrat ion in monkeys, despite the
presence of park insonism (Halliday et al., 2009). Nonetheless, as "~ 11 be further discussed
below, there is an intricate relationsh ip between Lewy bodies and PUFA.
3. The MPTP Model of Parkinson's Disease

There has been a great var iety of animal models of PD, mostly tox in-induced, including
reserpine (Carlsson et al., 1957, reviewed in Betarbet et al. (2002) and Gerlach and Riederer
(1 996)), 6-Hydroxydopamine (6-0HDA) (Ungerstedt, 1968, reviewed in Hirsch (2006);
Dauer and Przedborsk i (2003); Betarbet et al. (2002) and Gerlach and Riederer (1 996)),
pest icides paraquat and rotenone (reviewed in Hirsch (2006); Dauer and Przedborski (2003)
and Betarbet et al. (2002)) and lvlPTP (Dauer and Przedborski, 2003 ). Newer generat ion
models also include gene mutations (Dauer and Przedborski, 2003). The discovery of
neurotoxicant MPTP in the early 1980s boosted PD research significantly. A group of young
chemists injected themselves with a street preparation of meperidi ne (Demerol), wh ich was
contaminated with MPT P. Th is resulted in a parkinson ian syndrome that was, clinica ll y,
indist inguishable from idiopathic PD ( Langston and Ballard ( 1983) and reviewed in Dauer
Omega-3 Pol yunsaturated Fatty Acid Treatment in Park inson's Disease...
and Przedborsk i (2003)). At a neuropathological leve l, the human and primate mo dels mimic
most maj or features of PD neuropathology (Herrero et al., 1993; Moratalla et al., 1992;
Herkenham et al., 199 1). The overall validity and value of the MPT P human and non-human
primate model cannot be disputed ( Dauer and Przedborski, 2003).
The first papers on the MPTP mouse model appeared in the mid eight ies (Pileb lad and
Carlsson, 1988; Pil eblad et al., 1984; Hall man et al., 1984; Heikk ila et al., 1984 ). it was soon
discovered tha~ similar to the primate model, the mouse model also features simil ar
neuropathology as in humans, includi ng loss of striatal DA and select ive degenerat ion of
nigrostriatal neurons. S imil ar to human PD, MPT P in mice may also affect non-dopaminergic
and extra-str iatal neurotransmitters, including 5-HT in the striatum (Luchtman et al., 20 12;
2009; Rousselet et al., 2003; Rozas et al., 1998) an d frontal cortex (Luchtman et al., 2009;
Vuckovic et al., 2008), DA in the frontal cortex (Luchtman et al., 2009; Vuckov ic et al.,
2008; Rousselet et al., 2003; Fredriksson et al., 2001); and NA in the striatum (Luchtman et
al., 2012; 2009; Rousselet et al., 2003) and frontal cortex (Rousselet et al., 2003). Simil ar to
the primate model, the lv! PTP mouse model does not readil y induce the presence of LBs
(Shimoj i et al., 2005; Dauer and Przedborsk i, 2003), although indications of a ltered asynuclein processing have been reported in several studi es (J in et al., 2005; Meredi th et al.,
2002 and Vil a et al., 2000) . MPTP, or its active metabolite, M PP+, can also be applied in exvivo or in-vitro sett ings, such as cell li nes, primary cells or brain slices (lvleng et al., 20 10).
3.1. M l'Tl' Mechanisms of Action

The lipoph ili c pro-neurotoxicant MPTP rapidly crosses the BBB and becomes metabo lized
to 1-methyl-4-phenylpyr idinium (MPP+). l'v!PP+ is far less lipophilic than MPTP and needs,
similar to 6-0H DA, the membrane transporter to enter neurons (Dauer and Przedborsk i, 2003).
The fact that MPP+ has varying degrees of af'!'inity to the DAT, noradrenaline transporter (NET)
and serotonin transporter (SERT) exp lai ns that the toxin affects multiple neurotransmitter
systems, sim il ar to PD (Jonsson et al. ( 1986); Pill et al. ( 1996) and reviewed in Dauer and
Przedborski (2 003)) . Once inside neurons, lv!PP+ can have several targets, as reviewed in detail
in Dauer and Przedborski (2003). The pr imary target of Iv! PP+ is the mitochondrion, in
particular comp lex- I of the e lectron transport chain. Mitochondrial dysfunction, although toxic
by itself, may cause secondary effects, such as ox idative stress and apoptosis. MPTP can also
cause inflammation, possibly as a secondary factor.
3.1.1. Oxidative Stress

The oxidat ive stress hypothesis postulates that react ive oxygen (ROS) and react ive
nitrogen species (RNS) cause oxidat ive damage to cell ular macromolecu les (Drechsel and
Pate!, 2008; Dauer and Przedhorski, 2003) . MPP- causes co ll apse of the mitochondrial
membrane potential ((A 'I m), leadi ng to an increase in ROS production (Schapir a, 20 I 0;
Drechsel and Pate!, 2008; Dauer and Przedborski, 2003). Pro-oxidant enzymes
NADPHox idase and cyclo-oxygenase (COX)-2 also play a role in neuronal or glial ROS
production, wh ile nitric oxide synthases (NOS) catalyze the production of nitric oxide (NO).
The role of these enzymes in MPTP neurotox icity has been demonstrated in mice Jacking
them that have increased resistance to the neurotoxin (Anantharam et al., 2007; Hoang et al.,
2007; W ang et al., 2005; Teismann et al., 2003; Wu et al., 2003; Dehmer et al., 2000).
Interestingly, these enzymes, including NOS, have been largely associated w ith acti vat ion of
microglia, but act ivation of their neuronal forms appears deleterious as well (Anantharam et
al., 2007; Hoang et al., 2009). As discussed further in sections below, COX-2 with cytosotic
phosphoplipase A2 (cPLA2) also plays a rote in metaboli sm ofn-6 PU FA arach idonic acid to
potentiall y pro-inflammatory molecules call ed eicosanoids. A deplet ion of major ant i-oxidant
glutath ione (GS H) in the SNpc and impai red function of ant i-oxidant enzymes further
aggravates the oxidat ive damage (Th omas et al., 2008; Zeevatk et al., 2007; Dauer and
Przedborski, 2003; de la Torre et al., 1996; Martt ila et al., 1988). In part icu lar P UFA may be
suscept ible to oxidat ive stress, wh ich can lead to membrane damage, the fom1at ion of
nonenzymatic adducts and crossli nk ing of proteins, as further discussed below . The presence
of a methylene group between 2 double bonds renders the fatty acid-sensitive to ROS-induced
damage, and their sensitivity to oxidat ion increasing exponentiatty as a funct ion of the
number of doub le bonds per fatty acid molecu le, DHA th us being the most susceptib le target.
(Dalf6 et al., 2005).
3.1.2. Inflammation
Increasing evidence suggests a role of inflammat ion in PD (Depino et al. (2003); Boka et
al. (1 994); Lawson et al. (1 990) and reviewed in Nagatsu et al. (2000ab); Minghett i (2004)
and Teismann and Schu lz (2004)). Th is may be particu larly relevant to therapy w ith n-3
PU FA due to their potent ant i-inflammatory effects . Microglia and astrocytes, wh ich are the
primary mediators of inflammation in the brai n, can be activated by tissue damage, wh ich was
observed in humans after lv!PT P intoxicat ion ( Liu and Hong, 2003; Langston et al., 1999) .
The SNpc is the brain region w ith the highest density o f rest ing microglial cells (Laws on et
al., 1990) and robust microglial act ivat ion has been reported consistently in PD pat ients
(Teismann and Schulz, 2004). M icroglia mediate toxi c inflanm1atory react ions, such as the
release of pro-inflammatory mediators, including cytokines, prostaglandi ns, RNS and ROS
(van Noort, 2006 ; Liu and Hong, 2003; Teismann et al., 2003). Upregulation of major
histocompatibility complex (MH C) molecules (McGeer et al., 1988), increased production of
pro-inflammatory mediators, including interleuk in- 1[) (lL- 1[)), IL-6, interferon- y (TFN-y) and
tumor-necrosis factor-et (TNF-a ), and increased act ivation of COX-2, with resultant increases
in ROS and pro-inflammatory arach idonic acid derived prostaglandin E2 (PGE2), have al l
been reported in the SNpc of PD patients (Depino et al. (2003); Boka et al. (1 994) and
reviewed in lvti ngheui (2004)), all indicat ing inflammatory changes in PD. Robust microglia
act ivat ion and cytokine production has also been observed in primates and rodents after
MPT P intoxicat ion (for a review, see Teismann and Schu lz (2004)). The importance ofTN FCt in MPT P neurotoxi city was indicated by the fact that TN F and TN F-a receptor (TN FR I)
ablation made mice resistant to lv!PT P (Leng et al., 2005; Ferger et al., 2004). Moreover,
genet ic delet ion or pharmacological inh ibition of COX-2 and cP LA2 were found to attenuate
?,1PT P toxi city to dopaminergic neurons (Okuno et al., 2005; Hunot et al., 2004; Teismann et
al., 2003; Ktivenyi et al., 1998), indicat ing an important rol e of these fatty acid metabolism
enzymes in PD pathogenesis. Fi nall y, anti -inflammatory mediators, such as non-steroid antiinflammatory drugs (NSA !Ds), were associated w ith a reduced risk of developing PD
(Wahner et al., 2007) and attenuated MPTP toxici ty (Asanuma and Miyazaki, 2008; Koprich
et al ., 2005).
Omega-3 Pol yunsaturated Fatty Acid Treatment in Parkinson's Disease. ..
3.1.3. Apoptosis
Post-mortem evidence suggests that apoptosis may be the primary fom1 of cell death in
PD, although confl ict ing resu lts have emerged. One of the reasons for th is is that apoptotic
cell lo ss in PD may occur over many years, and the clearance of apoptotic cell s is a rapid
process, making it hard to measure apoptois (see Dauer and Przedborski (2003) and V ila and
PrLedborski (2003) fo r reviews of apopwsis in PD). Invest igat ions at molecular level in postmortem brain t issue of PD patients have revealed cells posit ively staining for Bcl-2 ant i-and
pro-apoptot ic proteins, and caspases, suggesting that apoptosis does occur in PD.
Furthem10re, abundant ev idence suggests that lv!PT P triggers apoptosis, although it depends
on dosing regimen. Convincing evidence f()r a role of apoptosis in lv!PT P was shown by the
finding that pro-apoptotic bax ablat ion prevents dopaminergic cell death in mice (Vi la et al.,
200 l ) and that over-expression of ant i-apoptotic bcl-2 can protect mouse neurons from M PT P
neurotoxicity (Yang et al., 1998; Often et al., 1998). A link between inhibition of complex- !
inhibit ion, oxidat ive stress and act ivation of bax was shown to be crucial in M PT P
neurotoxicity (Perier et al., 2007) .
4. PD Therapy
Despi te massi ve investments in basic and clinical research and deve lopment, effect ive
and safe treatments of PD remain elusive. Prevent ion or cure is current ly not possi ble. LDOPA, the most common treatment of PD for over 30 years, although effective at
suppressing Park insonian symptoms, produces aversive side-effects in 75% of pat ients,
especiall y ail er 5- 10 years of cont inued treatment. L-DOPA also does not retard or cease the
progressive neurodegeneration. Other drugs include DA agon ists, as well as COMT and
MA.OB inhibitors.
There has been increasing interest in pro-drugs, to increase the bioavai lab ili ty and
pham1acological activity of anti-parkinson ian drugs (see Sozio et al. (2012), Ll edo (2000) and
Rezak (2007) for further details about L-DOPA or other pham1acotherapy). Deed brain
stimu lat ion (DBS) is another fom1 of therapy effective at symptom reduct ion, but is invasive
and can on ly be peribrmed in cogn it ively intact indi viduals, as they need to be ab le to
understand the procedure, its risks and benefi ts (Massano and Garrett, 20 12). Due to the proinflammatory and pro-oxidant nature of PD, anti-inflammatory and ant i-oxidant drugs have
been tested for therapeut ic efficacy. However, anti-i nflammatory treatments have been shown
to have some adverse side effects. For example, COX-2 inh ibitors increase the risk of
cardi ovascular disease ( Peura and Goldkind, 200 5). Furthermore, while effect ive at
suppressing inflammat ion, these d rugs are not des igned to effect ively modulate th e reso lut ion
of inflanmmt ion, an important component that allows the return from inflammat ion to
homeostasis in a finely tuned manner (Serhan et a l., 2008).
Although several anti-oxidants fail ed to protect against lv!PTP induced neurotoxicity
(Dhanasekaran et al., 2006), other anti-oxidant and anti-inflammato ry compounds have been
shown to be beneficial (C ieren et al., 2005; Yang et al., 2005).
lvlore useful wou ld be a treatment that can be admin istered chronicall y and produces
subtle changes in the neuronal environment well before the disease develops, without causing
side-effects. Polyunsaturated fatty ac ids (PU FAs) are very promising candidates for such a
Dirk W. Luchtman, Jeflrey A . Zidichousk i and Cai Song
therapy. They can be administered chronically, w ith vi rtuall y no side-effects, and mediate
myriad of benefical cellular and systemic effects, di rect ly relevant to PD neuropathology and
pathogenesis. Interestingly, omega-3 (n-3) PU FAs have been reported to improve psyc.h iatric
diseases, such as depression an d sch izophren ia, but also neurodegenerat ive disorders,
including AD and HD (Puri et al. 2008; Green et al., 2007; Cole and Frautschy, 2006; ;
lvlartin et al., 2002 and reviewed in Cole et al. (2005); Young and Conquer (2005) and Song
and Zhao (2007)). The scient ific exploration of the potential beneficial effects of n-3 fatty
acids in PD has on ly just begun. To date, recent studies invest igating the relat ionship between
brain n-3 PUFA and PD (Fabelo et al., 2011; Ju li en et al., 2006), or the effects of n-3 PUFA
in (experimental) PD (Luchtman et al., 2012; Yakun in et al., 2012; Shchepinov et al., 2011;
Ozsoy et al., 2011; De Franceschi et al., 2011; Bousquet et al., 2011; Riedel et al., 2011;
Tanriover et al., 2010; Meng et al., 2010; Delalire et al., 2010; Muntane et al., 2010; De
Franceschi et al., 2009; Bousquet et al., 2009;; Kabuto et al., 2009; Liu et al., 2008; Bousquet
et al., 2008; Samadi et al., 2006; Broersen et al., 2006) are current ly Jjm ited (reviewed in
Bousquet et al., 2011 ).
Furthermore, only a few recent epidemiological studies investigating the relationsh ip
between dietary intake of n-3 PUFA and PD have been reported (Aden et al., 2011 ; Miyake et
al., 20 I 0; de Lau et al., 2005; Chen et al., 2003 and others (see below)). No randomized
controll ed cli nical studies have yet been perfom1ed, except one by da S ilva et al. (2008) who
investigated the effect of n-3 PUFA against depression in PD. A ll the evidence w ill be discussed
in the sections below, preceded by a general account ofPUFA metabolism in the brain.
5. PUFAs
Omega-3 and omega-(n)-6) fatty acids are vital fbr th e brain, constitut ing about 30-35%
of total brain fatty acids. In the mammalian brain, lipids const itute 50-60% of the dry weight,
and the major brain li pid class is phospholipids (Youdim et al., 2000). In phosphol ipids, the
most abundant n-3 and n-6 P UFA are respect ively docosahexaeno ic acid (DHA: C22:6, n-3)
and arachidonjc acid (AA: 20:4, n-6) (Y oudim et al., 2000). See Figure 1 fbr a simple
di agram of brain fauy acid composit ion. Important ly, n-3 similar to n-6 PUFAs are dietary
essential, as th e body cannot synthesize them itself, and are critical for th e development and
function of mammali an brain and, in case ofDHA, also ret ina (Yo udim et al., 2000). DHA,
the major n-3 PU FA in phospholi pids and eicosapentaenoic acid (EPA, C20:5, n-3), an
another important membrane n-3 PU FA, although far less abundant, are long chain n-3 PUFA
that can either be directly obtained from dietary sources, in part icu lar fish oils (fauy fish), or
enzymaticall y derived from al pha- linolen ic acid (A LA, C J8:3, n-3), which is an n-3 fa!!y
acid der ived from vegetable sources, such as flax, soy, rapeseeds and walnuts. The major n-6
PUF A is AA, wh ich can he deri ved di rectly from meat and dairy product s, or enzymaticall y,
from lino leic acid (LA, C l 8:2, n-6) in vegetable sources. In Western soc iety, the di etary
intake of LA is typ icall y 5-20 times higher than that of A LA (Calder, 2012; Youdi m et al.,
2000). See Figure 2 for a si mple overview ofP lJFA metaboli sm.
Omega-3 Pol yunsaturated Fatty Acid Treatment in Parkinson's Disease...
Brain lipid profile
Figure I. Grain lipid profile (rat). Grain li pid profile, adapted fro m Palsdoccir et al. (2012) and
Luchunan and Song (20 12). C l8:0: stearic acid; Cl8:2, n-6: linoleic-acid (LA); C20:3, n-6: Dihomogamma-linolenic acid (DG LA); C20:4, n-6: arachidonic acid (AA); C22 :5, n-6: Docosapemaenoic acid
(DPA); Cl8:3, n-3: Alpha-linolenic acid (ALA); C20:5, n3: Eicosapemaenoic acid ( EPA); C22:5, n-3:
Docosapemaenoic acid (DPA); C22 :6, n-3: Docosahexaenoic acid (DI-IA).
P UFA metabolism
1 a~~:e J
t''''''' ~;..3
Linoleic acid (LA) C18:2 n-6
A lo ha-linolenic acid (ALA) C16:3n-3
Stearido nic acid (SDA) c 18:4n -3
Gamma-Linolenic acid (GLA)

C18:3n-8
Eicosatelraer.o ic acid C20:4 n-3
Oihomo-gamma linolen ic acid (D G LA) C20:3n-6
E icosapent aenoic a-cid (EPA ) C20:5 n-3
A rachidonic acid (AA) C20:4n-6
tt,C
'''''' ,1
H, C
Oocosalelraenoic acid C22:4 n-6
COOH
Oocosapentaeooic acid (OPA ) C22:5 n-3
l
Docosah exaenoic acid rDHAl C22:6 n-3
Oocosapen taenoic acid (DPA) C22:5n--6

H,)C
A , COOH
Figure 2. Overview of fatty acid metabolism. Schematic represemacion of the chemical scrucmre and
conversion of omega-6 PUFA linoleic acid (C I8 :2, n-6) and omega-3 PUFA alpha-linolenic acid
(C I8:3, n-3) to their longer-chain mecabolices (adapted from Luchunan and Song (2012), Calder (2012)
and Youdim et al. (2000)). Note chat the final metabolic seep from DPA (C22:5, n-3) to DHA has also
been suggested eo involve delta-4 desamrase and beta-oxidation (\'larcinez et al., 2010).
10
Dirk W. Luchtman, Jeflrey A . Zidichousk i and Cai So ng
5.1. Uptake and MetaboHsm of Omega-3 PUFAs

The importance of PUF A in neuronal health and function is suggested by the rigid
maintenance of a un ique membrane fatty ac id composition w ith high levels of palmitate and the
polyunsaturated fatty acids (PU FA), includ ing DHA and AA, but very low levels of other
PUFA, including EPA, ALA and LA (Youdim et al., 2000). In order to maintain relatively
constant levels of PUFA, the brain relies on PU FA uptake from plasma, which may be supplied
from the diet and/or the liver (Demar et al., 2005). Recent efforts have shed light on the means
of transport by wh ich PUFA are transported to the brain, wh ich until recently was a matter of
controversy and thought to invo lve lipoproteins or plasma proteins such as albumen. Using the
in situ brain perfusion techn ique, Ouellet et al. (2009) demonstrated that both EPA and DHA in
free form are rap idly transported through the BBB as freely di ffusible lipoph ilic drugs.
S ince di fferent PU FA appear to be equall y well absorbed in the brain, th is cannot explain
the un ique profi le of fatty acids in neuronal membranes. For instance, brain phospho lipids are
much more enriched in DHA than EPA (Chen et al., 2011 ). 1v!uch of this unbalanced
di stribut ion of membrane PUFA has to with the unequal the part iti on ing of these fatty acids
between degradat ion and esteri fication processes. For instance, wh ile A LA can rapidl y
di ffuse from plasma to brain, it was demonstrated in adult rats fed a diet enriched in both
DHA and A LA that the majority of ALA in the adult brain is recycled through P-oxidat ion;
the remaining is incorporated unchanged into ph ospho lipids (I 0%) and tr iglycer ides (2%) or
as new ly synthesized DH A (< 0.2%) (Del\,lar et al., 2005). Simi larly, more than 86% of
uptake plasma LA is [l -ox idized in the brain and on ly a small fract ion (< I%) is used to
synthesize AA (DeMar et al., 2006). A simi lar iate was reported for EPA. For instance, Chen
eta/. (2011) reported that of intracerebroventri cularly (i.c.v) infused radi o lebe led EPA, afl er
4 days, on ly 9% of intact EPA was recovered, the remaining wasP-ox idi zed or lost by deesteri fication from phospholipids. Yet AA (Green et al., 2010) and DHA (Del\,lar et al., 2004)
are much better retained in the bra in, making a case f()r direct intake of these long-chain
PUF A rather than their shorter-chained precursors, if one were to ensure more efficient
accumulat ion of these PUFA in the brain. !t should be noted that in rats fed inadequate
amounts of DHA, conversion of A LA to DHA in the liver is upregulated, thereby establishing
nom1al brain levels of DHA, as long as A LA is provided (DeMar et al., 2006). Thus, in cases
of liver damage, fail ure or age-related impairment of liver funct ion, one may need di etary
intake of DHA to establish nom1al leve ls of DHA in the brain. Furthem10re, neuronal
membrane n-3 PUFA Ctlntent may decrease with age and neurodegenerative diseases (Yehuda
et al., 2002). L-DO PA treatment in PD patients was also shown to decrease brain DHA
content (Ju lien et al., 2006).
5.2. Function of Omega-3 PUFAs

Once in the brain, PU FAs ( includi ng AA and DHA) are converted by an acyi-CoA
synthetase in acyi-CoAs and are then esterified into glycerophospho li pis at the sn-2
(stereospeciiic numbered) position via the acyi-CoA transferase. The effects of PU FA on
cell ular function are complex but, summarized, include but are not limited to: I) modulating
the structure and function of li pid rail s and neuronal membranes, inc ludi ng raft an d
membrane-incorp orated proteins, such as channels, recepto rs and signalingproteins; 2) act ing
Omega-3 Po lyunsaturated Fatty Acid Treatment in Park inson's Disease.. .
I!
as direct ligands to transcript ion facto rs of genes that play a role in a w ide variety of
processes, including fatty acid metabo li sm, neurogenesis and synapto genesis, di fferentiation,
infl ammat io n and ox idat ive stress; 3) act ing as precursors to biosynthesis of lipid medi ators
which are invo lved in regu lat ion of many cell and t issue responses, particularly aspect s of
infl anm1ation. Dietary intake of PUFA and the ratio of ingested n-6: n-3 PUFA can in turn
atr ect how PUFA afr ect a ll these cell ular funct ions (Ca lder, 20 11 ).
5.2.1. Mem brane Fluidity

Long-chain esteri fied PUFA play an important ro le in modulat ing the structure of
neuronal membranes. The lipid bilayer tends to exist at an optimum transition po int between
gel and li quid crystal, and the maintenance of this state, o Ren referred to as fluidity, is of
physio log ical importance and can be strongly infl uenced by fatty acid composit ion.
Transmembrane and peripheral proteins of various shapes, mo lecular masses and charges
(receptors, ion chann els, and membrane bound-enzymes) regulate important cellular funct ions
but their position, integrat ion and funCl ioning are all affected by membrane fl uidity and thus
fatty acid composit ion.
Especiall y the double bonds in PUFA, wh ich are exclusively in the cis confornmt io n,
have strong infl uence and the replacement of even a single doub le bond in these PlJ FAs is
suffi cient to exert a profound e ffect on the physical properties of the membrane (Frisardi et
al., 20 11 ; Youdi m et al., 2000). Fatty ac id composition of neuronal membranes can be
directly altered by di etary modi !I cations. Membrane fl uidity also changes w ith age and
neurodegenerat ive di seases, for various reasons, including increased content of cho lesterol
and ROS (Yehuda et al ., 2002). Rep Jenishment of PUF A by means of di etary intake may
compensate for these neu rodegenerat ive changes (Yehuda et al., 2002), although the risk of
peroxidabil ity of PU FA is a matter of debate.
5.2.2. Gene Expression

PlJ FAs are known to medi ate w id espread e lrects on (dopami nergic) gene expression in
the CNS (Calder, 2011 ; Kuperste in et al., 2008; 2005; Kitaj ka et al., 2004; Rojas et al.,
2002), either as free ligands, or th rough their eicosano id metabo li tes. Putat ive targets of n-3
PU FA in regulat ing gene expression include ligand-activated transcript ion facto rs, includ ing
the ret ino ic acid receptors (RA Rs), retino id X receptors (RXRs), and perox isome pro li feratoract ivated receptors (PPA Rs). Reti no id signali ng pathways have been impli cated in regu lating
synaptic plast icity and learn ing and memory in rodents (Dyal et al., 20 I 0; Dyal and M ichaeiTitus, 2008; K itaj ka et al., 200 4). Another important tr anscript ion factor invo lved w ith n-3
PUF A is cA MP response element binding protein (CRE B), wh ich is important f()r synapt ic
integrity and synaptic transmission and largely be longing to the CRE B l transcript ion
pathway. CRE B !-d ependent gene expression plays an important ro le in learn ing and memory
in mammals and is invo lved in hippocampal long tem1 potent iation (LTP), the
neurophysio logical process underlying learn ing and memory (S idhu et al., 2011 ; Cao et al.,
2009). Thus, these w idespread effects of PU FA cou ld be relevant to the cogn it ive and
neuronal impairment associated w ith PD . Brain-der ived neurotroph ic factor (B DNF) is a
major neurotroph ic factor transcribed by CRE B I and plays an important rol e in neuronal
survival (Rao et al., 200 7). Through these effects on gene expression, PUFA play a ro le in
12
neurogenesis and synaptogenesis (reviewed in Suet al., 2010). PPA Ry, wh ich PUFA can
bind to, is ab le to di rectly downregulate inflanm1atory gene expression, but it also interferes
" i th the activat ion of N FKB, a maj or in fl ammatory transcript ion factor responsible for the
induct ion of pro-i nflammatory cytokines and enzymes such as COX-2 and iNOS (Calder,
2011, Bordet, 2006). Interestingly, it was observed that PPARy agon ist piogl itazone
allenuated the MPTP-i nduced gli al activat ion and prevented dopaminergic cell loss in th e
SNpc (Bre idert et al., 2002), indicati ng the importance of PUFA-mediated P PAR act ivat ion
in neuroprotect ion.
5.2.3. Eicosanoid Production and Inflammatio n

Wh ile the lipid composition of neurons is rigid, it can be modi fied to a certain degree by
di etary alterations. For instance, increased intake of EPA over the course of 6-8 weeks can
sign ificantly increase brain content of EPA and metabolite DPA (C22: 5, n-3) in mice, even
though these are trace components of neuronal membranes (Luchtman et al., 20 12; lvleng et al.,
20 I 0). Pro longed intake of DHA w ill modestly increase brain DHA content, w ith a reciprocal
decrease in brain n-6 content, in particular DPA (C22:5, n-6) (Bousquet et al., 2008).
Conversely, comp lete long tem1 di etary n-3 PUFA deprivation w ill dep lete the brain of
DHA, but with a rec iprocal increase in brain n-6 con tent, in part icu lar DPA (n-6). In th is way,
the degree of unsaturat ion is more or less maintained, but the functi on o f these fatty acids can
be drast icall y di fferent. For instance, high levels of DPA (n-6 ) instead of DHA w ill lead to
cogn it ive impaim1ent, even tbougb these molecu les di ffer on ly by a single double bond
(Fedorova and Salem, 2006). Because of the increased amount of n-6 fatty acids in Western
di et (increased use of cereals, vegetab le oil s and meats), the ratio of n-6 to n-3 PUFA has
increased substantiall y during the last 150 years to 15:1- 17: I (reviewed in S imopou lus
(2002)), w ith potentiall y far reaching consequences for menta l and neurological health.
A maj or way by whjcb the rat io of n-3 and n-6 PUFA influence brain function is by
afTecting eicosanoid and docosanoid product ion. [t is beyond the scope of this chapter to
di scuss the e icosanoids derived from PUF A in detail , as there are excell ent reviews on this
complex topic (e.g. Calder, 2011; 20 10; Frisardi et al., 20 11 ; Russo, 2009; Serhan et al.,
2008; Farooqui et al., 2007; Youdi m et al., 2000). Th is section summarizes some of the most
important aspects.
Eicosanoids, wh ich include prostanoids (prostaglandins, tromboxanes, prostacyclins) and
leukotrienes are fast acti ng bioactive molecu les that are key medi ators and regu lators of
inflammat ion and immun ity and are generated from 20-carbon PUFAs, including AA and
EPA, by the metabolic processes summarized in Figure 3. Because of the relatively high
amount of AA in membrane phospho lipids compared to for instance EPA, this fatty acid is
iypicall y the major precursor for eicosanoid medi ators, wh ich are produced in great ly
increased amounts upon cellular stimu lat ion. Aside inflammat ion and inmmn ity, eicosanoids
also play a rol e in s leep, synapt ic plasticity and neurot ransmission. Rather than potent iall y
act ing at di stal sites, eicosanoids act in an autocrine or paracrine fashion (Tasson i et al.,
2008). Because of their amphiph ilic nature, eicosanoids can cross cell membranes and act on
their receptors on the ne ighbouring cell s (Frisardi et al., 2011 ).
Arachldonk acid (AA)
2-rtes
pro..MIIdt
e.s.
PGf,
.(.series
kllllolllf~tt~es
v.g.
Docosahexaenolc acid (DHA)
IOO{e ~nflammc;~toty
nu.
l
,_rillS
Elco...,enlaenolc acid (EPA)
p tO.SCIIIIMds
llpottiM
$."''"
leublrie~e~~
Arm. mllammatory
and I 1SOIVI ng
Less lnnammaty
o.""*
IYins
E.s.rie.
rftGMns
r-
lXA,
Arcl inf!aml'fi;S(Of)'-
and neutOptotecuw
u.g.
13
D~
'"-Cii;l
NP01
Figure 3. Eicosanoid!docosanoid production. Simplified diagram of eicosanoid!docosanoid symhesis

&om AA, EPA and DHA. PGE 2 : prostaglandin E2 ; LTB. : leukmriene B.; LXA. : lipoxin A.; NPD I:
"!europrotectin D I (adapted from Luchunan and Song, 2012).
The process of eicosanoid production may be init iated when a cell is stimu lated by
inflammatory stimuli, such as cytokines. This can trigger the action of phosphol ipases to
hydrol yze fatty acids from the neuronal membrane. Following detachment from phospholipids
by phosphol ipases, free fatty acids can be ox idized by cyclo-oxygenases (COX- I, a constitutive
enzyme and COX-2, induced by mostly, inflammatory conditions) and lipoxygenases (LOX).
Through COX metabolism, prostanoids are produced, whereas through lipoxygenases, the
Jeukotrienes are synthesized. These eicosano id~ a ll regulate the inflammatory response
(including the process of blood clotting), but their inflammatory nature depends on the fatty acid
source. Essent iall y, n-6 der ived eicosanoids have more potent and more pro-inflammatory
effects than n-3 derived eicosanoids. For instance, the activity of COX on AA leads to the
synthesis of 2-series prostanoids (e.g. prostaglandins E2 (PGE 2) prostacyclin 12 and
thromboxane A 2~ whereas COX activity on EPA generates 3-series prostanoids (e.g.
prostaglandins E3 , prostacyclin 13 and thromboxane A3) , which are less potent and less
inflammatory. Dietary modi fication can influence the production of eicosano ids. For instance,
PG E1 production is increased by AA feeding and decreased by EPA or DHA feeding. However,
the role of AA-derived eicosanoids as solely pro-inflammatory is an over-simpli fication as
PGEz was shown to have both pro- and anti-infl anm1atory effects. Furthem1ore, another
eicosanoid derived from AA, lipox in A., is anti-inflanm1atory.
Resolution of inflammat ion was defi ned in molecu lar terms relat ively recent ly but it is
now considered to be a dist inct process fr om ant i-i nflammatory processes, in that it is more
concerned w ith the "clean ing up" stage, such as the removal of apoptotic cells and
microorgan isms. Thus, whil e pro-resol ving molecu les are not trul y immune suppressors, they
act ivate specific mechanisms to promote retum to homeostasis. Lipox ins, but also EPA and
DHA-derived resolvins play an important role in th is process (Serhan et al., 2008). DHA can
also give rise to neuroprotect ins (docosanoids), biosynth esized via a li poxygenase-medi ated
14
Dirk W. Luchtman, Jeflrey A . Zidichouski and Cai So ng
pathway, one of wh ich, neuroprotect in D l (N PD I), documented by Bazan and coll eagues
(Bazan, 2009), is particular potent, can be induced by tox ic conditions, such as oxidat ive
stress, and plays a role in resolving inflammation and promot ing cell survival. N PD I can
inhibit N FKB and COX-2-mediated inflammat ion as well as ailenuate bcl -2 mediated
apoptosis. The protect ive role of N PD I is PD is not yet known, but has been documented in
AD (Bazan et al., 20 11 ).
Thus, through production of resolvins and protectins and promot ing return to homeostasis
!hllowing inflammat ion, n-3 PUFA may o!Ter important bene tits compared to for instance,
select ive COX-2 inhibitors, which merely suppress infl ammat ion and may even impair the
process of resol ut ion (Serhan et al., 2008). In sum, there are two major ways by wh ich n-3
PUF A modulate intlanunation and inmllln ity; by act ing on the transcription of inflammatory
genes, largely through PPA R act ivat ion, and through the modulat ion of eicosanoid and
docosano id product ion. Furth em1o re, d ocosanoi ds, such as N PD I can provide di rect
neuroprotection.
5.2.4. Syuaptic Function and ~eurochemistry

PUFA have w idespread e ffects on synapt ic function, integrity and neurochemistry, as
touched upon in previously. The exact mechanisms are unknown, but likely involve a
complex interp lay of synergistic effects on neuronal membrane structure and function, gene
expression and metabol ism to eicosanoids!docosanoids.
The effects of PU FA on synapt ic function and integrity have been extensively
documented in the seliing o f brain develo pment (reviewed in Luchtman and Song, 2012).
Indeed, a number of recent in-vitro and in-vivo studies have directly related DHA de fic iency
or supp lementat ion to major aspects of synapse develo pment and funct ion, including neurite
outgrowth (Robson et a .., 20 I 0; Cao et al., 2009; Calderon and Kin, 2004); neuron size
(Ahmad et al., 2002); and the expression of pre- and post-synaptic proteins involved in
vesi cle tra!licking and recycling processes, as well as synapt ic transmission (S idhu et al.,
2011; Cao et al., 2009 ; Cansev and Wurtman, 2007).
Research over the past decade has demonstrated that quantitat ive changes in n-3 tatty
acids in the body are often parall eled by quant itati ve changes of the monoamine concentrat ion
in the brain, in particu lar the frontal cortex, nucl eus accumbens and the str iatum, but also the
hippocampus (Cha lon, 2006; Zimmer et a/., 1998; Chalon et al., 1998 ; Del ion et al., 199 7;
Del ion et al ., 1994).
Changes in PU FA intake, for instance by dietary dep letion during pre- and post-natal li fe,
but also throughout adulthood correlated with neurochemical al terat ions, including downregulation of the vesicular monoamine transporter (VMA T-2) and a deplet ion of VJ'viA Tassociated vesicles in the hippocampus (Chalon, 2006; Kuperstein et a l., 2008). A deplet ion
of pre-synapt ic vesicles can explain the neurotransmi ller depletion in condi tions ofn-3 PU FA
defici ency. Some of these neurochemical effects of n-3 PU FA dep letion can be restored by
di etary rep letion if done early in neonatal li fe (Cha lon, 2006 ). Furthermore, supp lementat ion
\\ith n -3 PUFA, including EPA, has substantial effects on monoamine and metabolite levels
in the brain of adult rodents (e.g. Luchtman et al., 20 12; Meng et al ., 20 11 ).
15
6. Omega-3 PUFA and PD: Hu man Studies

The previous sect ions on the general neuro logical effects of n-3 PUFA clearly ill ustrate
the enormous potent ial these faHy acids have in not only neuroprotection, but also general
improvement of brain fun ction, cogn it ive impai m1ent and elevat ing mood, the Jailer which
can be disturbed by neurochemical imbalances.
However, the investigat ion of the beneficial effects of n-3 in PD is only in its infancy.
Preceding an extensive review of evidence from experimental animal studies, this sect ion
explores evidence f:Tom studi es done so far in the human populat ion. Table I provides an
overview of observat ional and cli nical studi es involving di et and PD.
6.1. Findings from ObservationaJ and Clinjcaf Studies

1vlost studi es conducted in the ni net ies focused on total fat, or animal fat intake, w ithout
discriminating for types of fai. Nonetheless, some these studies do report a posit ive
assoc iat ion between (animal) fat intake (An derson et al., 1999; Johnson et al., 1999;
Logrosci no et al., 1998; Logrosci no et al. , 1996) or saturated fat intake (Johnson et al., 1999)
and risk of PD. Other studi es (lvli yake et al., 2010; De Lau et al., 2005; Chen et al., 2003;
Hell enbrand et al., 1996ab; Davies et al ., 1994) do not report such a relation. In tem1s of
PUFA and PD, the following observations were made. De lau et al. (2005) found total fat
intake, incl uding monounsaturated fatty acids and PU FAs to be correlated to a lower risk of
PD. Miyake et al. (2010) found arach idonic acid and cholesterol to be posit ive associated
\vi th PD, while other PUFAs, includi ng n-3 PUFAs and the rat io of n-3: n-6 PUFAs were not
related. Aden et al. (20 11 ) found PUFA intake in PD patients to be comparat ively low. While
these fi ndi ngs overall do point to a role of animal (saturated) Jat and n-6 PUFA in PD, the
results are inconsistent. A problem w ith these studi es is that they are observat ional and are
plagued by methodological problems such as retroact ive and select ion biases. PD patients are
asked to judge their diet ary habits, but the problem with such desi gns is that the disease itself
may a) affect dietary habits and b) affect memo ry of previous dietary habits due to cogn it ive
impairmeni. Prospective studi es are therefore better in that di etary hab its are assessed at
baseli ne and the selected populat ion then followed up, allow ing for some degree of causality
inference. In the prospective studi es per formed, Chen et al. (2003) did find a relat ionship
between PD and di etary intake of any type of macronutr ient, whereas de Lau et al. (2005) did
fi nd high PU FA intake to be related to lower risk of PD. Randomized doub le-bli nd controll ed
clinical trials (RCTs) are far superior for scientifi c explorat ion of causality, but no studies
have been perfom1ed yet to assess the relationship between dietary habits and PD.
One doub le-bli nd RCT pilot study by da Silva et al. (2008) invest igated the effect on n-3
PUF A supp lementation against major depression in PD (Table I) . Patients taking or not
tak ing ant i-depressant medicatio ns received four capsules of fi sh oil dail y for 3 months,
which resuJted in an increase of red blood ce ll membrane DHA and EPA content as well as an
improvement in standard depression scales (in all supplemented groups). Even if n-3 PUFA
were not e~fecti ve at preventing or curing PD, their well establi shed mood-i mproving e~fects
could be high ly usefu l to PD patients.
Dirk W. Luchtman, Jeflrey A. Zidichouski and Cai So ng
16
Table 1. Epidemiological and clinical studies investigating t he r elation between dieta r y

PUFA intake and PO (symptoms)
Design
Case.
c omrol
or
~Popula ti on
subj ects
15 patiems/ 5
age/se.x.matched
COIHI'O!S
Case
110 patients I 28:7
CO!lti'OI
COnti'OI.s
Case.
3 42 patiems/ 342
comrol
comrols
84 patiems/ 336
comrol!; {ftom
Pmspe.ctive 8006.man
H onolul u Hean
Studycohon )
Case.
comrol
Case.
CO!lti'OI
J 04 patiems/ 352
103 patients/ 156
Case.
126 patiems/ 432
comrol
comrols
... - 9 PD
cases m
. totat (thousands of
Prospe.cuve b'
su jtS \VIt 11 110

.>)
- . )
PO at balo e1m..
51 PO cases in
Prospective total (5 .289 with
no PO
at baseline)
Dietary measures
Outcome
Citation
Increased calorie, fib er, vitamin C and

7 day diary for assessmem of
folate intake, but no o thet differences.
Oavies et aL,
calories. mact'Onutriems.
Patients had towet body weig lu an d bod)' 1994
vitamins and minerals
fat
Increased ca!orie and macronutriem
imake.
Recall of dietary habits o f last
logroscino
Increased fat and ani n1a! fat, butnm
1-10 years.
e al., 1996
vitamins were associated with PD.
No di fference in bod>' weight patients.
Increased calorie {ca1bohydrate, bmnm
SU'ucmred imervie\v and a
protein m fat)} imake bm lo\ver body
Hellenbrand
self-adminisreted foodmass index {Bt\U) in patients.
et al.,
frequency queslionnai1e about Nodifference in f1uit/vegetable
1996ab
habits priOI'lOPO diagnosis c.onsumption, but patiems consume more
raw n1eat.
Dietary vitamin E intake was
While legume consumption was
assessed by food frequeJlCY
M01ens et
.
.
.
associated \Vith PO absence. vitamin E
quesuonnatres m a 1arge
.
.
:
al., 1996
sample and followed ~p 27 _30 uuake was not assoctated \VIlh PO
occurrence
years.
Semi-quantitative foodNo relationship between iron and PO, but
frequency questionnaire for
high animal fat imake and tow
logi'Oscino
ii'On and animal fat imake, as
transferring saruralion was associated
e al., 1998
\ve!t as measure of
with PO, suggesting lipid peroxidation
transfer1ing saruratiobn
45-m in interview of dietary
No !'e lation between fruitlvegetabte, imn
Andel'son et
habits during most of aduil
Ol'ami..oxidam imake and PO, but animal
al., 1999
fat was associated.
life
Significam associations between PO and
Food frequency questionnaire fat, saturated fat, chole.sterol, lmein and
Johnson et
to evaluate dietary intake
iron. H
al., 1999
dul'ing past >'ea1
Highe1ene1gy imake in patients, despite
!owe1 BM I.
food fi
.
No signifieant association between PD .
1-equencvquesuonnaue
. k d
.
\vas taken al b;seline, then PD ~nelrgd~ 11113 e l~ macronmnents,

Chen et al.,
m e u mg tota 1at.
assessed in 12 -18 year follow.
~
2003
No 1'elat10n between PO and fat t~pes,
up
.meIu d'mg nl0noun.saru1ated and PUFA .
A lo\ver risk of PD in the sample was
associated with total fat, monounsaturated
Complete dietary assessment
fa uy acids and Pllf As.
D e lau et
at baseline, then 6 year follow
Imake of dietary saturated fat, cholesterol, al., 2005
up.
or tran.s-fals \vere not associated to risk of
PO.
PO patiems had tov..er BMf, bmno
Casecomrol
2 49 patiems/ 368
COIUI'O IS
differemcalorie imake.
AA and cholestei'OI were positi vety
f.ood frequency questionnaire associated \Vith PD, butnm total fat,
saturated farry acids, monounsan11ated
fany acids, n-3 PUFA or the l'atio of n-3
lO
n-6 PUF A .
t\..1iyake et
al., 2010
Omega-3 Polyunsaturated Fatty Acid Treatment in Park inson's Disease.. .
17
T able 1. C o ntinued
Design
Case.
comrol
Rcr pit01
study
Population or
subjects
8 7 patients, 28
comrols
3 1 patients
Dietary measures
24-h dietary t-ecall h~s t to
assess dietary intake during
last 24 hours and 3-day
Outcom e
Citation
PO patients ' energy imake was

comparatively to\v in PUF As (while total Aden et al.,
fa t intake did nOL diffet) and carotenoid, 2011
while high in cat'bohydmtes.
diemty regisuation
Depressed patiems \vere
double-blind separated in 2
groups (fish oil Ot' placebo for Depressed symptoms improved in all fish
3 months} and e ach group was oil treated groups along with an in et-ease Da Silvaet
sub-divided in 2 nev.. groups: in erythrocyte DHA (and EPA nonone taking ami-dept-essant
significantly) levels
medication and anmhet one
not mking it
al., 2008
A>\= arachidonic acid; EPA= cicosapcntacnoic acid; DHA= docosahcxacnoic acid.
In parti cu lar E PA appears effective at restoring mood, compared to DHA (Song and
Zhao, 2007; Peet and Stokes, 2005). A lso, cogn it ively impaired or even demented PD pat ients
may benefit from n-3 PUFA treatment, as the lip ids are known to improve cognition in
cogn it ively impaired elderly (Cole et al., 2009).
Interestingly, as indi cated in Table I, a number of studies have cons istent ly reported
increased calorie intake in PD pat ients (.lohnson et al., 1999; Logroscino et al., !996;
Hell enbrand et al., 1996ab; Davies et al., 1994), or increased calor ie intake from carbohydrates
(Aden et al., 2011 ), desp ite sign ificant ly reduced body mass index (BM I), body weight and
body fat (M iyake et al., 2010; Johnson et al., 1999; Hellenbrand et al., l 996ab; Davies et al.,
1994). A lthough little is reported on energy expendi ture and sarcopen ia in PD patients
(D'Amelio et al., 2006; Poeh lman et al., 1995), these results suggest that PD pati ents may suffer
from these conditions. Omega-3 PUFA, in particu lar EPA may be effective at combating such
conditions, partially due to their anti- inflammatory eflects (Murphy et al., 2011 ).
6.2. Findings from Post-Mortem Studies

C lues about the role of PUFA in PD can be obtained from post-mortem fatty acid
analyses of PD brains, although in these cases, PD has already been established and thus a
di rect li nk between di etary P UF A intake and brain li pid changes as well as risk of PD cannot
be inferred from such studies. However, Dalfo et al. (2005) investigated changes in fatty acid
pron les and li p id peroxidati on in brains from non-treated individuals in early nonsymptomat ic cases but w ith verified incidental Lewy body disease-related ch anges (preclini cal or incidental PD). Remarkab ly, in such early stages, they already found increased
lipid peroxidat ion compared to contro ls in SNpc, but also neocortex (frontal cortex) and
amygdala. Total n-3 PUFA in the SNpc was decreased wh il e total n-6 P UFA remained the
same in th is area. In the cortex and amygdala, however, n-3 content, inc ludi ng h igh ly
perox idizable DHA was increased, whil e in n-6 content was decreased. These re-sults were
part ially corroborated in a more recent study by Fabe lo et al. (2011) who also reported
increases in high ly peroxidi zable DHA and AA in the fronta l cortex of PD patients,
suggest ing that the peroxidability index is increased in th is area . Juli en et al. (2006),
compared the temporal cortex fatty acid content between bra ins of 12 L-DOPA treated PD
18
patients and 9 aged-matched controls. Interestingly, higher levels of cerebral AA and total n-6
PUFAs were observed in PD pat ients w ith motor comp li cat ions, co mpared to patients without
these side effects or age-matched controls. They also rendered monkeys park insonian by
MPTP treatment and treated a subset of the animals with L-DOPA. Wh ile MPT P itself did
not have an effect on fatty acid profile, they did report an increase in AA, a decrease in DH A
and a decrease in the n-3:n-6 rat io in L-DOPA treated monkeys w ith motor complications.
Since the mo nkeys had similar dietary n-3 PU FA intake prior to MPT P treatment, th e authors
reasoned that the observed changes are due to disrupt ion of brain fatty acid metabol ism, and
that in part icu lar the increase in AA may be responsible for the motor comp li cat ions seen
with L-DOPA treatment.
Interest ingly, Samad i et al. (2006) reported that L-DOPA induced dyskinesias in MPT P
treated monkeys can be prevented by DHA treatment pr ior or after L-DOPA. Wh il e these
studi es overall do show somewhat inconsistent results, it is cl ear that PD already in an ear ly
stage involves changes in brain lip id profil e and metabol ism, which could be enhanced by LDOPA treatment. The exact mechanisms of changes in brain lipids in PD are unknown and
need to be further invest igated, but lipid peroxidat ion may be one mechanism (Fabelo et al.,
2011) . A lso changes in fatty acid metabol ic enzymes, inc ludi ng phospholipases, are li kely
involved ( Fabelo et al., 2011 ). For instance, as ment ioned above, mice lacking cPLA2 are
resistant to MPT P neurotoxicity (Kii venyi et al., 1998).
7. Omega-3 PUFA a nd PD: A nimal Studies

The number of animal studies invest igat ing the effect o f n-3 PU FA on experi mental PD
are steadil y increasi ng, most ly with posit ive results, although w ith variation as well. T able 2
provides an overv iew of studies performed to date, including all the measures (includi ng
behavior, nigrostriataVextra-nigrostriatal neurodegeneration, oxidative stress, inflammat ion,
and apoptosis) perfo rmed in those st udies. The next sections review fi ndi ngs from these
studies for each measure seperaturely.
7.1. Motor Symptoms and Complica ting Factors with the MI'T I' Model
lvlotor symptoms may be the most important aspect of an exper imental PD model, in
terms of valid ity as well as therapeut ic relevance, yet very litt le bebavioral studi es have been
perfom1ed to invest igate the effects of n-3 P UFA on motor behavior in experimental PD,
perhaps due to a number of methodo logical facto rs that makes the lviPTP model int imidating.
Achieving sign ificant and reproducible motor impaim1ents in the MPTP-treated laboratory
rodent is very hard to achieve compared to non-human primates ( Luchtman et al., 2009;
Sedelis et al ., 200 I; Rousselet et al., 2003; Meredith and Kang, 2006) and even if motor
impairment does take place, bebavioral recovery may follow w ithin 24 ho urs. MPT P and
MPP+ are much mor e rapidl y cleared from the brain in mice than in monkeys and humans . In
rats, metabolic c learance of MPT P and lv!PP+ is even more rapid, which may explai n why
rats are even less sensit ive to MPT P th an mice (Giovanni et al., 1994 ). W ithin the mouse
species, there are strain di fferences in MPT P suscept ibility. For instance, C57BL/6 mice clear
l'vi PT P and metabolites less rapidl y fr om the brain than white mice, which may explain why
19
the black mice are more susceptible to MPT P than their white counterparts (R iachi et al.,
1988). Even if mouse strains are kept consistent, other variation in experimental factors, such
as the dosing regimen used, could influence the extent of neuropathological damage as well
as the ability of the brain to employ compensatory mechanisms and engage recovery. In
addit ion, the type of dosi ng regimen used may infl uence whether non -dopaminergic and
extra-str iatal neurotransmitters, such as NA are affected (l uchtman et al., 2009;
Rommel fanger et al., 2007; Meredith and Kang, 2006). Thus, i fa study investigated the effect
of n-3 PUFA agai nst motor impairment, the result shou Id be interpreted carefull y and retested in other dosing regimens and behavioral tests. Ideall y, in the experimental PD society,
there shou ld be progress towards a standardized lvfPT P model of PD w ith standardized
behavioral tests for measuring motor impairment.
Indeed, another important aspect in testing motor behavior with the M PT P model is the
behavioral tests chosen. W hen a rodent init iates movement, motor input from the cortex, afier
strong modulat ion by the basal ganglia, projects through the pyramidal tract and spinal cord
to the musc les in the limbs. Importantly, in the rodent, un like the human and pr imate, the
pyramidal tract regu lates mainly distal forepaw movement and di git use rather than hind paw
and trunk movements.
Thus, when invest igat ing the effect oflvPTP in mice, tests that measure skill ed forepaw
use w ill produce the most reliable results w ith the highest sensit ivity (lvleredith and Kang,
2006). These tests include the beam traversal task, pole test, grid test and furelimb placement
test, but much less so the open fie ld and rotorod (lvleredith and Kang, 2006). However, even
in the sensitive pole tests, variation in bebavioral response may di trer across dosing regimens,
despite simi lar nigrostriatal neuropathology ( l uchtman et al., 2009 ). This may be d ue to
factors such as di fferent extent of nigrostriatal DA loss as well as confounding behavioral
factors such as anxiety. The open field and rotorod may be more sensi t ive to MPT P-i nduced
al terat ions in non-dopaminergic and extra-striatal neurochemistry (Luchtman et al., 2009;
Rozas et al., 1998; Fredr iksson et al., 2001; Rousselet et al., 2003; Rommelfl:mger and
Weinshenker, 2007; Vuckovic et al., 2008), which are major aspects of PD and should be
considered when evaluat ing the effect of a therapeut ic.
We recent ly conducted a study taking account all these factors (Luchtman et al., 20 12).
We used a model applied to male, 6 week old C57B J/6 mice that causes severe nigrostriatal
dopaminergic neuropathology, but also significant extra-nigrostriatal and non-dopaminergic
neuropathology and inflammat ion (l uchtman et al., 20 12; 2009 ; Schintu et al., 2009) . This
model, called the chronic MPTP-probenecid (lvPTP-P, reviewed in lvleredith et al. (2008))
involves I 0 subsequent subcutaneous (s.c.) inj ections of MPT P (25 mglkg) combined with
intraper itoneall y (i.p. ) injected probenec id (250 mglkg) (experimental group), o r saline s.c.
comb ined w ith probenecid i.p. (control group), 3.5 days apart . Probenecid is used to decrease
the renal excret ion of lvi PT P and its metabolite i'viPP+, thereby maintaining the effects of the
neurotoxicant during the 3.5 days injection interval , while not interfering w ith neurochemistry
or behavior (l au et al., 1990; Petroske et al., 2001).
Prior to MPT P-P inj ect ions, our mice were fed for 6 weeks a di et consist ing of regular
powdered chow supplemented w ith 0.8% ethyi-EPA (Amarin Neuroscience ltd, UK) or 0.8%
palm oil. T he fatty acid composition of the diets as well as general diet preparation
proced ures are detai led in luchtman et al. (20 12). During the fi rst week fo llowing MPT P-P
inj ections, th e rotorod, pole test and open fi eld were conducted. Several interest ing findings
were made: lviPT P-P impaired rotorod and pole test perfum1ance, suggest ing that the mice
20
sufFered bradyk inesia, as well as postural imbalance and a bnom1al gait (lv!eredith and Kang,
2006). More importantly, per formance was only impaired in palm-oil treated mice and in the
pole test, E-EPA treatment of mice comp letely attenuated the impairment. In the open field,
MPTP-P increased locomoto r activity, a co mmon finding with chronjc high-accumu lat ive
dosing regimens (Luchtman et al., 2009; Chia et al., 1996; Sedel is et al., 200!, Rousse let et
al., 2003; Qu inn er al., 2006). it is unknown what exact ly caused th is hyperact ive response,
but it was un likely anx iety-related, as I\1PTP-P treated mice d id not d iffer from sal ine-P
treated mice on t ime spent in the cen ter of the open field, a measure of anx iety. E-EPA d id
not attenuate the hyperact ivity induced by I\1PTP-P but it was f ound that E-EPA by itself
increased locomotor act ivity in the open fie ld, which was most likely due to its anxiolyt ic
effects, indicated by increased time spent in the center of the open fiel d and reported by
others as well (Yehuda et al., 2005; Federova and Sale m, 2006 ). Our findi ngs were th e fi rst to
show that n-3 PUFA can prevent M PTP-induced motor impaim1ent.
Table 2. Overview of effects o f n-3 P U.FA (pre)-treatment i n experimental PO

Animal/cell
Model:
,-3PUFA trea tment
Dependent \'91'iables 1 outcomes
Citation
Brain PUFA: EPA i ncreased coni cal EPA and

JJPA bm not JJHA
B ehavior: E-EPA pl'evented I'Otor od and pole test
In-vim
Chronic MPT P-
probenecid ( 10 x
25mg/kg MPTP
mouse. male.
..
be . d
.
s:c. + pto neca
C57!JI/6
6 weo-
.. klo
'50
... .t p, .>,)
.. _ mg.;t<g
d apan) or
saline/probenecid
C57BI/6
ln-v l\'O
Sub-acme MPTP
mouse, nlille, 20mglkg i.p. ot

10 momhs
saline 4 times, 12
hours apan .
In-vim
Sub-chmnic
MPTP: 7 i.p.
. . .
f
.
111JecUOI1S 0
fa t-J tntce, 6 1:\ i PTI' , 0 n iL ,
.' . . - m,.g.
d
momtI o.Id
UlJecUons/ ay on
the fitst 2 days, 12
hours apart, then
1/day for 3 days
E- motot in-)pairmem and improved procedural

.
. I "
.
')
menx)l'y .unpatnnem
111 t le tY
IOrrts
\Vater maze.
Ep "orpamot
1
1
N"
.
I
EEP
d"d
.
. I
.
od
t tgrostnata :
A 1 not prevenr ntgrosmata
add eod d at1VlO r ent J) ' I
b
d ed .
.
. I IJA d
chov.. and fed for 8
A oss, ut re uc tncte ases tn smata
an
5-HT turnover
\veeks priot to
Exlla-nigrostriatal/non-dopaminergic: E-EPA did
MPTP-P injections.
not ptevem conical Ot' hippocampal OA depletion,
Diet continued until
but ptevemed hippocampal increa;;e in DA
e.-.;petiment fi nished
tul'llOVet
Inflammation, oxidative stress, apoptosis: EPA
anenuated increases in striatal pto-intlamn'll\tO!l-'
c ytokines and midbtain bax:bcl-2 ratio
Brain PUFA: N/A
Behavior: OHA did not prevent pole test
impairment
DHA (36mg!kglday) Nigrosuialal: OHA panially prevemed loss of
daily b)' gavage fot 4 SNpc TH stain ing.
\veeks
Extr~Hligrostriatal/non-dopaminetgic: N/A
Inflammation, oxidative stress, apoptosis: DHA
did not prevent a decrease in brain SOD and did
not prevellt inctease in brain TtiARS
Brain PllfA: Corticai OHA and total n-3 PllfA
increased, while DPA (C2 2:5, 11 6) decreased.
Increase in n-3: n-6 t'atio.
Behavior: N!A
.
None. Fat-1 mtce
Nigrostriatal: f at-1 did nott-esult in protection
~ .
against depletion of nigrostri.atat OA, DOPA C.
endogenoul>t~
6PUF"A
ron \en nl> HVA, Ot' striatal TH pratein. HO\\!ever, numbet' of
imo n-3 PUF As
nigra! TH J>OSit ive neurons, Nurl'1 and OAT
mRNA correlate \Vith DHA romenL
Extta-ni gmstriatalfnon-dopamin ergic: N/A
Inflammation, oxid.ative stress, apoptosis: f al-1
resulted in anenuation of striatal asrro gtiosis.
n. "Q'-
v.o ,.
I-EP
1V
ll
A(
l uchtman
et al. 2012
Ozso~ er al.
201I
Bousquet et
al., 2011
Omega-3 Polyunsaturated Fatty Acid Treatment in Parkinson's Disease.. .
21
Table 2. Contin ued

Animal!cell
M odel:
n3 PUF A rrea rment
Dependent \'ar iables, outcomes

Brain JlUFA: Fish oil increased con ical and nigra!
Citation
DHA
Behavior: Fish oil induced lilllall but significant

Jn-YI\1()
Daily oral
reduction in rotational behavior. No effect in open
field,
or jaw uemor.
Wistal' rats, 3 4!!S 6-0 HOA
supplememation of
\veeks old
injection into the 4gtkg fish o il
Nigro:miatal: Fish o il did not prevent nigro:miatal
male
medial fo1-ebrain (120mg EPN I. Omg DA or nigra! T H l oss and fun iH~1 i ncreased strialal
DH A } fo1 10 \veeks OA turnover.
bundle
EXtl'a.nigmstriatallno n.d.op.a mine1gic: NIA
Inflammation, oxid:n ive stress, apoptosis: DHA
prevemed increase in nignll TEARS
Brain PUFA: Conical OHA increased, while DPA
( C22: 5, ll-6) as well as ll6:n3 l'atio decreased.
Behavior: N/A
Nigmstriataf: OHA pal'tiatly anenuated suiaml DA
and DOPAC depletion; prevented a decl'ease in
Daily oral
nigl'al TH immunoteactiviry and irH itu nigl'al
in-vim
Nurl'1 e.-.: pression, and prevented a drop in ni gra!
ad n1in i:aration of
C 57BI/6
Sub-chronic
4 24mg/kg
DAT mRNA {expressing cells). DHA did not
mouse, male, MPTP: same as
prevent a dt'Crease in striatal T H- and OAT.
encapsulated n -3
2 momhs
Housquet et al.,
PUFA, mainly DHA, positive fibel's.
2011
Ext ra.nigrostl'iatallnon-dopaminergic: N/A
for 10 momhs
Inflammation. oxidative stress, apoptosis,
neurotl'ophic suppon: OHA increased stl'iatal TtkB
pl'o tein expression in MPTP treated mice. Striatal
BDNF and TtkB mRNA e.xpression cOI'related
\Vith bl'ain DHA cement in MPTP m~ ated mi-ce.
B1ain PUFA: hlct-eased striatal OHA
In-vivo
Daily i.p. inj ections Behavior: N/A
Single iml'a
of ethyi-DHA up to Nigrostl'iata!: DHA fun he1 increa;;ed loss of Di\
and metabolites in the suiarum.
IRC mice, ce1ebrovemricutar 500mglkg body
(l.c.v.) injection \\eight fol' 7 days
E.xua.nigt'Ostriatallnon.d.opanlinergic: N/A
n.lilte
wit h 6-0 HDA
priM to 6-0 HOA and Inflammation, oxidative .stress, apoptosis: DHA
(60!lg).
7 days after
imetacted wit h 6-0 HOA and funher increa;;ed
Oelanre er
al. 2010
Bousquetel
al., 2008;
2009
Kabuto er
al., 2009
nigral TBARS
Btain PU F A : N/A
In-vivo
Daily oral (by
Behavior: DHA pt'evemed catepsy and pole test
MPTP was
impair mem
gavage)
Adult male
Nigmstd atal: IJHA prevemed loss ofTH positive
adtHinistration of
infused into the
Wistar l'ats
medial fotebl'ain 36mglkg DHA fo r
nemotts in the SNpc.
E.-.:tra.nigrostl'iatal/non-dopaminergic: NIA
3 0 da~s
bundle
Inflammation. oxidative stress, apoptosis: N/A
Brain PUFA: Diet increased cortical EPA and
DPA coment Behaviol': NIA
0.8% Eth yi-EPA (E.t.vfw>
Nigl'oStl'iatal: .EPA did not prevem a striatal DA
EPA) o l'palmoil
but improved stl'iatal D.-\ tumovel'.
C 576116
MPP+ 20!J..M was
added daily to rodent
mouse, male, added to stl'iatal chow and fed for 6 E.xtl'a.nigmstriatat/non.dop.aminel'gic: E-EPA did
6 weeks
and fmmal rone.x
not prevema conical NA depletion.
weeks prior to
Inflammation, oxidative stress, apoplosis: E-EPA
slices fOI' 4 houl's
euthanasia of mice.
anenuated an increase in conical AA and LA
comem, but did not prevent an incl'e.ase in cPLA2
Tanirovet
era/., 20 10
Meng et al.,
2010
22

Ta ble 2. C o nt in ued
Animal!cell
SH-SY5Y
cells and
pl'imary
midbrain
neurons
Model:
n-3 PUF A trea rment
Dependent \'ariables, outcomes

Oxidalive stress: E'PA did not attenuate toss of
mitochondrial membrane pmemial. bm did
auenuate an increase in ROS and RNS, as well as
an i ncrea;;e i n NADPHoxidase (P4~) mRNA

_ . , _
, ~ and protein expressi on, and COX-2 mRNA
In-vitro
Ep A -ll 00!Uhl lla1t \'o: l.\ )1
.
ba ed .
expresston. EPA al so j)revemed an mcrease m
MPP+ 50-100 uM mcu t Wil1l Lle
.
.
.
1
r
antt-oxtdant glmatluone (GSH).
was added to cell cells fot 48 hours,
culrure media fot

48 hours
si muh aneous i~
'I" IPP+.
\Vith
lnflammalion: fPA anenuated an inctea;;e in

cPLA2 and COX-2 mRNA expression
Apoptosis: EPA attenuated an increase bax:bcl2
Citation
l uchtman
et al. ( nm
published
to date)
tatio (mRNA) and reduced cytochrome-c re! ease.

Neurmrophi c suppon: I n primary mesencephalic
neurons, EPA prevemed an inc1-ease i n T rkB
mRNA expression.
A"- arachidonic ad d; L"- linoleic-acid; EI'A- eicosapcntacnoic acid; DHA- docosahexacnoic acid;
DPA= docosapcntacnoic acid; SOD = supcroxidc dismutase; cPLA2= cytosolic phospholipase A2;
COX-2= eyclooxygenase-2; BD:-.JF= brain-derived neurotrophic factor; TBARS: thiobarbituric acid
reactive substances; DA= dopamine; OAT= DA transporter; TH=t)rosinc hydroxylase; DO PAC= 3,4dihydroxyphenylacetie acid; HV A= homovanilic acid 5-HT= serotonin.
Ozsoy et al. (20 11 ) treated C57BL/6 mice (I 0 months old, weigh ing 25- 30 g) w ith DHA
(36mglkg!day) by gavage for 4 weeks and then injected MPT P 20mglkg i.p. or saline four
times, 12 hours apart. Th is sub-acute regimen caused po le-test perfom1ance impaim1ent, but
DHA did not reverse th is impairment, possibly due to the older age of the an imals. Nonetheless,
gavage may be a superior techn ique (than the regu lar food pot feeding we employed) for
ensuring sufficient n-3 intake and it may provide better protection against ox idation. Delattre et
al. (20 I 0) used 6-0H DA as the experimental model of PD and treated male 3-week old W istar
rats w ith 4glkg fish oil ( 120mg EPAII 80mg DHA, orally) fbr 10 weeks, fo llowed by un il ateral
or bilateral infusion of 4pg 6-0HDA into the medial forebrain bundle. They evaluated motor
behavior using a bright ly-lit open f ield, a rotational behavior test (6-0HDA treated rodents tend
to rotate to the site contralateral to the brain damage when given apomorphine to induce
rotation) and assessment of jaw tremor. Fish oil modestly but sign ificantly reduced this
rotational behavior, but did not affect the other behavioral measures. [t should be noted that a
br ightly-lit open field may not be su itab le as a measure of locomotor activity per se, as it affects
anxiety as well. Tanirover et al. (2010) treated adult male W istar rats with DHA (36mglkg) fbr
30 days, then MPT P was infused into the median forebrain bundle. Th is treatment did attenuate
pole test perfom1ance impaim1ent and catalepsy.
7.2. 1\on-Motor and Cog nitive Performance

This domain of parkinson ian clinical symptoms is perhaps where n-3 PU FA w ill have
provide most beneficial effects, as the general mood-and cogn itive enhancing effects of n-3
PlJFA are well established, clinically (Cole et al., 2009) as well as experimentall y (Fedorova
and Salem, 2006). However, th is has not yet been thorough ly investigated in experimental PD
sett ing. Randomized cli nical trials (RCTs) with aged, mild-cogn it ively impaired and
demented (AD) individuals suggest that most benefit can probab ly be obtained in early stages
23
of cognitive impairment (luchtman and Song, 20 12; Cot e et al., 2009) . One parti cularly
chal lenging aspect in approaching parkinsonian cogn it ive impaim1ent therapeut icall y is the
heterogeneity of presentat ion. Cogn it ive impai m1ent in PD can range from execut ive deficits
to visuospat ial and memory deficiency (Kehagia et al., 20 10).
Experimentall y, only one study has been conducted to evaluate the cogn itive-enhancing
effect of n-3 PUFA in PD. In the same study described above (l uchtman et al., 20 12), we
also conducted a l\tlorris water maze test .fo llow ing the battery of motor tests. Th is test is
su itable to test implicit (procedural, non-ep isodi c memory) as well as spatial (declarat ive,
episodic memory) memory. Spat ial and explicit memories generall y involve the medial
temporal lobe structures, including the hippocampus, wh ereas imp licit leaning of habit and
ski ll s has been associated with basal ganglia functi on (H ay et al. (2002); Vakil and
Herisbanu-Naaman ( 1998); and reviewed in Thompson and Kim ( 1996)). Thus, it comes as
no surprise that MPT P treated mice are generall y only impai red at the procedural component
of th is task (Da Cunha et al. (2001); Miyoshi et al. (2002) and reviewed in Da Cunha et al.
(2002)) as we found in MPT P-P treated mice as well. Interestingly, we found that E-EPA
treatment of mice improved procedural memory sign ifi cant ly (luchtman et al., 20 12). We did
not fi nd impairment on the spati al version of the task. On the other hand, mice with
Alzheimer di sease neuropathology are impaired at the spat ial memory version o f the water
maze, or other tests that invol ve spat ial memory or indi rect measures of spatial memory, such
a~ hippocampal LT P. IV!ost important ly, n-3 P UFA treatment can si gn ificant ly improve
perfom1ance in these mice (Hashimoto et al., 2006; 2005ab; 2002;), including EPA only
(H ashimoto et al., 2009; T aepavarapruk and Song, 2010; Minogue et al., 2007; Lynch et al.,
2007; Lonergan et al., 2004; Kavanagh et al., 2004). Si nce declarative memory impaim1ent
and hippocampal atrophy has been observed in PD patients (Farlow and Cummings, 2008), n3 PU FA may th us be beneficial to these patients.
7.3. Dopaminergic, 1\on-Dopaminergic and Extra-1\igrostriata l

1\eurochemistry
Neurochemical findings in PD and the MPT P model of PD follow highly comp licated
pattern s. In most studies using th e MPT P model, nigrostriatal dopaminergic neurochemistry is
the main target, and arguably the most important. However, as mentioned above, behavio ral
changes may correlate to non-dopaminergic neurochemistry, so in evaluating the effi cacy of
therapeutics, one should preferably not restrict to nigrostriatal dopaminergic neurochemistry
only. We took th is approach in our recent study (Lucbtman et al., 2012) in which, following
assessment of motor and non-motor behavior, we took striatum, frontal cortex and
hippocampus t issue follow ing behavioral measures and assayed monoamines and metaboli tes
" i th HPLC. Perhaps th e major findi ng was that E-EPA treatment of mice did not affect the
MPT P-P induced stri atal DA dep let ion, despite the improvements that were seen o n
behavioral measures. No nethe less, striata l DA and 5-HT turnover, which were both increased
by MPTP-P, were respectively partiall y and full y normalized in E-EPA treated mice. Both of
these measures were co rrelated to motor impaim1ent, suggest ing that through modulat ing
neurochemistry, E-EPA may have had some beneficial effects on motor impai m1ent. In the
frontal cortex, E-EPA by itself increased DA, but did not reverse an MPTP-P induced DA
dep letion in th is region, neither in the hippocampu s. ln the hippocampus, however, E-EPA
24
did attenuate an increase in the DOPAC/DA rat io, altogether suggesting that E-EPA acted on
metabolism rather than neurotransmitter content in MPTP-P treated mice. A simi lar effect we
found in MP P+ treated brain slices from C57B V6 mice that were treated w ith 0.8% E-EPA
for 6 weeks (Meng et al., 20 10). M PP+ 20~tM for 4 hours caused a significant DA depletion
in striatal slices and wh il e prior treatment of mice w ith EPA d id not au enuate th is deplet ion, it
did improve DA turnover. Interestingly, in both studi es, the E-EPA di et significant ly
increased bra in EPA and DPA (C22:5, n-3) content, while DHA or n-6 PUFA were not
affected, suggesting that EPA (or DPA), despite its low content in the brain, can have
functional effects.
Other studies focused more on striatal dopaminergic neurochemistry and reported
somewhat inconsistent fi ndings. Bousquet et al. (20 ll) used Fat- ! mice, a transgenic model
expressing an n-3 faity acid desaturase converting n-6 PU FAs into n-3 PUFAs and tested 6month o ld mice against MPT P treatment. The ad vantage of using this model instead of a di et
is that confounding factors such as total di etary intake and oxidat ion can be avoided. They
used a moderate lvlPTP dosing regimen consisting of7 i.p. inject ions of a l\IIPTPIH C I solution
(20mg/kg), with 2 injections per day on the first two days, 12 hours apart, and then once a day
for the three following days. The Fat- ! model was successfu l in that it increased brain DHA,
" h i le strongly decreasing DPA (C22: 5, n-6), and also increased total brain n-3 PUFA and the
n-3:n-6 PUFA rat io compared to wild-type contro ls. However, no protection was measured
against nigrostriatal DA deplet ion. On the oth er hand, th e same group also conducted studies
u.~i ng 2 month o ld C57B I/6 mice fed di ets either low or high in n-3 PUFA (part icularly DHA)
for 10 months and then exposed these mice to the same lvlPT P regimen. The high n-3 PUFA
diet ( 424mg/kg of encapsulated n-3 PUF A dail y) resulted in a much higher n-3:n-6 rat io than
in the Fat- ! mouse. More important ly, the diet resulted in significant dopaminergic
protection, as DA and DOPAC were significantl y less reduced by M PT P in the high n-3
PUF A group. It shou ld be noted that the control, low n-3 PUFA di et, had a very high n-6:n-3
(1 0 1.79: 1) rat io, which is unreali stic compared to the typical western diet n-6:n-3 ratio (1 5:1,
Simopou los et al. 2002 ). Theref(lre, the etrects of th e high n-3 PUFA may have been
exaggerated in th is study. Interestingly, Shchepinov et al. (2011) used a diet of saturated,
monounsaturated and PU FA (equal proport ion n-3 an d n-6), but in their experimen tal group,
PUFA were modifi ed to be stabilized from oxidat ive stress (deuterated PUFA). Compared to
control PU FA, stabilized PUFA did offer better protection against M PT P (single i.p. inject ion
of 40mg/kg) induced DA and DOPAC dep letion than control PUFA, suggest ing that
protective potential of n-3 PUFA could be enhanced if protected from oxidat ive stress.
Delattre et a/. (20 10 ), who tested whether 10 week fish oil treatment could protect against a 60H DA les ion in Wistar rats, found no protection against deplet ion of striatal DA and
metabolites, but did fin d that fish oil further increased DOPAC/DA and HVA/DA rat ios in 60H DA treated rats. These findings are consistent w ith our study (Luchtman et al., 20 12)
showing that EPA mainly acted on metabolism ofneurotransmitters.
There are also findings that the comb inat ion of n-3 PUFA and park inson ian neurotoxin
can aggravate neurotoxicity. Kabuto et al. (2009) used a rather unusal experimental design by
treating male IRC mice with i.p. inj ect ions of ethyi-DHA up to 500mg!kg body weight for 7
days, then treated these mice intra-cerebroven tricu lar (i.c.v.) w ith 6-0HDA (60.u g), followed
by another 7 days of DHA treatment. The high dose of DHA significant ly increased DH A
concentrations in the st iatum (other lipids were not measured), but also aggravated the loss of
DA and metabolites in th is brain region . Levels of lipid perox ides were also increased in the
25
DHA+ 6-0HDA condition compared to DHA o r 6-0 HDA alone. Possible causa l factors
underlying th is enhanced toxici ty by DHA, including oxidative stress and aberrant asynuclein processing, are further discussed be low.
7.4. TH, DAT an d Other Markers of Dopamine rgic 1\euron Integrity

Ozsoy et al. (2011) who treated 10-month old mice with DH A and did not find protection
against MPTP-induced pole test impaim1ent, did find a partial protect ive effect of DH A
against loss of TH immunopositive neurons in the SNpc. However, DHA itself also reduced
TH posit ive neurons, possibly due to increased ox idat ive stress. On the other hand , Bousquet
et al. (20 I I) who used Fat - I mice that convert n-6 PU FA to n -3 PU FA found cortical DH A to
posit ively correlate w ith TH -posit ive neurons, as well as nigra I Nurrl and DA T mRNA
expression. Nurrl is involved in th e development and maintenance of the dopaminergic
phenotype in neurons of the mesencephalon (SN and VT A) (Bousquet et a., 2008). The same
group (Bousquet et al., 2008) found that long-tem1 (I 0 month) treatment of C57B l!6 mice
'"ith fish oil not on ly partiall y protec ted against DA dep let ion, as described above, but also
comp letely prevented loss ofTH immunopos itive cells in the SNpc and mRNA expression of
Nurr l. Surprisingly, the diet did not affect loss ofDA T and TH -posit ive fibers in the striatum.
Tan irover et al. (20 I 0), who found that DHA protected aga inst MPTP- induced motor
impairment m Wistar rats, showed that DHA a lso improved loss of TH
immunohistochemistry in the SN. On the other hand, De lam e et al. (20 I 0) who injected
DHA-treated rats w ith 6-0 HDA and did find protection against motor impaim1ent, did not
report any protect ion against loss of nigra! TH immunopositive cells. There are to date no
studi es investigating th e effect ofEPA on these markers.
7.5. ct-Synuclein Pathology

In Parkinson's disease (PD), aside progressive loss of dopaminergic neurons,
polymerizat ion of the cytoplasmic protein a-synuclein into fi lamentous inclusions may be
found in neuronal cell bodies and dendrites. As ment ioned above, the presence of Lewy
bodi es as a hall mark of PD neuropathology or pathogenesis is somewhat controversial. More
relevant to th is chapter, however, is that there is evidence of an intricate relationship between
a-synuclein and P UFA, perhaps detrimental. Several lines of evidence indicate that cell ular
~ p ids can trigger the ol igomerization of a-synuclein, with speci lie enhancement by PUF A at
physiological concentrations, and in a dose and t ime dependent manner, but inhibit ion by
saturated fatty acids. The enhancement of ol igomerizat ion appeared correlated to degree of
unsaturat ion as we ll as carbon chain length of fatty acids, suggesting th at DHA would have
the strongest effect. These PUFA-induced so luble oligomers are o f disease relevance in that
they are increased in the neuronal cytoplasm of PD pat ients and may precede the fom1at ion of
much larger, insoluble po lymers. In addi tion, the accumu lat ion of oligomers induced by
PUFA is accelerated in dopam inergic cell s expr ess ing the PD-caus ing A53T mutat ion.
Furthem10re, addition ofDHA-induced ol igomer ic rt-synuc lein aggregates to SH-SY5Y cells
causes th ese cells to undergo apoptosis, all indicat ing that P UFA induced a-synuclein
ol igomers may be relevant to PD pathogenesis. The exact mechan isms by which PUFA
26
interact w ith c1-synuclein are unclear (Riedel et al., 2011; Assayag et al., 2007; Sharon et al.,
2003; Perrin et al., 200 I; Sharon et al ., 2001; De Francesch i et al., 20 ll; 2009), but oxidative
stress as well as activation of RXR/PPARy by DHA may play a role (Y akun in et al., 20 12).
7.6. Oxidative Stress

The previous sect ion suggests that PU FA, partiall y due to their susceptibility to oxidat ive
stress, could have a pathogenic role in PD. However, this subject seems somewhat
controversia l, as anti-oxidant eflects of n-3 PUFA have been described extensive ly as well
(Okuyama et al., 2008; Richard et al., 2008; Bas et al., 2007;; Sarsil maz et al., 2003) and
there is evidence that n-3 PUF A can protect against MPT P/ MPP+ induced ox idative stress.
We invest igated (study unpublished to date) the anti-oxidant effects of EPA in l\1JPP+ treated
fu lly di fferent iated SH -SY5Y cell s. The human neuroblastoma cell line SH -SY5Y was
originall y derived from child neuroblastoma and frequent ly used as an in-vitro model of
dopaminergic neurons, especiall y when full y di fferent iated (Luchtman and Song, 2010; X ie et
al., 2010; Presgraves et al., 2004). EPA (50~tM) was added to the cell culture in sodium-sal t
fom1, wh ich facili tates dissol ut ion in the media, and prevented an MP P+ induced decrease in
cell viab il ity. Ant i-oxidant protection could be one mechan ism for the improved cell viability,
a~ EPA attenuated an MPP+ induced increase in ROS and NO production as well as the
mRNA and prote in expression of enzymes involved ROS product ion, including NA DPH ox idase and COX-2. EPA attenuated an increase in major ant i-oxidant glutath ione (GSH), but
did not affect the expression of ami -ox idant enzymes SOD, catalase and GSHpx, wh ich were
increased by MPP+, most likely as a cell ular defense mechan ism. EPA also did not
sign ificantly increase levels of lipid peroxides. Anti -ox idant eftects of exclusivel y EPA (n o
other n-3 PUFA) in neuronal context have rarely been studi ed, and th is was the fi rst study
show ing anti-ox idant effects of EPA in an experimental PD model.
Other studies focused more on fish oil or DHA. Ozso y et al. (20 11) who treated I 0month old mice w ith DHA, then w ith MPTP found that the neurotoxin significantly decreased
SOD levels but th is effect was not attenuated by DHA. Interestingly, the level of
th iobarbitur ic acid react ive substances (TBARS), by-products of lipid peroxidat ion, were
increased by both DHA and MPTP treatment, suggesting that the fatty acid caused li pid
perox idation, but th is was not aggravated in combination with MPTP. On the other hand,
Delattre et al. (20 I 0) showed that a 6-0H DA induced increase of TBARS can be reduced by
prio r fish oi l treatment of the rats. The fish oil treatment was much longer (1 0 weeks) and the
dose in th is study higher (4g/ kg dail y) compared to Ozsoy et al. (20 11 ), who gavaged mice
for on ly 4 weeks with a lower dose (36mglkg dail y). The di fference in results between these
studi es may also be due to use of fish oil (EPA + DHA) rather than DHA onl y in Delattre et
al. (2010). 6-0HDA may also have di fferent efrects on lip id peroxidation than MPTP.
Kabuto et a/ (2009) injected male IRC mice i.p. once dai ly w ith DHA (500mglkg) for a
week and then subjected the mice to 6-0HDA. This dosing regimen was effective at
increasing DHA in the striatum and did not cause an increase in TBARS, however it did
interact with 6-0HDA to increase TBARS sign ificantly. As ment ioned above, these authors
also found that dopaminergic tox icity was aggravated in DHA-treated m ice. Liu et al. (2008)
perfom1ed an experiment in wh ich DA was incubated w ith fatty acid hydroperoxides derived
from AA. and DHA in-vitro, leading to the fom1at ion of toxic DA-adducts. They also
27
harvested these adducts from 7 and 27-week old rat brains. These adducts were th en
incubated w ith SH-SY5Y cells and it was found that the one derived from AA were the most
potent ly toxic to the cell s, caus ing apoptosis, possibly ROS-mediated . Interesti ngly, these
tox ic effects (apoptosis and oxidative stress) were inhibited when suppressors of monoamine
transporters (DA T, NET and SERT) were used, suggesting that DA-adducts can be uptaken
by monoamine transporters. In cells lacking monoamine transporters (embryon ic fi broblasts),
these effects were not observed, convincingly illustrating that PUFA perox ide -derived DAadducts are toxic speci fically to neurons.
These potent ially pathogenic effects of PUF A may part icu larly pertain to highly abundant
PUFA such as AA and DHA. EPA, on the other hand, is on ly a trace component in neuronal
membranes and may as such not be as much of an oxidative threat (Liu et al., 2008). Indeed,
PUFAs may be unstable in an oxidat ive environment and one of the most important lipid
oxidation products is 4-hydroxy-2-nonenal (HN E), which has been implicated in the
pathogenesis of PD. HNE binds covalently to o.-synuclein, promoting to the formation of
ol igomeric species (Q in et al., 2007), thus providing a possible link between lipid peroxidation
and format ion of o.-synuclein oli gomeric species, w ith PUFA as the common denominator.
However, lvluntane et al. (20 I 0) showed that in transgen ic m ice expressing both w ild mouse
alpha-synuclein and the P D-soci ated A53 T mutated human alpha-synuclein, Jong-tem1 dietary
dep letion of n-3 PUFA did not result in changes to the park inosnian phenotype of the models,
even though the diets did cause a reduction in the content of brain n-3 and total PUFAs. While
the dietary depri vation of n-3 PUFA lead to sign ificantly lower doub le bond and
perox idizability indexes as well as to lower protein oxidative damage, th is apparently did not
afTect the sever ity of PD. This would argue against a toxic role of PUFA in PD.
7.7. Brain PUf A and lnllammation

Aside neurochemistry, brain PUF A and inflammation may be one of the areas where n-3
PU FA have the strongest effects, yet again very little is reported on the anti-inflammatory
efTects of n-3 PUFA in experimental PD. As mentioned above, there are essentially two major
ways by which n-3 PUF A can affect infl anm1ation; through the eicosanoids/docosanoids
pathway, and through modulating inflammatory gene expression. We recently conducted two
studies demonstrating that EPA may modulate infl ammation through both routes, albeit in
di fferent model systems. In one model system (Meng et al., 20 10), briefl y described above,
striatal brain slices were treated with MPP~ 20~tlvl for 4 hours, as a way to mimic the effect of
acute exposure to park inson ian neurotox in. The other mode l system is already described above
(Luchtman et al., 20 12) and involved the chron ic MPT P-P model. Interestingly, in the acute
brain slice model, both AA content and cPLA2 mRNA expression were sign ificantly increased
by MPP-, wh ile levels of n-3 PUFA, remained unaltered. On the other hand, in the chronic
MPTP-P model, n -6 nor n-3 PUFA were altered. The latter finding is consistent w ith studies
reporting no changes in brain fatiy acid profiles in patients or exper imental an imal models of
PD involving repeated i'v!PTP admin istration (Bousquet et al., 2008; Juli en et al., 2006). The
acute effect of l'v!PP- on n- 6 PUF A in brain slices may be trans ient, but nonetheless an
important initial step in PD pathogenesis, since the increase in AA contents, combined with
increased expression of cPLA2, could increase AA substrate for COXILOX metabolism, and
trigger inflammation. More importantly, dietary pre-treatment with 0.8% E-EPA sign ificantly
28
increased brain EPA and DPA (C22:5, n-3 ) but not DHA content, while auenuating the increase
in AA content, thus favor ing an environment less prone to inflanm1ation. Whil e chronic tviPT Pp did not increase brain AA content, it did sign ificantly increase levels of pro-i nflammatory
cytokines TN F-a and IFN-y in the vulnerable striatum. E-EPA pre-treatment of m ice increased
brain levels ofE PA and metabolite DPA, and al!enuated th is increase in cytok ines. The diet also
attenuated a m idbrain increase in IL-10. In the SH-SY5Y cu lture experiments introduced above,
we also found that EPA treatment of the cell s can al!enuate an MPP+ induced increase in
cPL.J\2 and COX-2 mRNA expression (data not pub lished to date) . This data altogether
demonstrated that E-EPA can attenuate tviPTP-induced inflammation in di fTerent model
systems. I! would be of interest to di rectly compare these anti-infl ammatory effects of EPA
against DHA, as EPA has often been considered as a more potent (than DHA) or even main
inflammation suppressor (Calder, 2009), possibly due to the fact !hat EPA, as opposed to DHA,
is the precursor to ant i-infl anm1atory eicosanoids. Our recent findings on the anti-i nflammatory
efTects of EPA in experimental PD are consistent with other studies from our group or other
groups in wh ich an inflammatory stressor was used to impair memory or LT P (Taepavarapruk
and Song, 20 I 0; Song et al., 2004; Song and Horrob in, 2004; Lonergan et al., 2004; Kavanagh
et al., 2004; tvlartin et al., 2002; Lonergan et al., 2002), as models of AD-li ke memory
impai m1ent or neurodegeneration.
7.8. 1\eurotrophjc Support

Decreased neurotrophic support in dopaminergic neurons in PD has been reported (Di ng
et al., 20 11; Baydyuk et al., 20 I 0; l\1(ogi et al., 1999). BDN F is among the maj or brain
neurotrophic factors and its primary recepto r is the trop omyosin-related k inase B (TrkB)
tyrosine kinase receptor. Part ial deletion o f this receptor leads to a reduced number of
dopminergic cell s in the SN and the fom1ation of a-synuclein aggregates in older mice,
support ing a rol e of BDNF signaling in PD (von Bohlen et al., 2005). In fact, BDN F has been
recogn ized as a potential neuroprotective agent for PD (Peterson and Null, 2008), but the
blood brain barrier (BBB) blocks the entry of BDN F and other neurotrophic factors into the
brain (Kast in et al., 2003), whi ch severely li mits clinical appli cation. Thus, a neu roprotectiv e
compound that has access to the brain and acts w ith in the brain to modulate neurotrophic
act ion would be of great value. Possibly through CRE B, n-3 P UF A can modulate BN DF
signal ing in the brain (Rao et al., 2007) . Long-tem1 exposures to diets dep leted in n-3 PUFA
can lower brain BDN F and Trk B signali ng, which could be restored by rep let ion of n-3 in the
di et (Bhatia et al., 20 11 ; Rao et al., 2007). We recently r eported that administration of EPA to
fully di fferentiated S H-SY5Y cell s can increase TrkB mRNA and protein expression (Kou et
al., 2008). In an unpub li shed study, we found that EPA can also increase BDN F mRNA in
primary mesencephalic neurons . M PP+ administration to these cell s increased both BDN F
and TrkB mRNA, possibly due to engagement of the cellular defense system, and E PA
attenuated the increase in TrkB expression. The fact that EPA allenuated th is MPP+ induced
increase in Trk B suggest that the n-3 PU FA stabilized the cell ular survival pathway due to
reduced tox icity. A long these li nes, we previously repo rted that EPA sign ifi cantly increased
nerve growth factor (NGF) expression in the rat brain and reversed an ll- 1-i nduced decrease
in NGF expressio n (Taepavarapruk and Song, 20 I 0).
29
As an exten sion to the study by Bousquet et al. (2008), in which C57BI/6 mice were fed a
diet h igh in n-3 PUFA for 10 months, then treated with MPT P, Bousquet et al. (2009)
measured brain BDN F signaling. Surpris ingly, MPTP did not affect striatal BDN F or TrkB
content, but the high n-3 PUFA di et did increase striatal BDN F content in control (no MPT P)
condit ions. l'vlore interest ingly, TrkB protein expression was significant ly increased in high n3 fed an imals, but only if these animals were treated w ith MPT P. Furthermore, both str iatal
BDN F and TrkB mRNA expression correlated w ith brain DHA content, but in the context of
MPT P treatment only, suggest ing that modulation of brai n BDNF act ion may be one
mechanism by wh ich the high n-3 PU FA di et provide protection upon neuronal toxicity.
7.9. Apoptosis
Most studies investi gat ing the effects of n-3 PUFA in PD have focused on end-point
measures such as loss ofTH and DAT immunostain ing in the SNpc and striatum, or the loss
of nigrostriatal DA . Whil e important, such data does not indicate the mode of cell death in
PD. Cells that have not di ed or been lost may be in the early stages of apopt osis, so measuring
these changes may be an important strategy to gauge the apoptotic status of remaining
neurons and detem1ine whether neuroprotect ive compounds can counteract the changes.
Novikova et al. (2006) studied apoptosis in the MPT P-P regimen and found apoptotic
neurons throughout the ventral t ier of the SNpc, which decli ned over time, while the striatal DA
and tem1inalloss persisted. This suggested that apoptot ic neurons were cleared by phagocytosis.
We found that ~~tPTP-P treatment of mice increased the midbrain bax:bcl -2 ratio by 60%, two
weeks follow ing the last MPT P-P inj ect ion (Luchtman et al., 20 12), which was reversed by
pr ior E-EPA treatment of these mice, suggesting that EPA may have provided some antiapoptotic effects. However, as ment ioned before, EPA did not prevent the nigrostriatal DA loss
that was caused by MPTP-P in th is study, questioning the relevance of this finding. In our
unpub lished study with MPP+ treated fu ll y differentiated S H-SY5Y cells, we f()und that MPP+
sign ificantly increased the bax:bcl-2 ratio, as well as caspase-3 mRNA expression and
cytochrome-c release, all indicat ing that MPP- promoted apoptosis in SH-SY5Y cells. More
importantly, while EPA did not prevent increased caspase-3 mRNA expression, it sign ificantly
attenuated the bax :bcl-2 rat io and reduced the release of cytochrome-c, thereby reducing the
potential formation of an apoptosome. These results are consistent w ith the anti-apoptotic
effects of EPA observed in-vivo (Lonergan et al., 2004; 2002).
Other evidence co ncern ing anti -apoptot ic effects of n-3 PUFA as a mechanism of
protection in PD is sparse. More data is available in the setting of experimental A D. Several
excellent studies are pub li shed reporting di rect evi dence for the protective eff ects against A Dli ke neurop athology through ant i-apoptotic effects (e.g. Zhao et al. 2011, Hashimoto et al.,
20 11; Florent et al., 2006; Akbar et al., 2005; Calon et al., 2004) .
8. S ummary and Concluding Re marks

The conclusion regardi ng the eflect o f n-3 PUFA in the prevention, delay or treatment of
PD is not stra ight forward for several reasons . First, whil e evidence from human studies does
show a relationship between patterns o fmacro nutrient intake and ri sk o f PD, these studi es are
30
mainly observational and insutllcient ly valid to draw sound conclusions about any
relati onship between n-3 PU FA and risk of PD. There are no clinical studi es support ing the
effectiveness of n-3 PU FA in treating PD, except for depression in PD, in which n-3 PUFA
were shown to have favorable effects (Da S il va et al., 2008). Further, from th e observational
studi es, it appears that PD may cause loss of body weight, possibly due to increased energy
expenditure or sarcopen ia, against which n-3 PUFA may have favorable outcomes, although
lh is w ill need to be invest igated more thoroughly. From post-mortem studies, there are
indicat ions that an increase in n-6 PUFA may be associated with PD, but increased contents
of highly peroxidizab le n-3 PUF A (DHA) have been observed as we ll.
Second, animal studi es, wh il e generall y demonstrat ing support for effect iveness of n-3
PUFA against lVfPTP or 6-0HDA-induced neurotoxicty, also Jack consistency in results in
that it depends on the measure of interest to what extent protect ion is observed. ln our in-vivo
studi es w ith EPA, the n-3 PUFA was found to protect against motor impaim1ent and
inflanm1ation, however no protection was found against the primary neurodegenerat ion,
including nigrostriatal DA deplet ion. Our in-vitro studies on the other hand provided evidence
lhat EPA has robust anti-oxidant, ant i- inflammatory and anti -apoptotic effects in cells treated
'"itb lv!PP+. T he study by Bousquet et al. (2008) showed a robust protective effect of longterm fish oil (mainly DHA) supp lementat ion against lv!PTP induced nigrostr iatal
dopaminergic neurodegenerat ion, possib ly mediated by upregulat ing striatal BDN F signal ing.
Unfortunately, they did not measure the behavioral consequences of th is remarkable
protective effect. Most other animal studi es foun d a mixture of protect ive effects on some
measures, whil e no protection on others. Lack of consistency in findings may be due to
variat ion in experimental models used as well as treatment of the di ets; wh il e we used careful
and dail y addit ion of fresh oil s to rodent chow, others used encapsu lated oi ls or di rect oral
administrat ion by gavage. These methods may detemune to what extent the oi ls are oxidized
prio r to admi nistration. Concen tration of EPA and/or DHA in the oi ls and durat ion of the
di ets also var ies substant ial ly across studies.
Third, two findings from literature stand out as disconcerting in terms of possible n-3
PUFA toxicity in PD. Fi rs~ the interaction w ith a -synuclein and second, lip id perox idation.
Studies have clearly shown that PUFA, including D HA and AA can trigger the fom1ation of
a-synuclein oli gomers that can be toxic to neurons. Lipid perox idat ion may play a rol e in th is
and contribute to the fom1at ion of potent iall y tox ic DA-adducts. No such fi ndings were made
for EPA (although not invest igated). In fact, EPA may be safer as therapeut ic compound than
DHA, because it is on ly a trace component in neuronal membranes and wou ld not result in
large amounts of ox idants, compared to the much more abundant AA and DHA.
How can al l these fi ndings be reconcil ed? T he fact that fish-oil was strongly protect ive
against MPT P in the Bousquet et al. (2008) study suggest that the beneficial effects of n-3
PUFA may overrule the toxic effects, if proper care is taken in suppl ying and stabi li zing the
n-3 PU FA. Indeed, Shchepinov et al. (20 11 ) reported that stabil izing fats against oxidat ion
can enhance the protect ive effects of n-3 PUFA. While EPA in our studi es did not prevent
nigrostriatal DA depletion, it did not aggravate MPT P-P neurotoxicity and prevented motor
impairment and in flanm1ation. Combined with the protective effects of n-3 PUFA found in
experimental AD and the overa ll beneficial effects of n-3 PUFA to the brain, these fi ndings
shou ld altogether warrant furth er research in th e therapeutic potent ial o f n-3 PU FA in PD.
Even in th e case that n-3 PU FAs do not prevent the core neuropathology, their mood and
cognit ion -enhancing effects may be highly beneficial to PD patients suffering from
31
depression, an xiety and cogn itive-impai rment. Furthem10re, DHA was shown to protect
against L-DO PA induced dyskinesias, suggesting that adj uvant n-3 fatty acids with DAreplacement/ agonist drugs may be a therapeut ic o ption.
Some final recommendations for cont inued research need to be made, fi rst w ith regards
to PD models and second with regards to n-3 PUFA . As stressed above, in section 7. 1, there
should be progress towards a standardized MPTP model of PD w ith standardized behavioral
tests fo r measuring moto r impairment, in the experimental PD sett ing. Current ly, there is an
excessi ve amount of vari ation in methodology used in MPT P research, impedi ng consensus
on the true neuroprotective potential of candidate neuroprotect ive compoun ds, as results are
di ffi cult to interpret when the severity and complexity o f neuropathology di ffers across
studi es. One major aspect of PD is non-dopaminergic and extra-nigrostriatal alterat ions in
neurochemistry, wh ich may account for some of the behavioral changes observed in the
MPT P model. As we found in our study (Luchtman et al., 2012), n-3 PUFA may attenuate
those non-nigrostriatal changes rather than the core neuropathology and this may underlie
behavioral recovery. Si milar standardizat ion should also be app lied to diets.
Aside studi es from our laboratory, there are currently no studies investigat ing the
therapeut ic potential of EPA, or its metabolite DPA . Both are trace components in neuronal
membranes, but neuroactive and proven eflect ive in the treatment of psychiatric disorders
(Song and Zhao, 2007).
it would serve the sci enti fic co mmun ity and publi c to conduct studies directly and
systemat icall y compar ing EPA and DHA for their neuroprotective effect iveness in
experimental (or cli nical ) PD. Other studi es could focus on the test ing for formu las containing
various proport ions ofD HA and EPA and detem1ine optimal effect iveness. The authors stress
the benefit of an overall improved systemat ic approach towards the test ing of n-3 PU FA in a
more standardi zed experimental PD setting.
References
Aden, E., Carlsson, M., Poortvliet, E., Sten lun d, H., Linder, J., Edstrom, M., Forsgren, L.,
Haglin, L. Di etary intake and olfacto ry function in patients with new ly diagnosed
Park inson's disease: a case-control study. Nutr. Neurosci. 20 11; 14: 25-3 1.
Ahmad, A ., Murthy, M., Greiner, R .S., Moriguch i, T ., Sal em, N. Jr. A decrease in cell size
accompanies a loss o f docosahexaenoate in the rat hippocampus . Nurr. Neurosci. 2002; 5:
103- 13.
Akbar, M., Calderon, F., Wen, Z., Kim, H .Y. Docosahexaenoic acid: a posit ive modulator of
Akt signaling in neuronal survival. Proc. Natl. Acad. Sci. US A. 2005; 102(3 1): 10858-63.
D Amelio, lvl., Ragonese, P., Morgante, L., Reggio, A., Callari, G., Sal emi, G ., Savett ieri , G.
Long-tem1 survival of Parkinson's disease: a popu lat ion-based study. ./. Neural. 2006;
253: 33- 7.
Anantharam, V .. Kaul, S., Song, C., Kanthasamy, A. and Kanthasamy A .G. Pharmacological
inh ibition of neuronal N.t \D PH oxidase protects against 1-methyl-4-phenylpyri dinium
(NfPP+)-i nduced oxidat ive stress and apoptosis in mesencephali c dopaminergic neuronal
cells. Neurotoxicology. 2007; 28: 988-99 7.
32
Anderson, C., Checkoway, H., Franklin, G.M., Beresford, S., Smith-Weller, T., Swanson,
P.D. Dietary factors in Parkinson's disease: the role of food groups and speci fic foods.
Mov. Disord. 1999; 14: 21 -7.
Asanuma, M., 1v!iyazak i, I. Nonstero idal ant i-inflammatory drugs in experimental
parkinson ian models and Park inson's disease. Curr. Pharm. Des. 2008; 14: 1428-34.
Assayag, K., Yakun in, E., Loeb, V., Sel koe, D.J. Sharon R. Pol yunsaturated fatty acids
induce a lpha-synuclein-related pathogenic changes in neuronal cel ls. Am . .f. Pathol. 2007;
171:2000- 11.
Bas, 0., Songur, A., Sahin, 0., Mollaoglu, H., Ozen, O.A., Yaman, M., Eser, 0., Fidan, H.,
Yagmurca, M. The protect ive effect of fish n-3 fatty acids on cerebral ischemia in rat
hippocampus. Neurochem. lnt.2007; 50: 548-54.
Baydyuk, M., Nguyen, lvi.T., Xu, B. Chron ic depri vation ofTrkB signaling leads to selective
late-onset nigrostriatal dopaminergic degeneration. E~p. Neural. 2011; 228(1): 11 8-25.
Bazan, N.G. Cellular and molecular events mediated by docosahexaeno ic acid-der ived
neuroprotect in D I signaling in photoreceptor cell survival and brain protection.
Pmstaglandins Leukot. Essent. Fatty Acids. 2009; 8 1 (2 -3): 205- 11.
Bazan, N.G., lvlolina, M. F., Gordon, W.C .. Docosahexaenoic acid signalolipidomics in
nutrit ion: sign ifi cance in aging, neuroinflammat ion, macu lar degenerat ion, A lzheimer's,
and other neurodegenerati ve diseases. Annu. Rev. Nutr. 20 11; 3 1: 32 1-5 1
Betarbet, R., Sherer, T.B., Greenamyre, J.T. An imal models of Park inson's disease.
Bioessays. 2002; 24: 308-18.
Bhatia, H.S., Agrawal, R., Sham1a, S., Huo, Y.X, Ying, Z., Gomez-Pini lla, F. Omega-3 fatty
acid deiiciency during hrain maturat ion reduces neuronal and behavioral plasticity in
adulthood. PLoS One. 20 11; 6(12):e2845 !.
Bing, G., Zhang, Y., Watanabe, Y., lvtcEwen, B.S., Stone, E.A. Locus coeru leus lesions
potentiate neurotoxic effects of lv!PT P in dopaminergic neurons of the substantia nigra.
Brain. Res. 1994; 668: 261 -5.
Boka, G., Anglade, P., Wallach, D., Javoy-Agi d, F., Agid, Y., Hirscb, E.C.
Immunocytochemical analysis of tumor necrosis factor and its receptors in Park inson's
disease. Neurosci. Lett. 1994; 172: 15 1-4.
Bordet, R., Gele, P., Duriez, P., Fruchart, J.C. PPARs: a new target for neuroprotect ion . .f.
Neural. Neurosurg. Psychially . 2006; 77: 285- 7.
Bousquet, l\'1., Calon, F., Cicchelli, F. Impact of omega-3 fatty acids in Parkinson's disease.
Ageing. Res. Rev. 2011; 10(4):453-63.
Bousquet, M., Gibrat, C., Saint- Pierre, M., Ju li en, C., Calon, F., Cicchett i, F. Modulat ion of
brain-der ived neurotrophic factor as a potential neuroprotective mechan ism of act ion of
omega-3 fatty acids in a park insonian animal model. Prog. Neuropsychopharmacol. Bioi.
Psychiatry. 2009; 33(8): 1401-8.
Bousquet, M., Gue, K., Emond, V., Juli en, P., Kang, J.X., Cicchetti, F., Calon, F. Transgenic
con vers ion of omega-6 into omega-3 fatty acids in a mouse model of Parki nson's disease .
.f. Lipid Res. 2011; 52: 263-71.
Bousquet, M., Saint-Pi erre, M., Juli en, C., Salem, N. Jr., Cicchett i, F., Calon, F. Beneficial
effects of dietary omega-3 po lyunsaturated fatty acid on toxin-induced neuronal
degenerat ion in an animal model of Park inson's disease. FASEB. J 2008; 22(4): 12 13-25.
Bjl)rklund, A., Dunnett, S.B. Dopami ne neuron systems in the brain: an update. Trends
Neurosci. 2007; 30: 194-202.
Omega-3 Pol yunsaturated Fatty Acid Treatment in Park inson's Disease. ..
33
Blandini, F., Napp i, G., Tassorelli , C., Martignon i, E. Funct ional changes of the basal gang lia
circuitry in Park inson's disease. Prog. Neurohiol. 2000; 62: 63 -88.
Breidert, T., Call ebert, J., Heneka, M.T., Landreth, G., Launay, J.M ., Hirsch, E.C. Protect ive
act ion of the peroxisome proli ferator-act ivated rec.e ptor-gamma agonist piogli tazone in a
mouse model of Parkinson's disease. J. Neurochem. 2002; 82(3):6 15-24.
Broersen, K., van den Brink, D., Fraser, G ., Goedert, M., Davletov, B. A lpha-synuclein
adopts an alpha-helical confom1ation in the presence of polyunsaturated fatty acids to
hinder micell e fom1at ion. Biochemi.w y . 2006; 45(5 1): 15610-6.
Calder, P.C. Mechan isms of action of (n-3 ) fatty acids . .!. Nutr. 20 12; 142(3): 592S-599S.
Calder, P.C. Omega-3 fatty acids and inflammatory processes. Nutrients. 20 I 0; 2: 355-74.
Calder, P.C. Fatty acids and inflammat ion: the cutt ing edge between food and pham1a. Eur. J.
Phannacol. 2011; 668 Supp l I :S50-8.
Calderon, F., Kim, H .Y. Docosahexaenoic ac id promotes neurite growth in hippocampal
neurons . ./. Neurochem. 2004; 90: 979-88.
Calon, F., Lim, G.P., Yang, F., Morihara, T., Teter, B., Ubeda, 0., Rostaing, P., Trill er, A .,
Sal em, N . .Jr., Ashe, K.H., Frautschy, S.A., Co le, G.M . Docosahexaenoic acid protects
from dendritic pathology in an A lzheimer's disease mouse model. Neuron. 2004;
43(5): 633-45.
Cansev, M., Wurtman, R.J. Chron ic administrat ion of docosahexaeno ic acid or
eicosapentaenoic acid, but not arachidonic acid, alone or in combinat ion w ith uridi ne,
increases brain phosphatide and synapt ic protein levels in gerbils. Neuroscience. 2007;
148: 42 1-3 1.
Cao , D., Kevala, K., Kim, J., lvloon, H.S., Jun, S.B., Lovinger, D., Kim, H.Y.
Docosahexaenoic acid promotes hippocampal neuronal deve lopment and synapt ic
function. J. Neurochem. 2009; Ill : 5 10-21.
Carlsson, A., Li ndqvist, lvl., Magnusson, T. 3,4-Dihydroxyphenylalan ine and 5-hydroxytryptophan as reserpine antagon ists. Nature. 1957; 180: 1200.
Cha lon, S. Omega-3 fatty acids and monoamine neurotransmission. Prostaglandins LeukDt.
Essent. Fatty Acids. 2006: 259-69.
Chalon, S., Delion-Vancassel, S., Belzung, C., Gu ill oteau, D., Leguisquei, A.M., Besnard,
J.C., Durand, G. Di etary fish oil a ffects monoaminergic neurotransmission and behavior
in rats . .f. Nutr. 1998; 128: 25 12-9.
Chen, C.T., Li u, Z., Bazinet, R.P. Rapid de-esteri fication and loss of eicosapentaenoic acid
from rat brain phospholipids: an iniracerebroventricu lar study. ./. Neurochem. 20 11 ;
116(3):363-73.
Chen, H., Zhang, S.M., Heman, M.A., Willett, W.C., Ascherio, A. Dietary intakes of fat and
risk of Park inson's disease. Am . .f. Epidemiol. 2003; 157(11):1007- 14.
Chia, L.G., N i, D.R., Cheng, L.J., Kuo, .I.S., Cheng, F.C., Dryhurst, G . E ffects of 1-methyl-4phenyl- 1,2,3,6-tetrabydropyri dine and 5, 7-di hydro-xytryptamine on the locomotor
act ivity and striatal amines in C57BLf6 mice. Neurosci. Lett. 1996; 2 18: 67- 71.
Cleren, C., Cali ngasan, N.Y., Chen, .1., Beal, lvi.F . Celastrol protects aga inst l'v!PTP- and 3nitropropion ic acid-induced neurotox icity. .f. Neu rochem. 2005; 94: 995-1004.
Cole, G.M., Frautschy, S.A. Docosahexaenoic acid protects from amyloid and dendritic
pathology in an A lzheimer's disease mouse model. Nutr. Health. 2006; 18: 249-59.
34
Dirk W. Luchtman, Jeflrey A . Zidichouski and Cai Song
Cole, G .lvl., Lim, G .P., Yang, F., Teter, B., Begum, A., Ma, Q ., Harri s-White, M. E.,
Frautschy, S.A. Prevention of A lzheimer's disease: Omega-3 fatly acid and phenolic antioxidant interventions. Neurobiol. Aging. 2005; 26: I : 133-6.
Co le, G .M., Ma, Q .L., Frautschy, S.A. Omega-3 fatty acids and dementia. Prostaglandins
Leukot. Essent. Fatty Acids. 2009; 8 1 :2 13-2 1.
Da Cunha, C., Gevaerd, 1vi.S., V ital, M.A ., 1\,l iyoshi, E., Andreat ini, R., S il veira, R., Takahashi,
R.N., Canteras, N.S. Memory disruption in rats w ith nigra! lesions induced by MPT P: a
model for early Parkinson's disease amnesia. Behav. Brain. Res. 200 I; 124( I) :9-18.
Da Cunha, C., Angelucci , M.E.M., Canteras, N .S., Wonnacoll, S., Takahash i, R.N. T he lesion
of the rat substant ia nigra pars compacta dopaminergic neurons as a model for
Park inson's disease memory disabili ties. Cell. Mol. Neurobiol. 2002; 22:227-3 7.
Da Silva, T.M., Munhoz, R.P., A lvarez, C., Nali waiko, K-, Kiss, A., Andreatini, R., Ferraz,
A.C. Depression in Park inson's disease: a double-blind, randomized, placebo-controlled
pilot study of omega-3 fatty-acid supp lementation . .f. Affect. Disord. 2008; Ill (2-3): 35 1-9.
Dalf6, E., Portero-Oti n, M., Ayala, V., Martinez, A., Pamplona, R., Ferrer, I. Evidence of
oxidat ive stress in the neocortex in inci dental Lewy body disease. J. Neuropathol. Exp.
Neural. 2005; 64(9):8 16-30.
Darios, F., Ruiperez, V., L6pez, 1., Vi ll anueva, J., Gut ierrez, L. M., Davletov, B. A lpbasynuclein sequesters arachidonic ac id to modulate SNA RE-mediated exocytosis. EMBO
Rep. 20 I 0; 11 (7):528-33.
Dauer, W., Przedborski , S. Parkinson's disease: mechanisms and models. Neuron. 2003; 39:
889-909.
De Franceschi, G ., Frare, E., Bubacco, L., Mamm i, S., Fontana, A., de Laureto, P. P.
Molecular insights into the interaction between alpha-synuclein and docosahexaenoic
ac id . ./. Mol. Bioi. 2009; 394( 1):94- 107.
De Francesch i, G ., Frare, E., Pi vato M, Relini, A., Penco, A., Greggio, E., Bubacco, L.,
Fontana, A., de Laureto, P.P. Structural and morphological characterizat ion of aggregated
species o f a -synuclein induced by docosahexaenoic acid . .f. Bioi. Chem. 20 11 ; 286(2 5):
22262-74.
de Lau, L. M., Bomebroek, M ., Witteman, J.C ., Hofman, A., Koudstaal, P.J., Breteler, M.M.
Dietary faily acids and the risk of Park inson disease: the Rollerdam study. Neurology.
2005; 64( 12):2040-5.
Dehmer, T., Lindenau, J., Haid, S., Dichgans, J and Schulz, .I. B. Deficiency of inducible nitric
oxide synthase protects against MPT P tox icity in vivo . ./. Neurochem. 2000; 74: 22 13-22 16.
Delion, S., Chalon, S., Guilloteau, D., Lejeune, B., Besnard, J.C., Durand, G . Agerelatedchanges in phospholipid fatty aci d compostt ton and monoaminergic
neurotransmission in the hippocampus of rats fed a balanced or an n-3 pol yunsaturated
fatty acid-deficient diet. .f. Lipid Res . 199 7; 38: 680-9.
Del ion, S., Chalon, S., Heraull, J., Guilloteau, D., Besnard, J.C ., Durand, G . Chronic dietary
alpha-linolenic acid de ficiency alters dopaminergic and serotoninergic neurotransmission
in rats . .f. Nutr. 1994; 124: 2466-76.
~lallre, A.M., Kiss, A., Szawka, R.E., Anselmo-Franci, J.A ., Bagat ini, P.B., Xavier, L.L.,
Rigon, P., Achaval , M. Evaluation of chronic omega-3 fatty ac ids supplementat ion on
behavioral and neurochemical alterations in 6-hydroxydopamine-Jes ion model of
Parkinson's disease. Neurosci. Res. 20 I 0; 66(3): 256-64.
35
Davies, K.N., King, D., Davies, H. A study of the nutrit ional status of elderly pat ients w ith
Park inson's disease. Age. Ageing. 1994; 23(2): 142-5.
DeM ar, J.C. Jr, Lee, H.J., lvla, K., Chang, L., Bell , J.M., Rapoport, S.l., Bazinet, R.P. Brain
elongat ion of li noleic acid is a negligible source of the arach idonate in brain
phospholip ids of adult rats. Biochim. Biophys. Acta. 2006; 176 1(9): 1050-9.
DeMar, J.C. Jr., l\1(a, K., Bell , J.M., Rapoport, S. l. Half-li ves of docosahexaenoic acid in rat
brain phospholipids are prolonged by 15 weeks of nutrit ional depr ivation of n-3
pol yunsaturated fatty acids . ./. Neurochem. 2004; 9 1(5): 1125-37.
Demar, J.C. Jr., Ma, K., Chang, L., Bell , J.M., Rapoport, S. l. alpha-L inolen ic ac id does not
contribute appreciably to docosahexaenoic acid with in brain phospholip ids of adu lt rats
fed a diet enriched in docosahexaeno ic acid. J. Neurochem. 2005; 94( 4): I 063- 76.
Dep ino, A.M., Earl, C., Kaczmarczyk, E., Ferrari, C ., Besedovsky, H., del Rey, A., Pitossi,
F.J., Oertel, W.H. ivl icrogli al act ivation w ith atypical prointlammatory cytokine
expression in a rat model of Parkinson's disease. Eur. .f. Neurosci. 2003; 18: 273 1-42.
Dhanasekaran, M., Uthayathas, S., Karuppagounder, S.S., Parameshwaran, K. ,
Supp iramaniam, V., Ebadi, M., Brown-Borg, H.M. Ebselen effects on MPTP-induced
neurotoxicity. Brain. Res. 2006; 111 8: 25 1-4.
Ding, Y .X., Xia, Y., Jiao, X.Y ., Duan, L., Yu, J., Wang, X., Chen, L.W. The Trk B-posit ive
dopaminergic neurons are less sensiti ve to lv!PT P insult in the substantia nigra of adult
C57/B L mice. Neurochem. Res. 2011; 36(10): 1759-66.
Drechsel, D.A. and Pate!, M. Ro le of react ive oxygen species in the neurotoxicity of
environmental agents impli cated in Parkinson's disease. Free. Radic. Bioi. Med. 2008;
44: 1873-86.
Dyall , S.C., Michael, G .J., Michaei-T itus, A.T. Omega-3 fatty acids reverse age-related
decreases in nuclear receptors and increase neurogenesis in old rats . ./. Neurosci. Res.
2010; 88( 10): 209 1- 102.
Dyall, S.C., M ichael-T itus, A.T. Neurological benefits of omega-3 fatty acids.
Neuromolecular Med. 2008; I 0(4): 21 9-35.
Fabelo, N., Jvlart in, V., Santpere, G., Marin , R., Torrent, L., Ferrer, 1., Diaz, lVI. Severe
al terations in lipid composition of frontal cortex lipid rafts from Parkinson's disease and
incidental Park inson's disease. Mol. Med. 20 I I; 17(9- 10): I I 07 - 18.
Farlow, M.R., Cummings, J. A modem hypothesis: The dist inct pathologies of dement ia
associated w ith Parkinson's disease versus A lzheimer's disease. Dement. Geriatr. Cogn.
Disord. 2008; 25: 301-8.
Farooqui, A.A., Horrocks, L.A., Farooqui, T. Modulat ion of intlammat ion in brain: a matter
of fat. .! Neurochem. 2007; 10 1(3): 577-99.
Fedorova, 1., Salem, N ., Jr. Omega-3 fatty acids and rodent behavior. Prostaglandins Leukot.
Essent. Fatty Acids. 2006; 75: 271 -89.
Ferger, B ., Leng, A., Mura, A., Hengerer, B., Feldon, J. Genetic ablation of tumor necrosis
factor-a lpha (TN F-al pha) and pharmacological inh ibition of TN F-synthesis attenuates
MPT P toxicity in mouse striatum . .! Neurochem. 2004; 89: 822-33.
Florent, S., Malaplate-Am1and, C., Youssef, 1., Kriem, B., Koziel, V., Escanye, M.C., Fi fre,
A., Sponne, 1., Leininger-Mu ller, B., Oli vier, J. L., Pillot, T., Oster, T. Docosahexaenoic
ac id prevents neuronal apoptosis induced by so lub le amyloid-beta oligomers . ./.
Neurochem. 2006; 96(2): 385-95.
36
Fredr iksson, A., Schroder, N., Er iksson, P., lzquierdo, 1., Archer, T. Neonatal iron potenti ates
adu lt MPTP-induced neurodegenerat ive and funct ional deficits. Parkinsonism Re/at.
Dis ord. 200 1; 7: 97- 105.
Frisardi , V., Panza, F., Seripa, D., Farooqui, T., Farooq ui, A.A .. G lycerophospholi pids and
glycerophospholi pid-derived lipid medi ators: a complex meshwork in A lzheimer's
disease pathology. Prog. Lipid Res. 20 11 ; 50( 4): 3 13-30.
Galvan, A ., W ichmann, T . Pathophysiol ogy of Parkinsonism. Clinical Neurophysiology.
2008; 11 9: 1459- 1474.
Gaspar, P., Febvret, A., Co lombo, J . Serotonergic sprout ing in primate l\H P-induced
hemipark insonism. E~p. Brain. Res. 1993; 96: I 00-6.
Gerlach, M., R iederer, P. An imal models of Parkinson's di sease: an empirical compariso n
with th e phenomenology of the di sease in man. .!. Neural. Transm. 1996; 103: 987 - 104 1.
Giovanni , A., Sonsall a, P.K., Heikkil a, R.E. Studi es on species sensitivity to the
dopaminergic neurotox in 1-methyl-4-phenyl- 1, 2, 3, 6-tetra-hydropyri di ne. Part 2:
Central administrat ion of 1-methyl-4-pheny lpyridini um. .f. Phannacol. Exp. Ther. 1994;
270: 1008- 14.
Green, J.T., Liu, Z., Bazinet, R. P. Brain phospholipid arachidonic acid half-l ives are not
al tered foll owing 15 weeks of N-3 polyunsaturated falty acid adequate or deprived di et. .!.
Lipid. Res . 20 I 0; 5 1(3): 535-43.
Green, K.N., Mart inez-Coria, H., Khashwj i, H., Hall, E.B., Yurko-Mauro, K.A ., Elli s, L.,
LaFerla, F.M. Di etary docosahexaenoic acid and docosapentaenoic acid ameli o rate
amyloid-beta and tau pathology via a mechanism involving presen ilin I levels. J.
Newosci. 2007; 27: 4385-95.
Groenewegen, H.J.The basal ganglia and motor control. Neural. Plast. 2003; 10: 107-20.
Halliday, G ., Herrero, M.T., Murphy, K., McCann, H., Ros-Bernal, F., Barcia, C ., Mori , H.,
Bl esa, F.J., Obeso, J.A . No Lewy pathology in monkeys with over 10 years o f severe
MPT P Parkinsonism. Mov. Dis ord. 2009; 24( I 0): ! 5 19-23.
Hall man, H., O lson, L., Jonsson, G. Neurotox icity of the meperidine analogue N-methyl-4phenyl- 1,2,3,6-tetrahydropyridine on brain catecholamine neurons in the mouse. Eur. .f.
Phannacol. 1984; 97: 133-6.
Hashimoio, M .., Tanabe, Y ., Fuj ii, Y ., Ki kuta, T., Shibata, H., Shido, 0 . Chronic
administrat ion of docosahexaenoic acid ameliorates the impairment of spat ial cogn it ion
learn ing abili ty in amyloid beta-infused rats . .!. Nutr. 2005; 135: 549-55.
Hashimoto, M., Hossain, S., Agdul, H., Shido, 0 . Docosahexaenoic acid-induced
ameli orati on on impairn1ent of memory learning in amyloid beta-infused rats relates to
the decreases of amyloid beta and cholesterol levels in detergent-insoluble membrane
fract ions. Biochim. Biophys. Acta. 2005; 1738: 9 1-8.
Hashimoto, M., Hossain, S ., Shimada, T ., Shido, 0 . Docosahexaenoic acid-induced protective
effect against impaired learn ing in amyloid beta-infused rats is associated w ith increased
synaptosomal membrane fl uidity. C/in. Exp. Pharmacal. Physiol. 2006; 33: 934-9.
Hashimoto, M., Hossain, S., Shimada, T ., Sugioka, K., Yamasaki, H., Fujii, Y ., lshibashi, Y.,
Oka, J., Shido, 0 . Docosahexaenoi c ac id provides protection from impairn1ent of learn ing
abili ty in A lzheimer's di sease model rats . .f. Neurochem. 2002; 8 1: I 084-9 1.
Hashimoto, M., Katakura, M., Hossain, S ., Rahman, A., Shimada, T., Shido, 0.
Docosahexaenoic acid w ithstands the AB(2 5-35)-induced neurotox icity in SH-SY5Y
cell s . .I Nutr Biochem. 20 11 ; 22( I): 22-9.
37
Hay, J. F., Moscovitch, M., Levine, B. Dissociat ing habit and recoll ection: evidence from
Park inson's disease, amnesia and focal lesion pat ients. Neuropsychologia. 2002; 40:
1324-34.
Herkenham, M., Litt le, M. D., Bankiew icz, K., Yang, S.C., Markey, S.P., Johannessen, J.N.
Selecti ve retent ion of MPP+ within the monoaminergic systems of the primate brain
foll ow ing MPT P admi nistration: an in vivo autoradiograph ic study. Neuroscience. 199 1;
40: 133-58.
Heikk ila, R.E ., Cabbat, F.S., lvlanzino, L., Duvoisin, R.C. Effects of 1-methyl-4-phenyl1,2,5,6-tetrahydropyrid ine on neostriata! dopamine in mice. Neuropharmacology 1984;
23: 71 1-3.
Hell enbrand, W., Boeing, H., Robra, B.P., Seidl er, A ., Vieregge, P., N ischan, P., Joerg, J.,
Oertel, W.H., Schneider, E., U lm, G. Diet and Parkinson's di sease. 11: A possible rol e for
the past intake of speci lie nutrients. Results from a self-administered food-frequency
quest ionnaire in a case-control study. Neurology. 1996; 47(3): 644-50.
Hell enbrand, W., Seidl er, A., Boeing, H., Robra, B.P., V ieregge, P., N ischan, P., Joerg, J.,
Oertel, W.H., Schneider, E., U lm, G. D iet and Park inson's disease. 1: A possible rol e for
the past intake of speci fi c foods and food groups. Results from a self-administered foodfrequency quest ionnaire in a case-control stu dy. Neurology. 1996; 47(3):636-43.
Herrero, lvi.T ., Barcia, C., Navarro, J.M. Functional anatomy of thalamus and basal ganglia.
Chi!ds Nerv. Syst. 2002; 18: 386-404.
Herrero, M.T., Hirsc-h, E.C ., Kastner, A., Ruberg, M., Luquin, M.R., Laguna, J., Javoy-Agid,
F., Obeso, J.A., Agid, Y. Does neuromelan in contribute to the vu lnerability of
catecholaminergic neurons in monkeys intoxi cated w ith MPT P? Neuroscience. 1993; 56:
499-5 11.
Hirsch, E.C. How to j udge animal models of Park inson's disease in terms of neuroprotect ion .
.f. Neural. Transm. Suppl. 2006; 70: 255-60.
Hoang, T., Choi, D.K., Nagai, M., Wu, D.C., Nagata, T., Prou, D., Wilson, G .L., V ila, lvl.,
Jackson-Lew is, V ., Dawson, V. L., Dawson, T.M ., Chesselet, M.F. and Przedborski, S.
Neuronal NOS and cyclooxygenase-2 contribute to DNA damage in a mouse mode l of
Park inson disease. Free. Radic. Bio/. ,Hed. 2009; 47: 1049- 1056.
Hunot, S., V ila, M., Teismann, P., Davis, R.J., Hirsch, E.C., Przedborski, S., Rakic, P.,
Flavell , R.A . JN K-mediated induct ion of cyc looxygenase 2 is required for
neurodegenerat ion in a mouse model of Park inson's disease. Proc. ll/atl. A cad. Sci. U S A.
2004; 101: 665-70.
Jankovic, J. Park inson's disease: cli nical features and diagnosis . ./. Neural. Neurosurg.
Psychiatry. 2008; 79: 368-76.
Jin, J., lvleredith, G.E ., Chen, L., Zhou, Y ., Xu, J., Shie, F.S., Lockhart, P., Zhang, L
Quantitative proteomic analysis of mitochondri al proteins: relevance to Lewy body
format ion and Park inson's disease. Brain. Res. Mol. Brain. Res. 2005; 134: 11 9-38.
Johnson, C.C ., Gorell , J.M., Rybicki, B.A., Sanders, K., Peterson, E.L. Adult nutrient intake
as a risk factor for Park inson's disease. /nt. .f. Epidemiol. 1999; 28(6):1102-9.
Jonsson, G., Nwanze, E., Luthman, J., Sundstrom, E. Effect of MPTP and its pyridinium
metabolites on monoamine uptake and on central catecholamine neurons in mice. Acta.
Physiol. Scand. 1986; 128: 187-94.
38
Juli en, C., Berth iaume, L., Hadj-Tahar, A ., Raj put, A.H ., Bedard, P.J., Di Paolo, T., Ju lien, P.,
Calon, F. Postmortem brain fa tty acid profile of levodopa-treated Parkinson disease
patients and park insonian monkeys. Neurochem lnt. 2006;48(5):404 - 14.
Kabuto, H., Amakawa, M., Mankura, M., Yamanushi, T.T., Mori, A. Docosahexaenoic acid
ethyl ester enhances 6-hydroxydopamine-induced neuronal damage by induct ion of lipid
peroxidation in mouse striatum. Neurochem. Res . 2009; 34(7): 1299-303.
Kehagia. A .A., Barker, RA., Robb ins, T.W. Neuropsychological and clinical heterogeneity
of cogn it ive impaim1ent and dement ia in pat ients w ith Park inson's disease. Lancet.
Neurol. 20 I 0; 9( 12): 1200- 13.
Kandel, E.R ., Si egelbaum, S.A., Schwartz, J.H . chapter 10- 15. In: Principles of neural
science, Eds: Kandel, E.R., Schwartz, J.H ., Jessell , T.i'v!. lvlcGraw-Hill Companies, Inc.
USA, 4th ed; 2000: 175-280.
Kast in, A.J., Akerstrom, V., Pan, W. Glial cell line-der ived neurotroph ic factor does not enter
normal mo use brain. Neurosci. Lett. 2003; 340(3):239-4 1.
Kavanagh, T., Lonergan, P.E., Lynch, lviA . Eicosapentaenoic acid and gamma-linol en ic acid
increase hippocampal concentrations of IL-4 and IL- 10 and abrogate lipopol ysaccharideinduced inhibition of long-term potentiation. Prostaglandins Leukot. Essent. Fatty Acids .
2004; 70: 39 1-7.
Ki tajka, K., Sinclair, A.J., Weisinger, R .S., Weisi nger, H .S., lvlathai , M, Jayasooriya, A .P.,
Halver, J.E., Puski.\ s, L.G . E ffects of di etary omega-3 polyunsaturated fatty acids on brai n
gene expression. Proc. Natl. A cad. Sci. 200 4; I 0 I : I 093 1-6.
Klivenyi, P., Beal , M. F., Ferrante, R.J., Andreassen, O .A., Wermer, M., Chin, M. R.,
Bonventre, J.V. Mice defici ent in group IV cytosolic phospho lipase A2 are resistant to
MPTP neurotoxici ty . .f. Neurochem. 1998; 71: 2634- 7.
Kou, W., Luchtman, D., Song, C. E icosapentaenoic acid (EPA} increases cell viability and
expression of neurotroph in receptors in ret inoic acid and brai n-derived neurotrophic
factor differentiated S H-SY5Y ce lls. Eur. .f. Nutr. 2008; 47(2): 104- 13.
Koprich, J.B., Reske-N ielsen, C., lvlithal , P., lsacson, 0 . Neuroin nammat ion mediated by ILl beta increases susceptibi Iity of dopamine neurons to degenerat ion in an animal model of
Park inson's disease . ./. Neuroinjlammation. 2008; 5: 8.
Kuperstein, F., Eilam, R., Yavin, E. A ltered express ion o f key dopaminergic regulatory
proteins in the postnatal brain fol low ing perinatal n-3 fatty acid dietary defici ency. J.
Neurochem. 2008; I 06: 662-71.
Kuperstei n, F., Yakubov, E., Dinem1an, P., Gil , S ., Eylam, R., Salem, N. Jr, Yavin, E.
Overexpression of dopamine receptor genes and their products in the postnatal rat brai n
foll owing maternal n-3 fatty acid di etary deficien cy . .f. Neurochem. 2005, 95: 1550-62.
Langsto n, J.W., Ball ard, P.A . Jr. Parkinson's disease in a chemist working w ith 1-methyl-4phenyl- 1,2,5,6-tetrahydropyridine. N. Engl. .f. M ed. 1983; 309: 3 10.
Langsto n, J.W., Forno, LS ., Tetrud, J., Reeves, A .G., Kap lan, J.A., Karluk, D. Evidence of
act ive nerve cell degenerat ion in the substantia ni gra of humans years alter 1-methyl-4phenyl- 1,2,3,6-tetrahydropyri dine exposure. Ann. Neural. 1999, 46 : 598-605.
Lau, Y.S ., Trobough, K. L., Crampton, J.M., Wilson, J.A. Effects of probenecid on striatal
dopamine dep letion in acute and long-tem1 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine
(MPT P)-treated mice. Gen. Pharmacal. 1990; 2 1: 18 1-7.
Lauterbach, E.C. The neuropsychiatry of Park inson's disease and related disorders. Psychiatr.
Clin. North. Am. 2004, 27: 80 1-25.
39
Lawson, L.J., Perry, V.H., Dri, P. Gordon S Heterogeneity in the distr ibution and morphology
of microglia Neuroscience. 1990; 39: 15 1- 70.
Leng, A., Mura, A., Feldon, J., Ferger, B. Tumor necrosis factor-alpha receptor ablation in a
chronic MPT P mouse model of Parkinson's disease. Neurosci. Lett. 2005; 28; 375: !07-11.
Li u, B., Hong, J.S. Role of microglia in inflammat ion-mediated neurodegenerati ve diseases:
mechanisms and strategies for therapeutic intervention . ./. Pharmacol. Exp . Th er. 2003;
304: 1-7.
Liu, X ., Yamada, N., lvlaruyama, W., Osawa, T. Fom1at ion of dopamine adducts derived from
brain pol yunsaturated fatty acids: mechanism for Park ins on disease. ./. Bioi. Chem. 2008;
283( 50): 34887-9 5.
Ll edo, A. Dopamine agonists: the treatment for Park inson's disease in the XXI century?
Parkinsonism Re/at. Disord. 2000; 7: 5 1-58.
Logroscino, G., Marder, K ., Cote, L., Tang, M .X., Shea, S., Mayeux, R. Dietary li pids and
antioxidants in Parkinson's disease: a populat ion-based, case-control study. Ann. Neurol.
1996; 39( I): 89-94.
Logroscino, G ., Marder, K ., Graziano, J., Freyer, G ., Slavkovich, V., Lojacono, N., Cote, L.,
lvlayeux, R .. Di etary iron, animal fats, and r isk of Park inson's disease. Mov Disord. 1998;
13 Suppl I : 13-6.
Lonergan, P.E., Mart in, D.S., Horrobin, D .F. , Lynch, M.A . Neuroprotect ive effect of
eicosapentaenoic acid in hippocampus of rats exposed to gamma-i rradiation . .!. Bioi.
Chem. 2002; 277: 20804-11.
Lonergan, P.E., Marti n, D.S.D., Horrobin, D.F., Lynch, M.A .. Neuroprotect ive act ions of
eicosapentaenoic acid on lipopol ysaccharide-induced dys funct ion in rat hippocampus. J.
Neurochem. 2004; 9 1: 20-9.
Luchtman, D.W., 1v!eng, Q ., Song, C. Ethyl-eicosapentaenoate ( E-EPA) attenuates motor
impaim1ents and inflammat ion in the MPT P-probenecid mouse model of Park inson's
disease. Behav. Brain. Res . 2012;226 :386-96.
Luchtman, D.W., Shao, D., Song, C . Behavior, neurotransmitters and inflammat ion in three
regimens of the l'v!PTP mouse model of Park inson's disease. Physiol. Behav. 2009;
98: 130-8.
Luchtman, D.W., Song, C. Cogn itive enhan cement by omega-3 fatty acids from child-hood to
old age: findi ngs trom animal and cli nical studi es. Neuropharmacology, In press.
Luchtman, D.W., Song, C. Why S H-SY5Y cells should be differentiated. Neurotoxicology.
20 I 0; 3 1(I): 164-5.
Massano, J., Garrett, C. Deep brain stimulat ion and cogn itive decl ine in Park inson's disease: a
clinical review. Front. Neurol. 20 12; 3: 66.
Lynch, A .lvl., Loane, D.J., lv!inogue, A.M ., C larke, R.lvl., Kil roy, D., Nall y, R.E., Roche,
O.J., O'Connell , F., Lynch, M.A. Eicosapentaenoic acid confers neuroprotection in the
amyloid-beta challenged aged hippocampus. Neurobiol. Aging. 2007; 28: 845-55.
Marttil a, R.J., Lorentz, H., and Rinne, U. K. Oxygen tox icity protect ing enzymes in
Parkinson's disease. Increase o f superoxide dismutase-like act i,ity in the substanti a nigra
and basal nucleus . ./. Neurol. Sci. 1988 ; 86: 32 1-33 1.
Martinez. M., lchaso, N., Setien, F., Durany, N., Qiu, X., Roesler, W. The ~4-desaturation
pathway for DHA biosynthesis is operative in the human species : di trerences between
normal controls and children w ith the Zellweger syndrome. Lipids Health Dis. 20 ! 0; 9: 98.
40
Mavridis, lv!., Degryse, A.D ., Lategan, A.J ., Marien, M.R., Colpaert, F.C. Effects of locus
coeru leus lesions on park inson ian signs, striatal dopamine and substant ia nigra cell loss
after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyr idine in monkeys: a possible role for the
locus coeruleus in the progression of Parkinson's disease. Neuroscience. 199 1; 4 1: 507-23.
McGeer, P.L., Jtagaki , S., Boyes, B.E., McGeer, E.G. Reactive microgli a are positive for
HLA-DR in the substantia nigra of Parkinson's and A lzheimer's disease brains
Neurology. 1988, 38: 1285-91.
Melamed, E., Pi karski, E., Goldberg, A., Rosenthal, J., Uzzan, A., Confort i, N. Effect of
serotonergic, corticostriatal and ka inic acid les ions on the dopaminergic neurotoxicity of
1-methyl-4-phenyl- 1,2,5,6-tetm-hydropyridine (M PT P) in mice. Brain Res. 1986; 399:
178-80.
Meng, Q. , Luchtman, D.W., El Bahh, B., Zidichouski , J.A ., Yang, J., Song, C. Ethyleicosapentaenoate modulates changes in neurochemistry and bra in lipids induced by
parkinson ian neurotoxin 1-methyl-4-phenylpyridinium in mouse brain slices. Eur. J.
Pharmacol. 2010; 649: 127-34.
Meredith, G.E., Kang, U..l. Behavioral models of Parkinson's disease in rodents: a new look at
an old problem. Mov. Disord. 2006; 2 1: 1595-606.
Meredith, G .E., Totterdell , S., Petroske, E., Santa Cruz, K., Call ison, R.C. Jr, Lau, Y.S.
Lysosomal mal function accompan ies alpha-synuc lein aggregat ion in a progress ive mouse
model of Parkinson's disease. Brain. Res. 2002; 22; 956: 156-65.
Meredith, G.E., Totterdell, S., Potashk in, J.A., Sum1eier, D.J. Modeling PD pathogenesi s in
mice: advantages of a chron ic M PTP protocol. Parkins onism Re/at. Dis ord. 2008; 14:
S ll 2-5.
lv!inghetti, L. Cyclooxygenase-2 (COX-2) in in flanm1atory and degenerative brain diseases . ./.
Neuropathol. Exp. Neurol. 2004; 63: 901- 10.
1-'linogue, A.M., Lynch, A.M., Loane, D.J., Herron, C.E., Lynch, lvi.A. Modulat ion of
amyloid-beta-induced and age-assoc iated changes m rat hippocampus by
eicosapentaenoic acid. J Neurochem. 2007; I 03: 9 14-26.
Miyake, Y., Sasaki, S., Tanaka, K., Fukushima, W., Kiyohara, C., Tsubo i, Y., Yamada, T.,
Oeda, T., Miki, T., Kawamura, N., Sakae, N., Fukuyama, H., Hirota, Y., Nagai, M.;
Fukuoka Kink i. Park inson's Disease Study Group. Dietary fat intake and risk of
Parkinson's disease: a case-control study in Japan . .f. Neurol. Sci. 2010; 288(1 -2): 11 7-22.
lv!iyoshi, E., W ietzikoski, S., Camp lessei, M., Silveira, R., Takahashi, R.N., Da Cunha, C.
Impaired learn ing in a spat ial work ing memory version and in a cued version of the water
maze in rats w ith MPT P-induced mesencephalic dopaminergic les ions. Brain Res. Bull.
2002; 58: 4 1-7.
Mogi, M., Togari, A., Kondo, T., Mizuno, Y., Komure, 0., Kuno, S., Jchinose, H., Nagatsu,
T. Brain-der ived growth factor and nerve growth facto r concentrat ions are decreased in
the substant ia nigra in Parkinson's di sease. Neurosci. Lett. 1999; 270(1 ):45-8.
Moratalla, R., Quinn, B., DeLanney, L.E., lrw in, !., Langston, J.W., Graybiel, A.M.
Different ial vuJnerab il it y of primate ea udate-putamen and stri osome-matri x dopamine
systems to the neurotox ic effects of 1-methyl-4-phenyl- 1,2,3,6-tetrahyd ropyri dine. Proc.
Nclll. A cad. Sci. US A. 1992; 89: 3859-63.
Muntane, G., Janue, A., Femandez, N., Odena, M.A., O liveira, E., Boluda, S., Portero-Ot in,
M., Naudi, A., Boada, J., Pamplona, R., Ferrer, L Modi ficat ion of brain lipids but not
Omega-3 Pol yunsaturated Fatty Acid Treatment in Park inson's Disease. ..
41
phenotype in alpha-synucleinopathy transgenic mice by long-term dietary n-3 faHy acids.

Neurochem. lnt. 20 I 0; 56(2): 3 18-28.
Murpby, R.A., Yeung, E., Mazurak, V.C., Mourtzakis, M. Infl uence of eicosapentaenoic acid
supplementat ion on Jean body mass in cancer cachexia. Br . .f. Cancer. 2011; 105(10):
1469 -73.
Nagatsu, T., Mogi, l\'1., Jch inose, H., Togari, A. Cytok ines in Parkinson's disease. Neural
Transm. Suppl. 2000; 58: 143-5 1.
Nagatsu, T., Mogi., M., lch inose, H., Togari, A. Changes in cytok ines and neurotroph ins in
Parkinson's di sease . ./. Neural. Transm .Suppi. 2000; 60: 277-90.
Novikova, L., Garris, B.L., Garris, D.R., Lau, Y.S. Early signs of neuronal apoptosis in the
substantia nigra pars compacta o f the progressive neurodegenerative mou se l -methyl-4phenyl- 1,2,3,6-tetrabydropyri dine/ probenecid model of Parkinson's disease.
Neuroscience. 2006; 140( I): 6 7-76.
Often, D., Beart, P.M., Cheung, N.S ., Pascoe, C.J., Hochman, A., Gorod in, S., Melamed, E.,
Bemard, R., Bemard, 0. Transgen ic mice expressing human Bcl-2 in their neurons are
resistant to 6-hydroxydopamine and 1-metbyl-4-phenyl-1,2,3,6- tetrahydropyridi ne
neurotoxicity. Proc. Natl. A cad. Sci. U S A. 1998; 95: 5789-94.
Okuno, T., Nakatsuj i, Y ., Kumanogoh, A., Moriya, M., Jch inose, H., Sumi, H., Fujimura, H.,
Kikutan i, H., Sakoda, S.. Loss of dopaminergic neurons by the induction of inducible
nitr ic ox ide synthase and cyclooxygenase-2 via C D 40: relevance to Park inson's disease .
.f. Neurosci. Res.2005; 8 1: 874-82.
Okuyama, H., Orikasa, Y., N ishida, T. Sign ificance of ant ioxidat ive funct ions of
eicosapentaenoic and docosahexaenoic acids in marine microorgan isms. Appl. Environ.
Microbial. 2008; 74(3): 570-4.
Ouellet, M., Emond, V., Chen, C.T., Julien, C., Bourasset, F., Oddo, S., LaFerla, F., Bazinet,
R.P., Calon, F. Di ffusion of docosahexaenoic and eicosapentaenoic acids through the
blood-brain barrier: An in situ cerebral perfus ion study. Neurochem. lnt. 2009; 55(7):
4 76-82.
Ozsoy, 0., Sevai-Celi k, Y., Hacioglu, G., Yargicoglu, P., Demir, R., Agar, A., As ian, M. The
influence and the mechan ism of docosahexaenoic acid on a mouse model of Parkinson's
disease. Neurochem. lnt. 2011; 59(5):664-70.
Palsdott ir, V., Mansson, .I.E., Blomqvist, M., Egecioglu, E., O lsson, B. Long-tem1 effects of
perinatal essent ial fatty acid defi c iency on anx iety-related behavior in mice. Beha v.
Neurosci. 20 12; 126(2): 36 1-9.
Peel, M., Stokes, C. Omega-3 fatty acids in the treatment of psych iatric disorders . Drags.
2005; 65: I 05 1-9.
Perier, C., Bove, J., Wu, D.C., Dehay, B., Choi, D.K ., Jackson-Lew is, V ., Rathke-Hartlieb,
S., Boui ll et, P., Strasser, A ., Schulz, J. B., Przedborsk i, S., V ila, M. Two molecu lar
pathways Jnit Jate mitochondria-dependent dopaminergic neurodegeneration m
experimental Park inson's disease. Proc. Natl. A cad. Sci. US A. 2007; 104: 8 !6 1-6.
Perrin, R.J., Woods, W.S., C layton, D.F., George, J.M. Exposure to long chain
polyunsaturated fatty acids triggers rapid mult imerizat ion of synucleins . .f. Bioi. Chem.
200 I ; 276( 45): 4 1958-62.
Peura, D.A., Goldkind, L. Balancing the gastrointestinal benefits and risks of nonselective
NSA!Ds. Arthritis. Res. Ther. 2005; 7: S7-13.
42
Dirk W. Luchtman, Jeflrey A . Zidichouski and Cai Song
Peterson A L, Nut! JG. Treatment of Parkinson's disease w ith trophic factors.

Neurotherapeutics. 2008; 5(2): 270-80.
Petroske, E., Meredith, G.E., Callen, S., Totterdell, S., Lau, Y.S. Mouse model of
Parki nsonism: a comparison between subacute MPTP and chronic MPTP/ probenecid
treatment. Neuroscience. 2001; 106: 589-601.
Piil , C., Homyk iew icz, 0., Giros, B., Caron, lvi.G. Catecholamine transporters and 1-melhyl4-phenyl-1,2,3,6 -tetrahydropyridi ne neurotox icity: studies comparing the c loned human
noradrenali ne and human dopamine transporter. .f. Pharmacal. Exp. Ther. 1996; 277:
1437-43.
Pil eb lad, E., Carlsson, A. Studies on the acute and long-term changes in dopamine and
noradrenali ne metabo li sm in mouse brain follow ing administration of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP). Pharmacal. Toxicol. 1988, 62: 21 3-22.
Pil eblad, E., N issbrandt, H., Carlsson, A. Biochemical and functiona l evidence tor a marked
dopamine releasing act ion of N-melhy l-4-phenyl- 1,2,3,6-telrahydropyridine (NlviPTP) in
mouse brain . .f. Neural. Transm. 1984; 60: 199-203.
Poeh lman, E.T., Toth, M.J., Fishman, P.S., Vailkevicius, P., Golllieb, S.S., Fi sher, M. L.,
Fonong, T. Sarcopenia in aging humans: the impact of menopause and di sease . .f.
Gerontal. A. Bioi. Sci. Med. Sci. 1995; 50 Spec No: 73-7.
Presgraves, S.P., Ahmed, T., Borwege, S., Joyce, J.N. Tem1inall y di fferentiated SH-SYSY
cell s provide a model system for studying neuroprotective effects of dopamine agonists.
Neurotox. Res . 2004; 5(8): 579-98.
Puri, B.K., Bydder, G.M., Manku, M.S., Clarke, A., Waldman, A.D., Beckmann, C. F.
Reduction in cerebral atroph y associated w ith ethyl-eictlsapentaenoic acid treatment in
patients w ith Huntington's disease. .f. !nt. Med. Res. 2008; 36: 896-905.
Purves, D., August ine, G.J., Fitzpatrick, D., Hall , W.C., Lamant ia, A-S., J.O., i'vlcNamara,
Wi 11 iams, S.M. Neuroscience, S inauer Asssoc iates, Inc. Sunderland, MA, 4th ed; 200 I :
chapter 6, 129- 161.
Qin, Z., Hu, D., Han, S., Reaney, S.H., Di Monte, D.A., Fink, A. l. Effect of 4-hydroxy-2nonenal modi fication on alpha-synuclein aggregat ion . .f. Bioi. Chem. 2007; 282(8):
5862- 70.
Quinn, L.P ., Stean, T.O., Chapman, H., Brown, M., Vidgeon-Hart, M., Upton, N., Bi llinton,
A., Virley, D.J. Further val idat ion of LABORAS using various dopaminergic
man ipulations in mice includi ng lvlPTP-induced nigra-striatal degenerat ion . .f. Neurosci.
M ethods. 2006; 156: 2 18-27.
Rabey, J.M. and Bums, R.S. Neurochemistry. In: Parkinson's disease: diagnosis and clinical
management. Eds. Weiner, W.J. and Factor, S.A. Demos Medica l Publi sh ing, New York,
New York, 2002: 195-2 11.
Rao, J.S., Ert ley, R.N., Lee, H.J., Del\lar, J.C. Jr, Amold, J.T., Rapoport, S.l., Bazi net, R.P. n3 polyunsaturated fally acid depr ivation in rats decreases frontal cortex BDN F via a p38
MA PK-dependent mechan ism. Mol. Psychiatry. 2007; 12( 1):36-46.
Rezak, M. Current pham1acotherapeutic treatment opt ions in Parkinson's disease. Dis. Mon.
2007; 53: 2 1422.
Ri ach i, N.J., Harik, S. l., Kalaria, R.N., Sayre, L.M. On the mechan isms underlying 1-methyl4-phenyl- l ,2,3,6-tetrahydropyridi ne neurotoxicity. 11. Suscept ibi lily among mammali an
species correlates with the tox in's metabolic pauems in bra in microvessels and liver. .f.
Pharmacal. Exp. Ther. 1988; 244: 443-8.
43
Richard, D., Kefi , K., Barbe, U., Bausem, P., V isioli, F. Pol yunsaturated fatty acids as
antioxidants. Pharmacal. Res. 2008; 57(6): 45 1-5.
Ri edel, M., Goldbaum, 0., Will e, M., Ri chter-Landsberg, C. Membrane lipid modi fication by
docosahexaenoic acid (DHA) promotes the format ion of a-synuclein inclusion bodies
immunopositive fur SUM0- 1 in oligodendroglial cell s after ox idative stress . ./. Mol.
Neurosci. 20 11 ; 4 3(3): 290-302.
Robson, L.G., Dyall , S., S idl of~ D., lv!ichaei-T itus, A .T. Omega-3 pol yunsaturated (ally ac ids
increase the neurite outgrowth of rat sensory neurones throughout development and in
aged animals. Neurobiol. Aging. 20 I 0; 3 1( 4):678-87.
Rojas, C .V., Gre iner, R.S., Fuenzalida, L.C, Mart inez, J.l., Salem, N. Jr., Uauy, R. Long-tem1
n-3 FA defi ciency modi fies peroxisome proli ferator-activated receptor beta mRNA
abundance in rat ocular t issues. Lipid.~. 2002; 37: 367-74.
Rommelfanger, K.S., Edwards, G. L., Freeman, K.G ., Liles, L.C., l'v! ill er, G.W., Weinshenker,
D. Norep ineph rine loss produces more profound motor deficits th an MPT P treatment in
mice. Proc. Natl. A cad. Sci. 2007; I 04: 13804-9
Rommel fanger, K.S., Weinshenker, D. Norep inephrine: T he redheaded stepchild of
Park inson's disease. Biochem. Pharmacol. 2007; 74: 177-90.
Rommel fanger, K.S., Weinshenker, D., Mi Jl er, G .W. Reduced MPTP toxi city m
noradrenali ne transporter knockout mice . ./. Neurochem. 2004; 9 1: 111 6-24.
Rousselet, E., Joubert, C., Call ebert, J., Parain, K., Tremblay, L., Orieux, G ., Launay, J.1v!.,
Cohen-Sal mon, C., Hirsch, E.C. Bebavioral changes are not directly related to striatal
monoamine levels, number of nigra! neurons, or dose of park insonian toxin MPT P in
mice. Neurobiol. Dis. 2003; 14:2 18-28.
Rozas, G., Liste, 1., Guerra, lv!.J., Labandeira-Garcia, J. L. Sprouting of the serotonergic
afferents into striatum after select ive lesion of the dopaminergic system by MPT P in
adult mice. Neurosci. Let!. 1998; 245: 15 1-4.
Russo, G. L. Dietary n-6 and n-3 polyunsaturated fatty acids: From biochemistry to cli nical
imp li cat ions in cardiovascular prevent ion. Biochem. Pharmacol. 2009; 77: 937-46.
Samadi, P., Gregoire, L., Rouill ard, C., Bedard, P.J., Di Paolo, T., Levesque, D.
Docosahexaenoic acid reduces levodopa-induced dyskinesias in 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine monkeys. Ann. Neural. 2006; 59(2): 282-8.
Schap ira, A .H. Complex 1: inhibitors, inhibit ion and neurodegenerat ion. Exp. Neural. 20 10;
224: 33 1-335.
Sarsil maz, M ., Songur, A., Ozyurt, H., Ku~, 1., Ozen, O.A., Ozyurt, B., S1igiit, S., Akyol, 0 .
Potent ial role of di etary omega-3 essential fally acids on some oxidant/ant iox idant
parameters in rats' corpus striatum. Prostaglandins Leukot. Essent. Fatty Acids. 2003; 69:
253-9.
Sch intu, N., Frau, L., lbba, M., Cabon i, P., Garau, A ., Carboni, E., Carta, A. R. PPARgamma-mediated neuroprotect ion in a chron ic mouse model of Parkinson's disease. Eur.
.f. Neurosci. 2009; 29: 954-63.
Sedelis, M., Schwarting, R.K., Huston, J. P. Behavioral pbenotyp ing of th e lv!PTP mouse
model of Parkinson's disease. Beha11. Brain. Res. 200 1; 125: 109-25.
Serhan, C.N., Chiang, N ., Van Dyke, T.E. Resolv ing inflammat ion: dual ant i-inflammatory
and pro-resolut ion lipid mediators. Nat. Rev. lmmunol. 2008; 8(5): 349-6 1.
44
Sharon, R., Bar-Joseph, I., Frosch, M.P., Walsh, D.lvl., Hamilton, J.A., Sel koe, D.J. The
fom1ation of high ly solub le oligomers ofalpha-synuclein is regu lated by fatty acids and
enhanced in Park inson's disease. Neuron. 2003; 37(4): 583-95.
Sharon, R., Go ld berg, M.S., Bar-Josef, 1., Betensky, R.A., Shen, J., Selkoe, D.J. alphaSynuclein occurs in lipid-rich high mo lecular weight comp lexes, binds fatiy acids, and
shows homology to the fally acid-binding proteins. Proc. Natl. A cad. Sci. U S A. 200 I;
98( 16): 9 11 0-5.
Shchepinov, M .S., Chou, V. P., Poll ock, E., Langston, J.W., Cantor, C.R., Molinari, R.J.,
Manning-Bog, A. B. Isotopic re inforcement of essential polyunsaturated fatty acids
diminishes nigrostriatal degenerat ion in a mouse model of Park inson's disease. Toxicol.
Leu. 2011; 207(2): 97-103 .
Shimoj i, M., Zhang, L., Mandi r, A.S ., Dawson, V. L., Dawson, T.M. Absence of inclusion
body fom1ation in the M PTP mouse model of Parkinson's disease. Brain. Res. Mol.
Brain. Res. 2005; 134: I 03-8.
Sidhu, V.K., Huang, B.X., Kim, H.Y. Effects of docosahexaenoic acid on mouse brain
synaptic p lasma membrane proteome anal yzed by mass spectrometry and (16)0/ (18)0
label in g . .f. Proceome. Res. 2011; I 0( 12): 54 72-80.
Simopou los, A.P. The importance of the rat io of omega-6/ omega-3 essent ial fatty acids.
Biomed. Pharmacother. 2002; 56(8): 365-79.
Song, C., Zhao, S. Omega-3 fatty acid e icosapentaenoic acid. A new treatment for psych iatric
and neurod egenerat ive diseases: a review of clinical invest igations. Expe1t Opin. I nvestig.
Dmgs. 2007; 16: 1627 -38.
Sozio, P., Cerasa, L.S., Abbadessa, A ., Di , Stefano, A. Desi gn ing prodrugs for the treatment
of Park inson's disease. Expert. Opin. Dmg. Discov., 2012; 7(5): 385-406.
Su, H.M. Mechanisms of n-3 fatty acid-mediated development and maintenance of learn ing
memory performance . .f. Nutr. Biochem. 20 I 0; 21: 364-73.
Taepavarapruk, P., Song, C. Reductions of acetylcholine release and nerve growth factor
expression are correlated with memory impai rment induced by interleuk in- 1beta
administrat ions: effects of omega-3 fatty acid EPA treatment. .f. Neurochem. 20 I 0; 112:
1054-64.
Tanriover, G., Sevai-Celik, Y., Ozsoy, 0. , Akkoyun lu, G., Savcioglu, F., Hacioglu, G.,
Demi r, N., Agar, A. The effects of docosahexaenoic acid on glial derived neurotroph ic
factor and neurturin in bilateral rat model of Park inson's disease. Folia. Histochem .
Cytobiol. 2010; 48(3): 434-41.
Tassoni, D., Kaur, G ., Weisinger, R.S., Si nclai r, A.J. The role of eicosano ids in the brain.
Asia Pac. .f. Clin. Nutr. 2008; I :220-8.
Te ismann, P., Tieu, K ., Choi, D.K., Wu, D.C., Naini, A., Hunot, S., V ila, M., Jackson-Lew is,
V. and Przedborski, S. Cyclooxygen ase-2 is instrumental in Parkinson's disease
neu rodegenerat ion. Proc. Natl. Acad. Sci. USA . 2003; 100:5473-5478.
Teismann, P., Schu lz, J. B. Cell ular pathology of Parkinson's disease: astrocytes, microglia
and inflammat ion . Cell Tissue Res. 2004; 3 18: 149-61.
Thanvi, B.R., Munsh i, S.K., Vijaykumar, N., Lo, T.C. Neuropsychiatric non-motor aspects of
Parkinson's disease. Postgrad. Med. ./. 2003; 79: 561 -5 .
Thomas B., Saravanan K.S. and lv!ohanakumar K.P. (2008). In vitro and in vivo evidences
that antioxidant action contributes to the neuroprotect ive effects of the neuronal nitric
45
oxide synthase and monoamine ox idase-B inhibitor, 7-nitroindazol e. Neurochem. Jnt. 52:
990-2001.
Th ompson, R.F., Kim, J.J. Memory systems in the brain and locali zation of a memory. Proc.
Not/. A cad. Sci. US A. 1996; 93: 13438-44.
de la Torre. M.R., Casado A., L6pez-Femandez. M.E., Carrascosa. D., Casado. M.C.,
Venarucci. D. and Venarucci. V. Human aging brain disorders: role of ant iox idant
en zyn1es. Neurochem. Res. 1996; 2 1: 885-888.
Ungerstedt, U. 6-Hydroxy-dopamine induced degenerat ion of central monoamine neurons.
Eur. .f. Pharmacol. 1968; 5: I 07- 10.
Vak il , E., Herishanu-Naaman, S. Declarative and procedural learn ing in Park inson's disease
patients having tremor or bradykinesia as the predominant symptom. Cortex. 1998; 34:
6 11 -20.
Van den Heuvel, D.M., Pasterkamp, R.J. Getting connected in the dopamine system. Prog.
Neurobiol. 2008; 85: 75-93.
Van Noort, J.M. Human gli al cell cu lture models of inflanm1ation in the central nervous
system. Drug. Disco v. Today. 2006; 11: 74-80.
Vil a, lvl., Jackson-Lewis, V., Vukosavic, S., Djaldett i, R., Liberatore, G., Offen, D.,
Korsmeyer, S.J., Przedborski, S. Bax ab lat ion prevents dopaminergic neurodegenerat ion
in the !-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Park inson's
disease. Proc. Natl. Acad. Sci. US A. 200 1; 98: 2837-42.
Vil a, M., Przedborski, S. Targeting programmed cell death in neurodegenerative diseases.
Nat. Rev. Neurosci. 2003; 4: 365-75.
Vil a, lvl., Vukosavic, S., Jackson-Lewis, V., Neystat, M., Jakowec, M., Przedborski, S.
Alpha-synuclein up-regu lation in substant ia nigra dopaminergic neurons following
administrat ion of the park inson ian tox in MPT P. J. Neurochem. 2000; 74: 72 1-9.
Von Bohlen und Halbach 0., Minichiello, L., Unsicker, K. Hap loi nsufticiency for trk B and
trkC receptors induces cell loss and accumulat ion of alph a-synuclein in the substantia
nigra. FASEB .f. 2005; 19( 12): 1740-2.
Vuckovic, M.G., Wood, R.l., Holschneider, D.P., Abernathy, A., Togasak i, D.lvl., Smith, A .,
Petzinger, G.M., Jakowec, M.W. Memory, mood, dopamine, and seroton in in the 1methyl-4-phenyl- ! ,2, 3,6-tetrahydropyridine-J esioned mouse model of basal gangli a
injury. Neurobiol. Dis. 2008; 32: 3 19-27.
Wahner, A .D., Bronstein, J.l'vl., Bordelon, Y .M., Ritz, B.: Nonsteroidal ant i-infl ammatory
drugs may protect against Park inson di sease. Neurology. 2007; 69: 1836- 1842.
Wang, T., Pei, Z., Zhang, W., Liu, B., Langenbach, R., Lee, C., Wilson, B., Reece, J.lv!.,
Mi ll er, D.S. and Hong, J.S. MPP+-induced COX-2 act ivat ion and subsequent
dopaminergic neurodegenerat ion. FASEB. .f. 2005; 19: 11 34- 11 36.
Wichmann, T., Del ong, M. R. Funct ional neuroanatomy of the basal ganglia in Park inson's
di sease. Adv. Newo/. 2003; 9 1: 9- 18.
Wu, D.C., Teismann, P., T ieu, K., V il a, M. and Jackson-Lewis, V. NADPH oxidase mediates
oxidat ive stress in the 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine model of Parkinson's
disease. Proc. Narl. Acad. Sci. USA. 2003; 100: 6 145-6 150.
Xie, H. R., Hu, L.S., Li, G.Y. SH-SY5Y human neuroblastoma cell line: in vitro cell model of
dopaminergic neurons in Parkinson's disease. Chin. Med. .f. (Engl). 20 I 0; 123(8): I 086-92.
Yakun in, E., Loeb, V., Kisos, H., B iala, Y., Yehuda, S., Yaari, Y ., Se lkoe, D.J., Sharon, R. Asynuclein neuropathology is controll ed by nuclear hormone receptors and enhanced by
46
Di rk W. Luchtman, Jeflrey A . Zidichousk i and Cai Song
docosahexaenoic acid in a mouse mode l for Park inson's disease. Brain. Pathol. 20 12;
22(3): 280-94.
Yang, L, Cal ingasan, N.Y., Chen, J., Ley, J.J, Seeker, D.A., Beal, M. F. A novel azulenyl
nitrone antioxidant protect s against MPTP and 3-nitropropionic acid neurotox icit ies. Exp.
Neural. 2005; 19 1: 86-93.
Yang, L., Matthews, R .T, Schulz, .1.8., Klockgether, T., Liao, A.W., l\l(art inou, J.C., Penney,
.I. B. Jr, Hyman, B.T., Beal, M. F. 1- Methyl-4-phenyl-1,2,3,6-tetrahydropyrid e
neurotoxicity is attenuated in mice overexpressing Bcl-2 . .f. Neurosci. 1998; 18: 8 145-52.
Yehuda, S., Rabinovitz, S., Carasso, R.L., Mostofsky, D.l. T he role of po lyunsaturated fatly
ac ids in restoring the aging neuronal membrane. Neurohiol. Aging. 2002; 23: 843-53.
Yehuda, S., Rabinovitz, S., IV! ostof.~ky, D.l. M ixture of essential fatly acids lowers test
anx iety. Nutr. Neurosci. 2005; 8: 265-7.
Youdi m, K.A, Martin, A., Joseph, LA. Essential fatty acids and the brain: possible health
implicat ions. lnt. J. Dev. Neurosci. 2000; 18: 383-99.
Young, G., Conquer, J. Omega-3 !ally acids and neuropsychiatric disorders. Reprod. Nutr.
D ev. 2005; 45: 1-28.
Zeevalk, G.D., Manzino, L., Sonsall a, P.K. and Bemard, L.P. Characterization of intracell ular
elevation of glutath ione (GSH) w ith glutath ione monoethyl ester and GSH in brain and
neuronal cultures: relevance to Parkinson's disease. Exp. Neurol. 2007; 20, 3 5 12-52.
Zhao, Y., Calon, F., Julien, C., Winkler, J.W., Petasis, N.A., Lukiw, W.J ., Bazan, N.G.
Docosahexaenoic acid-der ived neuroprotect in DJ induces neuronal survival via
secretase- and PPARy-mediated mechan isms in A lzhe imer's disease models. PLoS One.
2011; 6(1):e l 58 16.
Zi mmer, L., Hembert, S., Durand, G., Breion, P., Guilloteau, D., Besnard, J.C., Chalon, S.
Chron ic n-3 polyunsaturated fatty acid di et-defic iency acts on dopamine metaboli sm in
the rat frontal cortex: a microdialysi s study. Neurosci Leu. 1998; 240: I 77-8 1.
In: Eicosapentaenoic Acid

Edi tors: T.G. Bradley and F.P. Vargas, pp. 47- 73
ISBN: 978- 1-62257-480-3

20 12 Nova Science Publishers, Inc.
Chapter 2
E ICOSAPENTAENOIC ACID (EPA)

AND B ONE METABOLISM
Gerard G. Dumanca/ , Rangika S. Hikkaduwa Koraleg/,
Patrisha .f. Phanl and Elmer-Rico E. Mojica3
1
Department of Chemistry,
Oklahoma State University, StiUwater, OK, US
2
Dave C. Swalm School ofC'hemical Engineering,
Miss issippi State Un iversity, MS, US
>Department of Chemistry and Physical Sciences,
Pace University, One Place Plaza, New York, J\Y, US
Abstract
Polyunsarurated fatty acids (PUFAs) are precursors of a wide range of metabolites, for
example eicosanoids like prosmglandins and leukmrienes, which play critical roles in the
regulation of a variety of biological processes, including bone metabolism. PUFAs can be
classified imo ro-6 and ro-3 groups. Eicosapemaenoic acid (EPA) is an ro-3 PU FA obtained in
the human diet by the consumption of marine products. lt is also found in the human breast
milk. In the skeleton, there is evidence showing that altering the relative composition of
dietary fatty acids can affect bone cell metabolism. Several studies have shown the bene fits of
EPA in bone. An EPA-enriched diet prevemed loss of bone weight and strength in
ovariectomized (OVX) rats fed with a lower calcium diet. lt is also known to coumeract
weightlessness-induced bone loss by inhibiting ~F-KB, a transcriptional activator of many
genes, including some that lead m muscle atrophy and bone resorption. In another study, EPA
was shown to be effective for diabetic osteopenia. Despite these benefits of EPA, a study
indicated that supplememation of fish oil in large quamities and the associated increase in
dietary far comern can lead to substantial degradation of the morphological and mechanical
properties of cortical bone. This chapter will discuss the positive and negative effects of EPA
intake in relation to bone metabolism as well as the possible mechanisms that explain such
effects. In addition, the effect of ro-6/ro-3 ratios on calc ium balance and bone as well as the
recommended dietary imake of EPA will also be discussed.
E-mail address: gerard.dumancas@okstate.edu.
48
Gerard G. Dumancas, Rangika S. Hi kkaduwa Kora lege, Patrisha J. Pham et al.
List of Abbreviations
.1.\DA
AFFSA
AHA
ALA
.AJ\IIDR
Bcl-2
BD
BM
BMC
Bl'VID
CF
CHD
CLA
CO
CVD
CvPG
'
DH A
DRI
EFA
EPA
FA
FDA
FN-B MD
FO
FO
FOD
GO
Gl
GLA
HT
HSBO
IGF-1
IGF-1
ILS I
IOM
ISSFAL
KO
LA
LDSTZ-D
LSCs
LTBs
LTEs
MCR
American Dietetic Association

Agence Francaise de Secur ite Sanitaire des Aliments
American Hean Association
a-Linolen ic acid
Acceptable macronutrient distribution range
B-cell lymphoma 2
Bone density
Bone marrow
Bone mineral content
Bone mineral density
Chicken fat
Coronary hean disease
Conj ugated linoleic
Corn oil
Cardiovascular disease
Cyclooxygenase-deri ved cyclopentenone prostaglandin
Docosahexaenoic acid
Dietary Reference Intake
Essential fatty acids
Eicosapentaenoic acid
Faily acids
Food and Drug Admi nistration
Femoral neck-bone mineral density
Fish oil
Flaxseed oil
Fish oil diet
Gap 0
Gap I
y-Linolenic acid
Hom10ne therapy
Hydrogenated soybean oil
Insulin-Like Growth Factor I
Insulin-like growth factor- I
International Li fe Sciences Institute
Institute ofl\l[edicine
International Society for the Study of Fats and Lipid s
Krill oil
Linoleic acid
Low-dose streptozocin-induced diabetes
Leukemia stem cel ls
Leucotr iene Bs
Leucotr iene Es
l\l[etabolic clearance rate
Eicosapentaenoic Acid (EPA} and Bone Metabolism

JviO
NATO
NF-KB
N IDD
NSA lD
OE
ovx
PG
PG~
PGE;
PG I;
PGh
PUFA
RA
RANK L
SBO
so
STZ
TNF- a
TO
TRAC P
TXA,
UPR
VITA L
WHO
49
Menhaden oil
North Atlantic Treaty Organ ization
Nuclear factor kappa-light-cha in-enhancer of act ivated B cells
Non-insu lin-dependent diabet ic
Nonsteroidal anti-inflammatory drug
Ovar iectomy
Ovar iectomized
Prostaglandin
Prostaglandin E2
Prostaglandin E3
Prostaglandin 13
Prostaglandin h
Polyunsaturated fatty acid
Rheumatoid arthrit is
Receptor activator of N F-KB ligand
Soybean oi l
Salmon oi l
Streptozocin
Tumor necrosis facto r a
Tuna oil
Tartrate-resistant acid phosphatase
Thromboxane A 3
Unfolded protein response
V ITamin D and omegA trial
World Health Organ izat ion
Introduction
Bone is a mult ifunctional organ known to provide framework support for the body as well
as play an important role in maintaining calcium physiological homeostasis. !t is a dynamic
organ, wh ich is continually renewed or "remodeled" throughout adulthood. Human studies
indicate that the di etary intake of certain fatty acids may aid in the conservat ion of bone m ineral
density in the elderly [ I]. Of particular importance are the co-3 PUFAs which have been shown
to promote bone formation in grow ing animals undergoing rapid bone modeling [ 1]. EPA, an co3 fatty acid is of great interest in society nowadays due to its posi tive effects on bone
metabolism and functions, and, thus, w ill be discussed in th is chapter.
Dietary EPA and Bone Metabolism (Human and A nimal Studies)

Dieta ry Refe rence Intake of EPA
According to the 2002 Dietary Reference Intake (DRI) report on energy an d
macronutrients, the Inst itute of Medicine ( IOiv!) of the National Academies recommended an
acceptable macronutr ient distribution range (AM DR) level of 0.6 - 1.2 % of energy for a-
50
Gerard G. Dumancas, Rangika S. Hikkaduwa Kora lege, Patrisha J. Pham et al.
lino lenic acid (A LA) with EPA and docosahexaeno ic acid (DHA) contr ibut ing on ly up to 10
%toward mee~ ing the AiVIDR for A LA. This recommendation represents current mean intake
for EPA and DHA in the Un ited States ( l OO mg/day) {2]. A DRI for intake of EPA and/or
DHA was not esta blished because of the lack of data in support of an ro-3 PUF A requirement
for healthy indiv iduals. The Dietary Gu ideli nes for Americans support the IOM report as we ll
(3]. The guideli nes noted that there is evidence suggesting that consumption of approx imately
two servi ngs of fish per week (approximatel y 8 oz. (224 g) may reduce the risk of mortality
from coronary heart disease (CHD). Health benefits associated with both EPA and DHA
triggered numerous agencies and organ izat ions in the US, such as the American Dietetic
Association (ADA), Food and Drug Administration (FDA), and the American Heart
Association (AHA) to issue recommendat ions for EPA and DHA (EPA + DHA), as well as
for fish consumpt ion, for health promot ion, and decreased risk of many chron ic diseases {4] .
Present ly, the Un ited States and Canadian Dietary Recommendations for EPA are based
on primary preven tion of coronary diseases. The Diet itians of Canada recommend
500 mg/day EPA+ DHA [5], American Diabetes Association [6], AHA (7] and th e 2005
Dietary Guid eli nes for Americans (3] reports, all suggest that consuming 2 servings of
fish/fatty fish per week (approximately 8 oz. total) may reduce the risk of mortality from
CHD. Global recommendat ions from inst itutions such as the Australi an and New Zealand
Nat ional Health and Medical Research Counc il suggest dietary target for men and women
from ages 19-70 years to be 6 10 and 430 mg EPA + DHA/ day, respect ively (8]. Agence
Francaise de Securite Sanitaire des A li ments (A FFSA) recommends 500 mg/day of EPA +
DHA [9] and the National Heart Foundation of Austral ia ( 10], United Kingdom Scient ific
Advisory Commi ttee on Nutrit ion [ 11 ], the World Health Organization (WHO) [ 12]
recommend regular fish consumption of one to two servings per week, each serving should
provide an equivalent of 200 - 500 mg of EPA + DHA . The M inistry of Health, Labor and
Welfare of Japan recommends 0.9- 3 g total EPA + DHA per day for in fants and adults, both
male and female ( 13], the International Society for the Study of Fats and Lipids (ISSFA L)
advocates 500 mg/day [ 14] and the North Atlant ic Treaty Organ ization (NATO) recommends
80 mg!day ( 15].
According to the Nat ional Health and Nutrition Examinat ion (1 999 -2000) for the US
population (including all ages and both genders) th e mean intake of EPA and DH A is about
100 mg/day, much lower than many groups worldw ide are reconm1endi ng. i\lany in the
population are not meeting current recommendations for ro-3 PUFA intake part ly because
seafood consumed in the US is from fi sh low in ro-3 fatty acids (i.e. shrimp and other shell fish
and whitefish). However, there are other strateg ies for meeting the levels of EPA other than
the recommended d iet of fatly fish. These strategies include fort ified foods (fish oi I, designer
oi ls high in EPA) and commercial supp lements (fish, o il, algae). There is typically more DHA
than EPA in f ish, w ith DHA and EPA rat ios (0.3- 0.7) varying across species. Commercial
supplements, however, contain more EPA . Thus, individuals who try to meet the
recommendations of 500 mg/day of EPA+ DHA w ith fish versus commercial supp lemen ts
\vill Ctlnsume proportionate ly di ff erent quantit ies of EPA and DHA such that fish consumed
di rectly would have more of DHA whil e commercial supplements, EPA (3]. A workshop
report as pub lished in the Brit ish Journal of Nutrit ion concluded that the available infom1at ion
relating dietary EPA and/or DHA intake in children aged 2- 12 years to growth, development
and health is insu tll cient to derive d ietary intake recommendations fbr EPA and DHA.
However, the di etary advice for children shou ld be consistent w ith advice f(lr the adult
51
population, such as regular intake of one to two fauy fish meals per week providing EPA and
DHA assoc iated with the risk reduction of cardiovascular diseases (CVDs) [ 16].
Taking into account new EPA health research since the 2002 IOM report, the Technical
Commitiee on Dietary Lipids of the International Life Sciences Institute (ILS I), North
America assembled a workshop (June 4-5, 2008) in Washington, DC. The results of th is
workshop were published in the Journal of Nutrition {1 i } . The workshop re-evaluated the
rationale and process that governed the development of DRI for ru-3 PUFA in 2002 and
whether research suggesting higher intakes of both the short-chain ALA (a-linolen ic acid),
and the long-chain EPA and DHA were suflicient to establish a DR I fbr EPA + DH A.
Some of the conclusions drawn from th e workshop include (I) a nutritionall y achievable DR I
tbr EPA and DHA between 250 and 500 mglday as a result of consistent evidence
from muliiple research paradigms, (2) protective tissue levels of EPA and DHA can be
achieved on ly through direct consumption of these fatiy acids, (3) evidence from EPA and
DHA intake fur other chron ic diseases such as cogni ti ve decline are not sufficient to support
an intake level di ft"eren t from that needed to achieve CH D risk reduction and (4) th ere is no
evidence suggesting th at intakes ofEPA + DHA in these recommended ranges are harn1ful to
humans {4] .
The majority of the recommendations have been on the basis of cumu lative EPA + DHA
and not on each indivi dual ru-3 PUFA. There is a pressing need to establi sh DR is for each
indi vidual w-3 PUFA. Currently, total EPA and DHA implies th at they are all the same
biologicall y, which presently sum1ounting research on individual ro-3 PUFAs has shown to be
otherwise. Future studies will lead to new consumption and DR I recommendations in
response to new inforn1ation obtained through ep idemiological, cli nical and an imal
investigations. To date there are no dietary recommendations for EPA established using bone
health as indicator, on ly DRI for calcium and Vitamin D {18]. Di rect evidence of any
beneficial effect of di etary ro-3 fatty acids on human osteoporosis is still lacking. However,
experiments using animal and cell cultu re models, and ep idemiological data suggest
promising applications of ro-3 PUFA on th is widespread publi c health problem. Several
studi es evaluating w-3 PUFA dietary supplements ranging from 3 to 6 glday showed a modest
but rath er consistent beneficial effect of these fai!y acids in j oint diseases [ 19]. A report of the
Scientific Opinion on the substantiation of health claims related to DHA, EPA and y-linolen ic
acid (GLA} and contribution to norn1al cogn itive function and maintenance of norn1al bone
published in the European Food Saiety Authority Journal showed several studies on the
effects of DHA, EPA and GLA on bone loss, bone mass, bone mineral density (BMD) and
bone turnover in different an imal models of post-menopausal osteoporos is (e.g. OVX rats),
and a series of in vitro studi es wh ich used osteoblast/osteoclast cell lines. The panel however,
considered that evidence provi ded in animal and in vitro studies is not sufficient to predict the
occurrence of an effect of the consumpt ion of DHA, EPA and GLA on the maintenance of
bone in vivo in humans. In weighing the evidence, the panel "took into account that two of the
human intervention studi es from which conclusions cou Id be drawn fur th e scient i fie
substantiation of the claim did not show an effect of the food constituents on bone mineral
density. In addition, in a th ird study acute changes in markers of bone turnover do not predict
the occurrence of an effect on BMD and/or mass. The evidence provided in an imal and in
t1tro studies is not suffi cient to predict the occurrence of an effect of the consumpt ion of
DHA, EPA and GLA on the maintenance of bone in vi vo in humans". The panel concludes
52
that a cause and effect relat ionsh ip has not been establi shed between the consumpt ion of
DHA, EPA and G LA and maintenance of normal bone [20].
The National V ITAL trial (V ITamin D and omegA. trial ) is a national trial that w ill
enroll 20,000 men aged 60+ and women aged 65+ for randomizat ion to 4 groups: vitamin 03
(1 600 IU/ day) and fish oil (EPA + DHA, I g/day) also Vitamin D3 and fi sh oi l placebo;
placebo Vitamin D3 and placebo fish oil. The grant will test effects of the supplements on
risk of cancer and cardiovascu lar events, with future adj uvant muscu loskeletal health studies
(21 ]. The nat ional tria l may provide answers to questions pertaining to recommendat ions of
ro-3 PUFAs as a component in the prevent ion of osteoporosis and other bone metaboli sm
related events. Thus, establishing new EPA and DHA DR I's that go beyond coronary disease
risk reduction and triglyceride lowering will require more scient ific evidence and short and
long-term support ing research.
EPA as Dietary PUFA and Bone Mineral Dens ity ( BM D) Association

Although a great deal of altent ion has been given to the importance of calcium and
Vitamin 03 intake in bone health and development, not much is known about the effects of
other EP A on bone health . it has been shown that high-fat diets, in comb ination w ith other
di etary man ipulat ions, over short and long-tem1 feedi ng regimes, significantly affect both
bone quantity and quality {22]. The quali ty or type of di etary fat whether it is saturated fully
acids (SFA), ro-3 PU FA, and ro.Q PUFA are important detem1inants of the heal th outcomes
{23]. Candidate PU FAs with the potential to enhance bone mass include EPA. The addi tion of
w-3 PUFA like EPA to bone cell media has been shown to modulate cyclooxygenase-2
protein expression, reduce PGE 2 production, and increase alkaline phosphatase activity [ 1].
Th is provides evidence that it is biologica ll y plausible for changes in the fatty acid
composit ion of bone to alter the bone microenvironment and inlluence BtvfD and bone
strength. Blv!D or bone density (BD) as defined in clinical pract ice is the amount of mineral
per square centimeter of bone. Actual bone density is usuall y expressed in grams per
milliliter. 11 is most frequently measured by X-ray absorpt iometry or X-ray computed
tomography. However, dual-energy X-ray absorpt iomeuy (DXA) is currently the preferred
method of measurement. BMD is an important predictor for osteoporosis. Majority of the
studies of ro-3 PUFA w ith BMD association involve a mixed ro-3 PUFA diet [22]. Thus, it is
dinicult to isolate the effect of indi vidual ro-3 PUFA, EPA on BMD. Moreover, a vast
majority of work in th is field has been conducted in an imals invol ving a range of different
models, wh ich include growing, growing-ovariectomized, mature-ovariectomized, and
diabetic animals [24] . Human clinical studies are sparse.
An imal studies suggest th at EPA may have a posit ive role in the bone mineral
accrual/ acc:umulat ion process. A study on experimental tooth development in rats showed a
decrease in the number of osteoclasis by 60% and alveolar bone resorpt ion by 80% w ith the
infl uence of fish oi l supplementat ion, wh ich is rich in ro-3 fatty acids [25]. lv!ost of these
studies util ized supp lements containing a mixture of ro-3 PUFAs. Two studies however,
examined the effects ofEPA a lone on bone in OVX rats. In the fi rst study, ovar iectomy (OE)induced bone weight and strength loss was prevented by supplementation of OVX rats
receiving a low-calcium diet (0.01% calcium) with 160 mg of EPA/ kg body we ight/day. No
em~ct ofEPA was seen in rats receiving a % calcium-adequate diet [26]. The results suggest
Ei cosapentaenoic Acid (EPA} and Bone Metaboli sm
53
that an EPA-enriched diet can prevent loss of bone weight and strength, as in the case of
women during post menopause [I ]. In a second study, when the effect ofE PA on BtvfD was
studi ed for female Sprague-Dawley OVX fed w ith low (1 00 mg EPA/kg body weight) an d
high EPA ( 1,000 mglkg) supp lemented calcium adequate diet. The study revealed that a highdose EPA supplementat ion exacerbated the effects ofO E on BMD (2 7]. Findi ngs from these
two studies suggest that EPA may on ly be beneficial for preserving bone mass after OE when
di etary calcium is li miting although one study has reported that dietary supp lementati on using
hi gh-DHA oil was more effective th an supplementation with h igh-EPA fish oil in maintaining
bone mass a fter OE [28]. However, a grow ing OVX rat model was used for this study and not
OE. Table I shows a summary of studies of the etrect in OVX animals of EPA-enriched diet
on bone mass and metabolism (24]. A recent study w ith intact female mice indicated that
EPA improved structural and mechanical propert ies of cort ical bone in th e femur, w ithout
detectable effects on age-related loss of trabecular bone or bone mineral density (29] .
T able I. Summary of st udies in O VX animals with E PA diet
on bone mass and metabolism 1241
Animal
6- month-old
OVX rats
n= 60
Mice 2 months
old at trial
commencement
OVX at4
months o f age
Dunltion
EPA Supplement
EPA Diet
l.Dw and high

doses o f EPA ethyl
ester te sted:O. I
glkg and I g/kg rat
body weight/day
4.0-4.5% o f total fat

El' A for low dose and
40-4 7.5% EPA in diet
JOr high dose. Ratio o f
ro-6;ro-3 21 : I fo r low
dose and 1.26: I fo r
high dose.
9 weeks
S'lo o f diet was fish oil
2 months
pre-OVX and
16 weeks
post-OVX
Fish oil c ontaining

14.3% EPA and
8.7% DHA
Result
l.Dw dose had no
eifcet on bo ne mass
b.ll high dose
caused a signi fic.ant
reduction in Bt-.ID
Reduced bone
mineral density loss
n= 5
17-wcek-old
OVX rats
n= IO
11-week-old OE
rats
n= 60
El' A (free fatty

acid) with either
calcium-dcllcicnl
or c.aJciumadequate die t
1: I ratio of G LA
and EPA (diester)
Bone weighl and

strength higher for
11 % o f total fat as
EPA
5 weeks
those fed with low

calcium diet - EP A
versus low calcium

det without EPA
I g/kg body
weight/day.
Percentage o f
GLAIEPA in diet not
stated.
14 weeks
~ e ffect on bo ne
mass
Human S tudies on EPA Assoc iation with Bone Mineral Density

Human studies re lat ing ro-3 PU FAs specificall y w ith EPA alone w ith bone mineral
density or skeleta I health are few. Results from a smal l number of experimental studies of the
effects of ril-3 PUFA supp lementat ion on bone in humans are inconclusive {30]. A pi lot study
suggested that dietary supplementation w ith even ing primrose and fi sh oil rich in EPA may
54
Gerard G . Dumancas, Rangika S. Hikkaduwa Kora lege, Patrisha J. Pham et al.
decrease bone turnover and increase bone mineral density in elderly postmenopausal women .
Elderly post-menopausal women were given a mixture of fi sh oi l and even ing primrose oil
" i th a high content of EPA and G LA whereas the control group received coconut oil. In the
group w ith G LA and E PA, BMD at the lumbar spine was maintained and the B1v!D at the
femoral neck increased. In the control group, Blv!D decreased at both sites. However, it
cannot be concluded from th is study whether EPA or GLA was the fatty acid responsible for
such effects {3 1]. Furth er human studies indicate that the di etary intake of w-3 PUFA fatty
acids may he lp to conserve bone mineral density in the elderly [ 1]. Associat ions of di etary ro3 PUFA w ith BMD in elderly women have shown a posit ive relat ionsh ip between the dietary
PUF As and BMD at lumbar spine and in total body but not at the femoral neck. Further
anal ysis revealed that these results were due to associat ions among the women without
hom1one therapy (HT) at baseli ne {32]. Aerobic exercise training plus ro-3 PU FA
supp lementat ion (i.e. 180 mg EPA/ day) was effect ive in reducing chronic in flammat ion and
increasi ng BMD in postmenopausal women. These changes in inflammatory markers are
related to indi ces that !avor en hancement of Bl\ID in heal thy sedentary post-menopausal
women. Further studi es on the physiologica l eftects of w-3 PUFAs and exerci se training on
bone metabolism and bone quali ty to prevent or treat osteo porosis are needed (33]. A first
cohort study to investigate th e association between individual PUFAs, BMD , and bone
mineral accrual was accomp lished in young men (n =78,mean age = 16.7 years at baseline)
found that ro-3 fatty acids, especially D HA not EPA, are posit ively associated w ith bone
mineral accrual and, thus, with peak BI\ID (total body and the spine) [34].
Gender Stud ies on EPA an d Bone Metabolism

A report that descr ibes the biomechan ical bone strength properties, funct ional measures
of bone metaboli sm, in response to feeding di ets rich in ro-3 PUF As d uring act ive skeletal
growth and development in both mal e and female rats showed that in mal e rats, feedi ng di ets
enriched w ith ro-3 PUFAs (EPA + DHA) from wean ing through puberty neither posi tively
nor negat ively infl uenced BMD and biomechan ical bone strength of femurs and vertebrae. In
contrast, female rats experienc.ed a marginall y sign ificant negat ive effect on vertebral strength
and on length growth w ith fi sh oi l feeding. The fi ndings from the present study suggests that
feeding a diet enriched w ith fish oil to male rats during act ive bone growth has no positive or
untoward effects on li near skeletal growth , femur bone mass or biomechanical bone strength
of femurs and lumbar vertebrae. A lthough linear skeletal growth and vertebra peak load were
marginall y decreased in females fed the fi sh oi l diet, biomechanical strength propert ies of th e
femur were not affected by the fi sh oil diet [35]. In the Framingham Osteoporosis Stud y
which ai med to examine the associat ions between di etary PUFAs and fish intakes w ith hip
BMD at baseline of adults (n = 623, mean age 7 5 years) and with in a span of 4 years, no
si gn ifi cant associat ions were found between intakes of EPA and 4 years changes in femoral
neck bone mineral density (FN-BMD), hip sub region in women however in men, those with
greater intakes of EPA lost sign ificantly less FN-BMD over 4 years than those w ith lower
EPA intakes. T his highlights, that men and women are di fferent and that we cannot
e'lt rapolate research from one gender to another [36] .
Eicosapentaenoic Acid (EPA} and Bone Metaboli sm
55
EPA and Osteoporosis

Osteoporosis is defined as a condition characteri zed by decreased bone mass and
increased bone fragility. Women can be affl icted by osteoporosis at their postmenopausal
period and consequent ly place them at high risk for hip and vertebra l fractures. Modi ficat ion
of di etary PU FAs content, part icularly increasing the intake of ro-3 PUFAs, has been shown
10 minimize the decline in bone mass caused by menopause in women ovariectomy in an imal
models (24]. In general, direct evidence showing the posit ive etrects of di etary ro-3 fatty acids
on human osteoporosis is still lacking [ 1].
Osteoporosis is probably one of the maj or limitat ions to long-term glucocorticoid therapy
(37]. Prolonged exposure to glucocort icoids at high doses is known to induce osteoporosis
and is associated w ith an increased risk of bone fracture {38]. Although certain
biphosphonates, such as alen dronate, can counteract the negat ive effects of glucocorticoids on
bone [39], diet therapy and lifestyle changes that minimize bone loss wou ld be very he lpful to
decrease the necessity for certain drug therap ies 10 minimize or prevent osteoporosis [40]. A
Siudy was conducted to invest igate the effects of EPA on glucocorticoid-induced bone
changes in rats, and to compare them with those of alendronate. [t was found out that EPA,
especially, at the dose of 160 mglkg exerts beneticial effects on methylprednisolone-induced
bone changes in rats; these effects are simil ar o r somet imes even better than alendronate [4 1].
EPA and Calcium Excretion, Absorption, and Bone Mineralization

Calcium is the most abundant mineral in the body w ith 99% of it stored in the bones and
teeth where it supports its structure and function. The body uses bone tissue as a reservoir and
source of calcium, depositing and w ithdraw ing calcium based on the need of the body to
mainta in constant concentrat ions with in blood, muscle, and intercellular fl uids [42 ]. Bones
are dynamic structures that constantly undergo a remodeling process which involves bu ilding
and breaking down. The delicate balance between bone bui ldi ng (fom1ation) and bo ne
breakdown (resorpti on) determined whether the bone structure and density is healthy or not.
Al though there are many facto rs a ffect the ca lcium balance in bones (such as aging and
Vitamin D), PU FAs suc.h as EPA and DH A were found to play an important role in helping
mainta ining healthy bone mass (43-45]. Processes such as calcium excret ion, calcium
absorpt ion and bone minerali zat ion are affected by the level ofE PA and other PUFAs taken
by the bod y. In a review by Kruger and Horribin [46], essential fatty acids (EFAs} like EPA
have been shown to increase calcium absorpt ion from the gut in part by enhancing the e ffects
of V itamin D to reduce urinary excretion of calcium.
Calcium excret ion is defined as the amount of calc ium eliminated from the body in urine,
feces and sweat. Human and animal studi es found that EPA decreases calc ium excret ion in
general. Dietary fish oil was used in most of these studies as so urce of EPA. In one an imal
Siud y, urinary calcium excretion in recurrent, hypercalciur ic stone-former group was
significantly reduced w ith fish oil treatment over an eight-week period (47]. In another paper
(48], fish oi l inhibited experimental nephrocalcinosis induced by intraperitoneal calcium
gluconate in rat but no sign ifi cant changes in ur inary biochemistry was observed The same
paper [48] reported that in a clin ica l study on 18 hypercalciuric recurrent stone pat ients, tish
oil sign ificantly decreased urinary calcium excret ion. Another study using Sprague- Daw ley
56
rats supplemented with fish oil show a decreased in both urinary and fecal calcium excret ion
si gn ifi cantly leading to an increased calcium balance (p < 0.05) {49]. In addi t io n, rats fed w ith
fish oi l and tuna oil showed enhanced reduction in urinary calcium excret ion [44] . A
correlat ion between the increase in w-3 level and a decrease in ur inary calcium excretion was
establi shed in one study when rats were administered w ith di fferent rat io of gamma-li nol enjc
acid (G LA, w-6) and EPA {50]. Last ly, a recent study on patients w ith hypercalci uric stone
fbm1ers showed reduct ion in urinary calcium levels in 52% of the patients w ith the average
urinary calcium (mg/d) decreasing si gn ificant ly from baseli ne (329.2796.23 to
247.4 784.53, p<.OOO I) [5 1].
A clinical study using highly purified preparat ion of EPA was administered (1 ,800
mglday) to 88 pat ients w ith urinary stones for 3 months (short term) and 18 months ( long
term) and results showed that urinary calcium was sign ificant ly reduced in the hypercalciuric
but not in the no m1ocalciur ic group [ 13]. In addition, the results of the study suggest that EPA
not only reduced urinary calcium but it favorably affected urine co mposit ion that lowers th e
risk of cal cium stone format ion [52]. The same result was obtained in an earlier study where
in ethyl icosapentate (! ,800 mg/day) was administered to 5 7 pat ients with ur inary stones fbr 6
weeks (53] .
On the other hand, calc ium absorpt ion is defi ned as the di fTerence between the amount of
calcium taken by the body and amount of calcium excreted. it is equal to the amount of
cal cium that is absorbed by the body from the digestive tract going to circulation. There have
been several studi es on the effect of PU FA including EPA on calcium absorpt ion. Ano ther
stud y deali ng with dietary long chain PUFA shows a di rect relat ionsh ip between di etary
PUFA intake and intest inal calci um a bsorpt ion [44] . Conj ugate li no leic acid (C LA)
supp lemented w ith w-3 PU FA and fed to 4-week-o ld mal e rats resulted to a sign ificant ly
higher calcium absorpt ion in compari son to CLA diet with w-6 P UFA r ich diet {54]. The
add it ion of C LA supp lementat ion over 8 weeks appeared to enhance cal cium absorpt ion in
young grow ing rats fed w ith t0-3 P UFA-rich diet. When only CLA without the ro-3 PU FA in
the diet was fed to OVX rats, the intest inal calc ium absorpti on remains the same [55] .
Calcium absorpt ion even decreased as demonstrated by other studies that ut ilized co rn o il
{56], cotton seed oi I {5 7] and peanut oil [58]. These results only showed that the use o f PU FA
part icularly ro-3 fatty acids where EPA belongs, seems to enhance calcium absorpt ion. This
was suppo rted by the result obtained by C laassen et al [50] wh ich made use of diet containing
di fferent ratio of GLA and EPA that were ied to mal e rats . Results showed that intestinal
calci um absorpt ion (mg/24 h) increased by 4 1.5% in the 3: I supplemented group, compared
\vi th the control group {50].
An efficient calci um absorpt ion ensure over the long term the retention and used of
calcium for bone mineral ization. Bone is made in two stages; matrix is formed first, and about
2 weeks later (in children) it begins to minerali ze [59 ] and the delay is longer in the ad ult
skeleton [60]. The process of add ing mineral like calc ium to matrix is referred to as 'bone
minerali zation." However, only limited studi es have been reported on the effect of EPA on
bone mineralizat ion. The PUFA that has a di rect relatio nship w ith bone mjneralizat ion is
DHA which is an ro-3 j ust like EPA . In some studi es, ro-3 fatty acids like DHA were foun d to
potent iall y reduce bone demineral izat io n [44, 6 1]. One study which made use of cod Iiver o il ,
a rich source of DHA and EPA, concluded that supp lementat ion of C LA have a posit ive
effect on bone mineralization in rat [62]. In addit ion, the lack of essent ial w-3 fatty ac ids
intake in the diet of phenylketonuria patients might affect bone mineralization [63].
57
Die tary w-6/w-3 Ratio and Calcium Balance

Dietary ro-6 and w-3 fatty acids have been implicated as important regula tors o f bone
metabolism. Most studi es done on the effect o f unsaturated fats on bone metabolism are
expressed as ro-6/ro-3 ratios rather than the indi v idual fatiy acids. Di fferem dieta ry ratios o f (1)6/(J.)-3 fatty acids were tested in pig lets for their effects on growth and bone metabolism.
Results show that the elevat ion o f circulat ing ro-3 faliy acids was significantly related to a
lower level o f bone resorpt ion (64 ]. In another study, Baggio concluded that the prov ision o f
di etary (1)-6 and w-3 PUFA in an imals resulted in a reduct ion o f bone resorpt ion biomarkers
and bone demineralizat ion caused by fatty acid deficiency [65].
The rat io o f (1)-6 to (1)-3 fatty acids in the diet is considered important. Lowering the
di etary rat io o f w-6/ (1)-3 PUFA is known to increase bone marrow cellularity {66] and bone
strength in an imals [67]. Few studies have ev aluated the relat ionsh ip between di etary fats and
bone status in humans. One epidemiolog ical study has shown an inverse relat ionsh ip between
the dietary w-6/ ro-3 ratio and bone mineral density in o lder adults (30] .
In another study, it was concluded tha t the supplementation o f GLA and EPA in an
appropr iate rat io might be o f beneli t in enhancing the calcium balance and calcium content o f
bone. GLA and EPA are more effect ive in mod ulating calcium metaboli sm than their
precursors, LA and et- li nolenic acid (A LA} [68].
rvlodi ficat ion o f dietary PUF As content, particularly inc reasing the intake o f ro-3 PUF As,
has been shown to minimize the decli ne in bone mass caused by menopause in women and
overiectomy in an imal models [69]. Dietary intervent ion studies invest iga ting th e effect o f
PUFAs on bone health in postmenopausal women have y ie lded mixed resu lts. In elde rly
osteoporotic women (mean age o f 80 years), dail y supp lementat ion with 4 g fish oil
consisting o f ! 6% EPA and 11% DHA or a mixture of fish and even ing prim rose oi ls
containing 60% LA, 80% G LA, 4% EPA, and 3% DHA for 16 weeks resulted in decreased
serum al kaline phosphatase act ivity and increased serum concentrat ion o f proco llagen. A
higher serum osteocalcin concentration compared with supp lementation w ith olive oi l,
even ing primrose oil , or fish oil alone was also associated w ith the combined evening
primrose oi l/ fish oil supplement [70]. Osteocalcin is important because it is conside red a
bone-speci fie protein that is released into the blood during both new matrix format ion a nd
osteoclast ic breakdown o f ex isti ng matrix. Its circulating concentration is, thus, indicat ive o f
the rate o f bone turnover. Results show ambiguousness because decreased serum alkaline
phosphatase act ivity indica tes a reduction in the rate of bone format ion, whereas increased
procollagen suggests th e opposi te. Serum calcium levels were slight ly increased and urinary
calci um clearance sign ificantly increased in the fish oil supplemented group compared with
all other g roups [71 ]. Th is is indicative o f increased bone resorpt ion in this group and,
therefore, signifies a negative effect o f fish oil in calcium balance. Some studi es, however,
reported increased intestinal calcium absorpt ion after the ingest ion o f (1)-3 fatty acids o r f ish
oi l [30, 72]. The refore, the increased urinary calcium excret ion observed in elderly women
maybe renect ive o f increased intestinal calcium a bsorpt ion in the fish-oi l supplemented
group. Although these findings suggest th at di etary supplementat ion w ith PUFAs can alter
calcium balance and the rate o f bone metabo lism, it is unclear whether these resu lts can lead
to increased bone fom1at ion [69].
58
Findings from both in vitro and in vivo studies suggest that PUF As may promote intest inal
calcium absorption, thereby increasing overall calcium balance. The active calcium absorption
in the intestine is made possible by the enzyme Ca2- ATPase [24]. The w-6 PUF As may be less
effecti ve than the ro-3 PUFAs in promot ing calcium absorption because an increase in overall
calcium balance has on ly been observed w ith llsh oil supp lementation [73, 74].
In some studi es invol ving marine-dwelli ng EPA-synthesizing bacteria under oxygenli mited or anaerobic condit ions, it has been shown that EPA enr ichment of membranes
supports proton bioenergetics, allow ing both oxidat ive respirat ion and energy transduction
{75]. Th is aspect, however, has not been proven whether it has any sign i lican t relevance to
the mechanism by wh ich they regulate calcium balance or bone metabolism [69].
EPA and Cortical Bone Morphology

Bone morphology refers to the way the bone t issue is organ ized into a variety of shapes
and configurat ions adapted to the funct ion of each bone [76] . Cortical bone consists of bone
that is cont inuously resorhed on the endosteal surface and produced in the periosteal surface.
it is the cort ical bone enveloping the marrow cavit ies that fom1s the discern ible shafts of the
long bones and, w ith the except ion of vertebrae, is the predominant skeletal tissue seen o n
roentgenogram [ 77].
EPA is known to be the main component of llsh oils. A stu dy fo und out that 10% fish oi l
supplementat ion in th e presence of vitamin E supp lementat ion can have detrimental effects on
the skeleton of rap idly grow ing rabbits {78]. Although fish oil wh ich is rich in EPA can have
posit ive effects on calcium absorpt ion and bone metaboli sm compared w ith oth er various
sources of fat {79], a study by Judex et ol showed that supp lementation of fish oil in large
quanti t ies and the associated increase in dietary fat content can lead to substantial degradat ion
of morphological and mechanical propert ies of cortical bone. However, the underlying
mechan isms, the long-term effects of ro-3 PU FA, and whether simil ar skeletal consequences
are evident in humans tak ing fish o il supp lements remain to be invest igated [78].
On the other hand, a study by Bonnet et a/ invest igated whether a di et enriched in EPA
for the ent ire adull li fe of mice cou ld improve bone microstructure and strength. In th is stu dy,
th irty female mice rece ived a di et enriched in EPA or an isocaloric control diet from 3 to 17
months of age, trabecular bone volume in caudal vertebrae was improved by EPA at 8
months, but not thereafter. EPA was also found to improve femur cortical bone volume and
th ickness compared to controls. EPA hut not DHA, reduced age-related decli ne of
osteocalcin, a noncollagenous protein found in bone and dent in. EPA and DHA increased
leptin wh ich is a protein hom10ne responsi ble fur the regu lation of energy i nt~ke and energy
expenditure, including appetite and metabolism. However, on ly EPA further increased
insu i in-1ike growth factor- ! (IGF- 1) levels. Resul ts of the study indicated that the long-tem1
intake of ro-3 fatty acid, part icularly EPA, may modestly improve the structural and
mechanical properties of cortical bone by an increase in lept in and IGF-1 levels, w ithout
afTecting trabecu lar bone loss (80].
Studies show ing the effects of EPA alone or as part of ro-3 PUFA supplementat ion to
bone morphology are shown in Table 2.
59
EPA and Bone Diseases

EPA and Bone Cancer
Bone cancer is a mali gnancy of the bone. Primary bone cancer (cancer that begins in the
bone) is rare, but it is not unusual for cancers to metastasize (spread) to bone from other parts
of the body, such as the breast, lung, and prostate. The most common type of primary bone
cancer is osteosarcoma, wh ich develops in new t issue in grow ing bones. Targeting cancer
stem cells is of paramount importance in successfully prevent ing cancer [84] .
Table 2. Studies showing the effects of EPA (or as part of ro-3 PUFAs in fish oils)
to cortical bo ne morphology
Au t hor (s)
Judex et
al. (78]
Matsushita
eta/. (8 1]
Tr eat ment Gtoup s

Wcanling male :-.Jew Zealand
white rabbits
le d with basal diet rellecting
the nonnal dietary fat levels
and energy requirements o f
rabbits lor 40 days
I. control (n = I 0)
2. !Ish oil group ( n = 20)
6sh oil (mcnhadcn oil)
added to diet at
JOg/ I OOg fish oil
,
>. pair-fed group (n = 20)
energy intake limited to 95%
of the mean energy intake o r
the lish oil group to match
body mass o f the Jlsh oil
group
Female retired breeder SpragueDawley rats
I.
2.
3.
4.
5.
divided into live groups based

on body weight (n = 810/ group)
baseline, c.o ntrol/sham,
controliOVX le d with A l:-.193M purillcd diet
tlsh!sham and lish/ OVX le d
4% llsh oil diet (menhaden oil
at 40 g/kg)
baseline
control diet + sham-operated
(control/sham)
control diet ..:.. ovariectomized
(controi/ OVX)
!ish oil diet - sham-operated
(lish/sham)
!ish oil diet - ovariectomized
(li sh/ OVX)
Results
energy restriction alone (30%) did not induce
signi ficant changes in tibial middiaphyscal
morphology between the control and pair-led groups
but tibiallongiludinal growth was signi ficantly
impaired
most tibial mcchanic.a1 properties were signi ficantly
degraded by energy restriction
!Ish oil group had signi llcantly smaller middiaphyscal
are.al properties and shorter tibiae than pair-fed rabbits
tibial structural properties were signi ficantly reduced
in lish oil group, but tibia l stress at the proportional
limit (material property) was not signi ficantly affected
10% !ish oil supplementation in the prescnc.e o r
modest vitamin E supplementation can have
detrimental e ffects on the skeleton o r rapidly growing
rabbits
bone mineral c ontent ( BMC) of the whole lemur was
signi ficantly higher in the lish!OVX than in the
controi/ OVX, and the di fie rences were most
pronounced in the distal and proximal ends o f the
JCmur
tlsh/sham and the ccmtrol/sham did not di fle r in the
measures o f BMC
although the controi/ OVX had signi ficantly lower
cortica l area and greater endosteal perimeter compared
with the c.ontro1/sham, lhc di ffe rences were not
signi ficant between the lish!sham and the Jlsh!OVX
tish/ OVX had a signilicantly larger percent double labeled surfac.e and mineral apposition rate at the
cndocortieal sur face than the controi/ OVX
fish oil supplementation had a positive e l'leet on bone
metabolism and might be a possible intervention to
slow the loss o r bone observed lo llowing menopause
60

T able 2. C ontinued
Author (s)
Lukas et
al. (82)
Liu e: al.
(83)
Treatment Groups
Female SpragucDawlcy rats
randomly assigned n =
10/group to a high Jltt 12%
(wt) diet consisting o f either
corn oil (CO) or ro-3 I' UFA
rich, J1axseed (FO), kritl
(KO), mcnhadcn (MO),
salmon (SO) or tuna (TO) for
8 weeks
Japancse quail (Cotumix C.
Japonica)
laying hens (n = 20) were fed
a basal diet containing either
5% soybcan oil (SBO),
hydrogenated soybcan oil
(HSBO), chicken fat (CF), or
menhadcn fish oil (EPA-rich)
Results
TO had higher tibial mineral bone density and bo ne
mineral content and lower lipid pcroxidation
compared to the CO- fed rats
FO and MO improved bone microarchitccture
compared to rats fed with CO or SO
sen1m ostcoealcin higher in rats led FO compared to
rats led SO; scrum ostcocalcin was associated with
improved trabecular txme mkroarchitecturc

addition o f menhaden fish oil in the maternal diet
elevated concentrations o f EPA
addition o f menhaden ilsh oil or HSBO to the
maternal diet signilk antly lowered the ex vivo
prostaglandin E2 (PGE2) produe.tion of tibiae in newly
hatched quail compared to those Jiom hens given the
SBO or CF diets
the addition o f different lipids in the maternal diets did
not affect growth, tibial length, diameter or collagen

content o f the progeny
supplementing the maternal diet with 5 % menhadcn
Jish oil or HSBO increased the percent bone ash ,
increased bone pyridinium erosslinks o f collagen,
enlarged thecartilaginous proli lerativc and
hypertrophied zones, increased diaphyseal cortical
thickness of the tibiae in embryos, and subsequently
ine.r cascd tibial shear force, stiffness and improved
cortical thickness, density and trabecular density in
early growth and development o f progen y compared
to those from hens consuming the S BO or CF diets.
In addition to its well-known ant i-innammatory benefits, particularly in cardiovascular

and other inflammatory d iseases, EPA is also associated w ith cancer prevention. E PA has
shown promising results for its potential to reduce di fferenttypes of cancer includi ng prostate
cancer, colon cancer, breast cancer and cancer cachexia {85-88]. There are few studies
reported in the Iiterature for the prevent ion/ control of bone cancer by EPA and most of these
have shown the effect of EPA on leukemic stem cell s (89].
12
Shailaja Hegde et al. reported that t. -PGh, a nove l and naturall y produced
cyclooxygenase-derived cyclopentenone prostaglandin (CyPG) from the di etary fish-oil ro-3
PUFA, EPA, all eviates the development of leukemia in two well -studied murine models of
leukemia. In th is study, administration of t. 12-PGJ, to mice infected w ith erythroleukemia
virus or those expressing the chronic myelogenous leukemia oncoprotein BC R-AB L in the
hematopoietic stem cell pool have comp letely restored nom1al hematologic parameters,
splen ic histology, and enhanced the survival. Predominantly, leukemia stem cell s (LSCs) for
12
apoptosis in the spleen and bone marrow has been selectively targeted by t. -PGh
Furthemw re, the study showed the complete eradiation of LSCs in vivo, as demonstrated by
the inabil ity of donor cell s from treated mice to cause leukemia in secondary transplantat ions.
Eicosapentaeno ic Acid (EPA} and Bone Metabolism
61
12
Th ese findings provide evidence that t. -PGh derived from di etary w-3 PUFAs, has the
potential to serve as a chemo preventive agent in the treatment of leukemia {89] .
Anoth er in vitro study using human acute promyelocyt ic leukemia, HL-60 and chronic
myelogenous leukemia, K-562 cell lines, showed that EPA has inhibited th e proliferat ion of
these cell s and arrested cell cycle progression at GO/ G I phase, and induced necrosis in both
HL-60 and K-562 cells. Authors suggest that the mechan ism for retarded cell division could
be that the leukemic cells, under the inf1uence of E PA, might pass more slow ly through th e
G l checkpoint and accumulate in G l phase of the cell cycle. EPA was also found to be down
regu lating B-cell lymphoma 2 (bcl-2) prot ein expression in much greater extent suggest ing
that the depression of bcl-2 might be an important step dur ing the EPA-induced apoptosis in
Hl -60 cells (90].
Similar study by J. E. Slagsvo ld et al., on ant iproliferat ive effect ofEPA on HL-60 cells
by altering calcium homeostasis, suggested that the inhibitory efrect of EPA on HL-60 cells is
initiall y medi tated through al terat ions of the Ca2 -- homeostasis followed by activation of the
unfolded protein response (UPR), result ing apoptosis or growth arrest in H L-60 cells .
Fi ndings showed that the EPA concentrat ions of 20 and 35 .u lv! reduced the cell number to
approximately 50 and 60 %, respectively, after 48 hours, whereas treatment with 70 ~~M
abolished further pro Ii feration of H L-60 cells. EPA treatment also affected several complex
gene networks and cell signaling pathways [9 1].
The abovement ioned studi es on di fferent leukemic cell lines provide good evidence of
bone cancer prevent ion by EPA.
EPA and Diabetic Osteo penia

Diabet ic osteopenia is a major prob lem associated w ith diabetes me/litus which can result
in an increased rate of bone fracture, a delay in healing fractures and consequent deterioration
of the quality of life [92] and is observed in both humans and animals. T his can later develop
to the di sease call ed "osteoporosis". Most experimental studi es demonstrated the alterations
in bone and mineral metabolism due to diabetes mellitus, but the mechan ism fur diabetic
osteopen ia is sti ll unclear. !t has been found that EPA may have etTects on bone cells due to
its influence on prostaglandi n metabo lism which inhibits the osteoclastic bone resorption and
st imu late bone fom1at ion [93-95]. Due to the fact that diabetic osteopen ia is an associated
problem in diabetes mellitus, prevention of diabetes mellitus is also important in
reducingosteopenia.
Yamada et al. have studied the effect of EPA in diabetic osteopen ia, by evaluat ing the
bone fragility in streptozotocin (STZ) -induced di abet ic rats. EPA has prevented osteopen ia in
diabeti c rats fed w ith low zinc diet, which was a potent ia l accelerator of diabet ic osteopen ia
and also part ly prevented the fragili ty of femu r in diabetic rats. A lso in diabet ic rats fed EPA,
urinary phosphate excreti on and plasma inorganic phosphate concentrat ion were slight ly
lowered, suggesting that EPA may have an effect in suppressing phosphate release from
bones in diabet ic rats {96].
lv!any of the scient il"ic reports in literature show the potential effects of EPA in reducing
diabetes me/lit us, rather than the di rect correlation of EPA to diabetic os1eopen ia. In an in
vivo study Li nn et al. repo rted the effects of di ets high in w-3 fatty acids on the development
of diabetes in the low-dose streptozocin (STZ)-induced di abetes (LDSTZ-D) model. l'v!ale
62
C57BL/6J mice were fed w ith fish oil diet (FOD) as a source of w-3 FA for 8 weeks before
STZ inject ion. FOD mice had reduced level of blood glucose compared to control study an d
the immunoh istology showed reduced numbers of class n antigen-expressing cell s in
pancreatic islets followed by a decreased extent of insu lit is. These data indi cate a benefic ial
effect of FO D on the immune component of the mouse LDSTZ-D model {97].
In another study, the effect of dietary w-3 PUFAs on in vivo insu lin sens it ivity was
studi ed using six non-insulin-dependent diabet ic (N IDD) pat ients. Fi ndings from th is stu dy
showed that, an 8 weeks dail y supplementat ion of 3 g of the w-3 faliy acids, inc ludi ng E PA
resu lted in an increase in metabo lic clearance rate (MCR) o f glucose in all pat ients; from
3.930.55 - 4.690.74 mVkg/min,suggesting improved sensitivity of insuli n in vivo by the
di etary supplementat ion of w-3 fatty acids [98].
However the effects of w-3 !ally acids on glycemic control, part icularly in patients with
type-2 di abetes, remain a controversial issue. Adverse effects of ro-3 fatty acids on glycemia
were reported in several reviews such as [99- 104], in contrast several trials have shown that
EPA and o ther ro-3 fatty acids have advantageous effects on type-2 diabetes {96-98].
Confl icting results reported in the literature are attributable to methodological limitat ions
such as dj fferences in patient select ion, the quantity of w-3 fats given, failing to randomize
treatment, study design, studi es using small numbers of patients and the lack of considerat ion
of the background diet. Accordi ng to the available sc ientific evidence, the cumulat ive data
suggest that incorporat ion of w-3 fatty acids into the diet of diabet ic patients may gain
sign ificant benefits [I 05].
EPA and Weightlessness-induced Bone l oss

Weightlessness is a consequence of reduced gravitational force felt by humans and is
mainly associated with spaceili ghts. Muscle catabolism an d bone loss are the major effects of
"eight lessness [I 06]. Mechan ical environment plays a major role on the growth and
absorption of a bone. Changes in mechan ical loadings may affect the bone amount and
structure. As a result of weight lessness, human skeleton will experience a sign ificant ly
reduced amount of loading and may induce more bone absorpt ion than growth and thus result
in bone loss [ I 07]. Therefore understandi ng the nature and mechan isms of bone loss an d
fin ding ways to reduce bone mass reduction become important.
EPA, an ro-3 PUFA has shown promising results in reducing bone loss in both hu man and
mice in vitro studi es. A study reported by Zwart and eo-works [ I 06] revealed the capacity of
EPA to counteract weight lessness-i nduced bone loss by inh ib it ing the transcript ional activator
nuclear factor kappa-1 ight-chain-enhancer of activated B cell s (N F-KB). From earlier studies,
NF-KB was found to be act ivated by modeled weightlessness wh ile EPA inh ibited the
act ivat ion. Th is gives the idea that EPA cou ld inh ibit the N F-KB pathway and have a
protective effect on bone. In th is study, they have investigated the effects of EPA on
di fferentiat ion of RA W264.7 monocyte/macrophage cell s induced by receptor act ivator of
NF-KB ligand (RANKL) and on act ivation ofN F-KB by tumor necros is factor a (TN F- et) or
exposure to modeled weightlessness. They have proven that EPA inh ibited RAN KL-i nduced
di fferenti ation and decreased activat ion of N F-KB induced by TN F- cl for 30 minutes or by
modeled weight lessness for 24 hours. In the same study they evaluated the effect of NF-KB
act ivat ion in humans after short-duration spacefl ight as well as the re lat ionship between
63
intake of ro-3 fatty acids (from fish) and bone minera l density after long-durat ion spacefli ght.
Reduced loss of bone mineral density and less N-telopept ide (a biomarker used to measure
the rate of bone turnover) excret ion duri ng bed rest after consumpt ion of fish provided
substantial evidence of the potential for EPA to counteract bone loss associated w ith
spaceflight {I 06].
Anoth er in vivo study using female mice with di et-enriched with EPA for their entire
adu lt li fe shows an improvement of bone m icrostructure and strength . EPA improved th e
vo lume of trabecular bone of caudal vertebrae and the vol ume and the th ickness of femur
con ical bone. EPA was found to furth er increase the levels of IGF-1 (Insulin-Like Growth
Facto r 1). As evident in the study, long-term intake of EPA improved the structural and
mechan ica l pro perties of cort ical bone, without aifect ing trabecular bone loss [29].
In another study Sakaguch i et al. reported that EPA enriched djet has inhibited th e
dec<ease of bone weight in femora and tibiae. In th is study, four groups of ovar iecwmjzed
rats were fed w ith nom1al, low calcium, EPA enriched and EPA enriched-low calcium di ets.
Rats fed w ith EPA enr iched diet were able to prevent the decrease in bone weight and bone
strength by means of three possible mechanisms suggested by the authors . Preli minary
45
observations from th is study showed that EPA increased th e accumulat ion of Ca in the ce ll
layer in cultured osteoblastic cell s and thereby st imu lating bone forn1ation which was the fi rst
mechanism suggested in the paper. The second mechanism invol ves osteoclast forn1at ion in
bone marrow which was found to be related w ith the inhibit ion of bone resorption caused by
EPA and th irdl y, EPA has increased intest inal calc ium absorpt ion as l a-hydroxyvitamin 0 3
or reduced ca lci um exc~et ion [ I 08].
There is evidence that fish oil (FO), which contains an appreciable amount of EPA,
decreased bone loss in OVX mice because of inhibition of osteoclastogenesis. l.n th is study
sham-operated and OVX m ice were fed diets containing either 5% corn o il (CO) or 5% fish o il
(FO) and an in vitro study using selected ro-3 fatty acids, EPA and DHA M ice fed w ith CO
showed significant increase in bone m ineral density loss (20% in diSial left femur and 22.6% in
l.umbar vertebrae) in OVX mice, whereas FO-fed mice showed on ly I 0% and no change,
respectively. In vitro study showed that eicosapentaenoic acid caused a sign ificant decrease in
tartrate-res istant acid phosphatase (TRACP) activity and TR..t\CP- mult inuclear cell forn1ation
from bone marrow (81'.1) cells and also inhibited BM macrophage NF-KB activation induced by
RANKL. According to their data, inhibition of osteoclast generation and activation may be one
of the mechanisms by which dietary ro-3 fatty acids reduce bone loss [ I 09].
Findings from both in vitro and in vivo studies confi m1 that EPA has the potential to
counteract bone loss.
EPA Metabolites and Various Bone Diseases

EPA can be metabo lized through the cyclooxygenase and 5-lipoxygenase metabolic
pathways to forn1 end products with altered bio logical act ivity. Through cyclooxygenase
pathway EPA fom1 thromboxane A3 (TXA3 }, prostaglandin 13 (PGh1 a metabol icall y active
prostacyclin and prostaglandin E3 (PGE 3) and the 5-lipoxygenase metabolic pathway gives
leucotriene 8 5 (LTB 5) and leucotriene E5 (L TE 5) [ 11 0, Ill ] . TheE series PGs have important
funct ions in blood pressure regu lation, fertility, and modulation of the immune response.
Ingestion of w-3 fatty acids has been documented to have significant effects in all of these
64
areas and in cardi ovascular health and well-being [ 11 2]. Apart from these, metabo lites o f EPA
have also shown to have effects on bone.
A pos itive effect ofPGE, was shown in a cJjnjcal study by Kremer et al. using pat ients w ith
rheumato id arthritis (RA) and these patients were able to di scontinue the use of nonsteroidal
ant i-inflammatory drug (NSA ID) whil e receiving a source of w-3 fatty ac ids {I 13], suggesting
that the mode of ro-3 fatty acid action in RA pat ients could be related to eicosano id biosynthesis.
One explanation given for th is phenomenon is that the EPA metabo lite PGE, is much less
inflammatory compared with PGE2 [ I 14], thus lowering PGE2 in the di seased joint w ith diets
rich in long-chain ro-3 fauy acids could further benefit RA patients [ I 13].
However, despite these benefits, EPA and its metabo lites also show some adverse effects
on bone structure. In a study, invest igati ng the effects of PGE,and EPA on rat bone in organ
culture, Raisz et a/ reported that PGE, is a potent st imulator of bone resorption and there may
be a possibility that di etary EPA can affect the production of bone resorbing prostano ids in
man [ 11 5].
In another study, Judex et al. reported that, di etary fish o il supplementation adversely
afTects cort ical bone morpho logy and biomechanics in growing rabb its. They have
investigated how a d iet supp lemented with IOg/1 00 g fish o il affected t ibial cortical
morpho logy and mechanica l properti es in weanling rabbits. The fi ndings showed that the fi sh
oil-supp lemented rabb its had sign ificant ly sma ll er middiaphyseal area! properties and shorter
tibiae and the t ibial structural properties were significantly reduced compared to contro ls.
Th is data suggested that 10% fish oi l supplementation in the presence of modest vitamin E
supp lementat ion can have detrimental effects on the skeleton of rapidly grow ing rabbits [78].
In a separate study, Poulsen and eo-workers showed that high-dose of E PA
supp lementat ion exacerbated the effects of ovari ectomy on Bl\,ID . In this study, bone mineral
density (BM D), 25 -hydroxyvitamin D3 and plasma fatty acid profi le of OVX rats
supp lemented with 0.1 g (low) or 1.0 g (high) of EPA!kg body weight for 9 weeks, were
compared to those of OVX and sham animals fed a non-supplemented diet. BMD was
sign ificant ly decreased in all OVX but not shan1 rats and there was no di fference in BMD
between the low group and OVX contro ls. A lso BMD was sign ificant ly lower in the high
group compared to OVX and sham contro ls. Findings suggest that, a high-dose of E PA
supp lementat ion exacerbated the effects of ovar iectomy on BMD [ I 16] .
In conclusion E PA and its metabo li tes have both positive and negat ive e fTects on bone
structure.
Conclusion
Several studi es on EPA have been found interest ing as with regards to its re lat ion to bone
meta bo lism. However, most of these stud ies were done as w-3 PUFAs and not as EPA a lone.
Al though some studi es have shown the effect of ro-3 PU FAs in declining bone mass caused
by menopause in women ovariectomy in animal models li ke f()r examp le, in general there are
sti ll lacking evidences to generall y Ctlnclude that EPA o r ro-3 PU FAs can mitigate or have
posit ive efTects in osteoporosis. EPA as part o f ro-3 PU FA has also been shown to prevent
bone resorption and have positive associat ion w ith bone mineral density. In tem1s of PUFA
rat ios, lower ing the dietary rat io of ro-6/ro-3 PUFA is known to increase bone marrow
cell ularity and bone strength in animals. In relati on to cort ical bone mo rpho logy, although
Eicosapentaeno ic Acid (EPA} and Bone Metaboli sm
65
fish oil which is rich in EPA can have posit ive effects on calcium absorption and bone
metabolism compared w ith other various fat sources, supp lementation of fish oil in large
quanti ties and the associated increase in djetary fat content can lead to substantial degradation
of morphological and mechan ical propert ies of cort ical bone.
Numerous human and an imal studi es have been conducted to investi gate the role of EPA
in bone cancer, diabet ic osteopen ia, weight lessness induced bone loss and other bone
di seases. EPA has shown a great potential to contribute to its proposed health benefi ts an d
showed promising results in the research work di scussed. However, there are some studi es
which have been rep orted on its adverse effects on bone related di seases. lv!oreover the
underlying mechan isms, the long-term etTects of diets high in EPA and whether simil ar
skeletal consequences are evident for humans taking fi sh oil supp lements remain to be further
invest igated.
References
{I]
Watk ins, B. A., Li, Y ., Lippman, H. E., and Feng, S., Modulatory effect of omega-3
polyunsaturated fatty acids on osteoblast function and bone metabo lism.
Prostaglandins Leukotrienes and Essential Fatty Acids, 2003. 68(6): p. 387-398.
(2] Medicine, Inst itute of, Dietmy reference intakes for energy, carbohydrates, fiber, f at,
f atty acids, cholesterol, protein and amino acids. 200 2/2005, Washington, D.C.:
National Academy Press.
[3] Agricu lture, US Department of Health and Human Services and US Department of,
Dietwy Guidelines for Am ericans. 2005, Washington, D.C.: Government Print ing
Office.
[4] Kris-Etherton, P. M., Grieger, .1. A., and Etherton, T. D., Dietary reference intakes for
DHA and EPA. Prostaglandins Leukotrienes and Essential Fatty Acids, 2009. 8 1(2-3):
p. 99- 104.
[5] Kris-Etherton, P. lv!. and !nn is, S., Posit ion of the American dietet ic associat ion and
clietitians of Canada: Dietary fatty acids. Journal of the American Dietetic Association,
2007. 107(9): p. 1599- 16 11.
(6] Association, American D iabetes, Nutrition recommendat ions and interventions for
diabetes: a posit ion statement of the American Diabetes Associat ion. Diabetes Care,
2008. 3 1 (Supp l. I): p. S6 1- S78.
(7] Kris-Etherton, P. M., Harris, W. S., Appe l, L. J., an d Comm, Nutrit ion, Fish
consumpt ion, fish oil , omega-3 fatly acids, and cardiovascular di sease. Circulation,
2002. 106(2 1): p. 2747 -2757.
(8] Council , Australia and New Zealand Nat ional Health and Medi cal Research, Nutrient
Riference values for Australia and New Zealand including recommended dietG/y
intakes. 2006.
[9] Martin, A., ed. Apports nutritionnels conseilles pur la population Francaise. 3rd ed.
2001, Tech. and Doe. Lavoisier: France.
[ I 0] Australia, National Heart Foundation oj Plant Sterols, omega -3Jots and heart disease
in Australian Capital Territory.
{ 11 ] Nutrit ion, UK Scienti fic Advisory Committee on, Advice on Fish consumption:
benefits and risks. 2004.
66
{12] Report, FAO/WHO Techn ical, Diet, nutrition, and prevention of chronic diseases.
2003, World Health Organ izat ion: Geneva.
[ 13] Tanaka, H. Japanese Nutritional Requiremem-Dietary Reference Intakes PolicyMaking Committee. 2004: Nat ional Institute of Health and Nutrit ion.
{ 14] Cunnane, S.C.; Drevon, C.A.; Harris, B.; S ine lair, A .; Spector, A ., Recommendations
for Intake of Polyumaturated Fatty Acids in Healthy Adults. 2004, International
Society for the Study of Fatty Acids and Lipids: Tiverton, UK.
{ 15] S imopolous, A., Summary of the NATO Advanced Research Workshop on Dietary
omega-3 and omega-6 fatly acids: biological effects and nutrit ional essent iali ty.
Journal of Nutrition, 1989: p. 521 -528.
( 16] Koletzko, B., Uauy, R., Palou, A., Kok, F., Homstra, G., Eil ander, A., Jv!oretti, D.,
Osendarp, S., Zock, P., and !nnis, S., Dietary intake of eicosapentaenoic acid (E PA)
and docosahexaenoic acid (DHA) in ch ildren - a workshop report. British Journal of
Nutrition, 20 I 0. I 03(6): p. 923-928.
{17] Harr is, W. S., l\1[ozaffarian, D., Lefevre, M., Toner, C. D., Colombo, J., Cunnane, S. C.,
Holden, J. M., Klurfeld, D. M., Morris, M. C., and Whelan, J., Towards Establish ing
Dietary Referen ce Intakes for Eicosapentaenoic and Docosahexaenoic Acids. Journal
of Nutrition, 2009. 139(4): p. 804s-819s.
{18] Ross, A. C., Manson, J. E., Abrams, S. A., A loia, J. F., Brannon, P. M., C linton, S. K.,
Durazo-Arvizu, R. A., Gallagher, J. C., Ga ll o, R. L., Jones, G., Kovacs, C. S., Mayne,
S. T., Rosen, C. J., and Shapses, S. A., The 2011 Report on Dietary Reference Intakes
for Calcium and V itamin D from the Inst itute of Medicine: What C li nicians Need to
Know. Journal of Clinical Endocrinology and Metabolism, 20 11. 96(1): p. 53-58.
{ 19] Watkins, B. A., Li, Y ., Lippman, H. E., and Sei fert, M. F., Omega-3 polyunsaturated
fatty acids and skeletal health. Experimental Biology and Medicine, 200 I. 226(6): p.
485-497.
[20] Authority, European Food Safety, Scienti fie substant iat ion of a health claim related to
Docosahexaeno ic Acid (DHA) and Arach idon ic Acid (A RA) and support of the neural
deve lopment of the brain and eyes pursuant to Art icle 14 of Regulat ion (EC) No
1924/ 2006. The EFSA Journal, 2008. 794: p. 1- 11.
{21 ] Glowacki, J.; Manson, J. E.; LeBoff; M.S., Omega-3 Fatty Acids and Bone Health.
Orthopaedic Journal at Harvard Medical School On line, 2011: p. 58-6 1.
[22] Lau, B. Y., Fajardo, V. A ., McMeek in, L., Sacco, S. M., Ward, W. E., Roy, B. D.,
Peters, S. J., and LeB ianc, P. J., Influence of high-fat diet from different ial dietary
sources on bone mineral density, bone strength, and bone fatly acid composit ion in rats.
Applied Physiology Nutrition and Metabolism-Physiologie Appliquee Nutrition Et
Metabolisme, 20 I 0. 35(5): p. 598-606.
(23] Hu, F. B., Manson, J. E., and Will ett, W. C., Types of di etary fat and risk of coronary
heart disease: A crit ical review. Journal of the American College of Nutrition, 2001.
20(1): p. 5-19.
[24] Poulsen, R. C., Moughan, P. J., and Kruger, M. C., Long-chain po lyunsaturated fatty
acids and the regulat ion of bone metabo lism. Experimental Biology and Medicine,
2007. 232(10): p. 1275-1288.
[25] lwami- Morimoto, Y., Yamaguchi, K., and Tanne, K., Influence of di etary n-3
po lyunsaturated fatty acid on experimental tooth movement in rats. Angle 01thodontist,
1999. 69(4): p. 365-37 1.
67
[26] Sakaguch i, K., Mor ita, 1., and Murota, S., Eicosapentaenoic Acid Inhibits Bone Loss
Due to Ovariectomy in Rats. Prostagl.andins Leukotrienes and Essential Fatty Acids,
1994. 50(2): p. 81 -84.
{27] Poulsen, R. C. and Kruger, M. C., Detrimental effect of eicosapentaenoic acid
supp lementat ion on bone following ovariectomy in rats. Prostaglandins Leukotrienes
and Essential Fatty Acids, 2006. 75(6): p. 4 19-427.
{28] Li, Y., Greiner, R. S., Salem, N ., and Watk ins, B. A., Impact of di etary n-3 FA
deficiency on rat bone tissue FA composit ion. Lipids, 2003. 38(6): p. 683-686.
[29] Bonnet, N. and Ferrari, S. L., Effects of long-term supp lementation with omega-3 fatty
acids on longitudinal changes in bone mass an d m icrostructure in mice. Journal of
Nutritional Biochemistry, 2011. 22(7): p. 665-672.
[30] Weiss, L A., Barrett-Connor, E., and von Muhlen, D., Rat io ofn -6 10 n-3 fatty acids
and bone mineral density in older adults: the Rancho Bemardo stu dy. American
Journal ofClinical Nutrition, 2005. 81(4): p. 934-938.
[3 1] Kruger, lvl. C., Coetzer, H., de Winter, R., Gericke, G., and van Papendorp, D. H.,
Calci um, ganm1a-li nolen ic acid and eicosapentaenoic acid supp lementat ion in sen il e
osteoporosis. Aging-Clinical and Experimental Research, 1998. 10(5): p. 385 -394.
[32] Christensen, P.; Bartels, E.lvl.; Riecke, B.F.; Bliddal, H.; Leeds, A .R.; Astrup, A.;
Winther, K.; Chri stensen , R., Improved nutr it ional status and bone health after di etinduced weight loss in sedentary osteoarthritis patients: a prospective cohort study.
European Journal of Clinical Nutrition, 2012. 66: p. 504 -509.
[33] Tarti bian, B., Maleki, B. H., Kanaley, J., and Sadeghi, K., Long-term aerobic exercise
and omega-3 supplementat ion modulate osteoporosis through innammatory
mechan isms in post-menopausal women: a randomized, repeated measures study.
Nutrition and Metabolism, 2011. 8.
[34] Hogstrom, M., Nordstrom, P., and Nordstrom, A., n-3 fatty acids are positively
associated with peak bone mineral density and bone accrual in healthy men: the N02
Study. American Journal of Clinical Nutrition, 2007. 85(3): p. 803-807.
{35] S irois, 1., Cheung, A. i'v!., and Ward, W. E., Biomechan ical bone strength and bone
mass in young male and female rats fed a fish oil diet. Prostaglandins Leukotrienes and
Essential Fatty Acids, 2003. 68(6): p. 4 15-42 !.
{36] Far ina, E. K., Kiel, D. P., Roubenoft~ R., Schaefer, E. J., Cupples, L. A., and Tucker,
K. L., Protective effects of fish intake and interact ive effects of long-chain
polyunsaturated fatty acid intakes on hip bone mineral density in older adults: the
Framingham Osteoporosis Study. American Journal ofClinical Nutrition, 2011. 93(5):
p. 1142 - 11 5 1.
[37] Rang, H. P.; Dale, M. M.; Ritter, J. !vi.; lvl oore, P. K., Phannacology. 2003, London:
Elsevier Church ill Li vingstone.
[38] Wang, Y. J., Ohtsuka-lsoya, M., Shao, P., Sakamoto, S., and Shinoda, H., Effects of
methylprednisolone on bone fom1at ion and resorption in rats. Japanese Journal of
Pharmacology, 2002. 90(3): p. 236-246.
{39] Carbonare, L. D., Bertoldo, F., Valent i, M. T., Zenari, S., Zanatta, M., Sella, S.,
Giann ini, S., and Lo Cascio, V., Histomorphometric analysis of glucocort icoid- induced
osteoporosis. Micron, 2005. 36(7 -8): p. 645-652.
68
[40] Sun, D. X., Krishnan, A., Zaman, K., Lawrence, R., Bhauacharya, A., and Femandes, G.,
Dietary n-3 fatty acids decrease osteoclastogenesis and loss of bone mass in
ovariectomized mice. Journal of Bone and Mineral Research, 2003. 18(7): p. 1206- 121 6.
{4 1] Shomali , T., Rezaian, M., Rassouli, A., and Asadi, F., Effect ofEicosapentaenoic Acid
on Bone Changes due to Methylprednisolone in Rats. Basic and Clinical
Phannacology and Toxicology, 2009. I 05(1): p. 46 -50.
(42] Dietary Reference intakes f or Calcium and Vitamin D. 20 10, Food and Nutrit ion
Board, Institute oflvledicine: Wash ington, DC: Nat ional Academy Press.
{43] Kett ler, D.B., Can manipulat ion of the rat ios of essenti al fatly acids slow the rapid rate
of postmenopausal bone loss? Alternati ve Medicine Review: A Journal of Clinical
Therapeutic, 2001. 6: p.:6 1-77.
{44] Kruger, M.C. and Scholium, LM., Is docosahexaenoic acid more effect ive than
eicosapentaenoic acid for increasing calcium bioavai lability? Prostaglandins,
Leukotrienes and Essential Fatty Acids, 2005. 7 3: p. 327-334.
[45] Reinwald, S., Li, Y., l\I(Origuchi, T., Salem, N. Jr., and Watkins, B.A. , Repletion with
(n-3) fatty acids reverses bone structural deficits in (n-3)-defici ent rats The Joumal of
Nutrition, 2004. 134: p. 388-394.
(46] Kruger, M. and Horrob in, D., Calcium metaboli sm, osteoporosis and essent ial fatty
acids: a review. Progress in Lipid Research, 1997. 36: p. 13 1- 15 1.
(47] Buck, A.C., Davies, R. L., and Harrison, T., The protect ive ro le of eicosapentaenoic
acid (EPA) in the pathogenesis of nephroli th iasis. The Journal of Urology, 199 1. 146:
p. 188- 194.
{48] Rothwell , P..J., Green, R., Blacklock, N.J ., and Kavanagh, J.P. , Does fish oil benefit
stone formers? The Jounwl of Urology, 1993. 150: p. 139 1- 1394.
{49] Kruger, M.C., Coetzer, H., de W inter, R., and Claasen, N., Eicosapentanoic acid and
docosahexanoic acid supplementation increase calc ium balance. Nutrition Research,
1995 15: p. 211 -2 19.
[50] Claassen, N ., Coetzer, H., Steinmann, C., and Kruger, M., The effect of di fferent n-6/ n3 essent ial faily acid rat ios on calcium balance and bone in rats Prostaglandins,
Leukotrienes and Essential Fatty Acids, 1995. 53: p. 13- 19.
{5 1] Ortiz-Aivarado, 0., Miyaoka, R., Kriedberg, C., Leavitt, D.A., Moeding, A., Stessman,
M., and Monga, M., Omega-3 fatty acids eicosapentaeno ic acid and docosahexaenoic
acid in the management of hypercalciuric stone formers. Urology, 20 12. 79: p. 282-286.
{52] Yasu i, T., Tanaka, H., Fuj ita, K., lguch i, M., and Kohri, K., Effects of eicosapentaenoic
acid on urinary calcium excret ion in calcium stone formers. European Urology, 2001.
39: p. 580-585.
{53] !to, H., Kotake, T., and Nomura, K., Effect of ethyl icosapentate on urinary calcium
excret ion in calcium oxalate stone furmers. Urologi.a lnternalis, 1995. 54: p. 208-2 I 3.
{54] Kell y, 0., Cusack, S., Jewell , C., and Cashman, K.D. , The effect of polyunsaturated
faily acids, inc ludi ng conjugated linoleic acid, on calcium absorpt ion and bone
metabolism and composit ion in young growing rats. The British Journal of Nutrition,
2003.90: p. 743 -750.
{55] Kell y, 0. and Cashman, K. D., The effect of conjugated linoleic acid on calc ium
absorpt ion and bone metabolism and composit ion in adult ovariectomised rats.
Prostaglandins Leukotrienes and Essential Fatty Acids, 2004. 71(5): p. 295-30 I .
69
(56] Kane, G.G., Lovelace, F.E., and McCay, C.lvl., D ietary fat and calcium wastage in old
age. Journal ofGerontology, 1949.4: p. 185- 192.
(57] Knudson, A. and Floody, R .J., Fat as a factor in the healing of rickets with vitamin D.
The Journal of Nutrition, 1940. 20: p. 3 17-.
{58] Cal verley, C.E. and Kennedy, C., The effect of fat on calcium and phosphorus
metabolism in normal grow ing rats under a normal dietary regime. The Journal of
Nutrition, 1949.38: p. 165- 175.
{59] Glor ieux, F.H., Travers, R., Taylor, A., Bowen, J. R., Rauch, F., No rman, lvl., and
Parfi tt, A.M. , Nom1at ive data for iliac bone histomorphometry in grow ing ch ildren.
Bone, 2000.26: p . 103- 109.
{60] Parfitt, A .M., Han, Z.-H., Paln itkar, S., Rao, D.S., Shib, lvi.-S., and Nelson, D., Effects
of ethnicity and age or menopause on osteob last funct ion, bone minerali zat ion and
osteoid accumu lat ion in iliac bone. Journal of Bone and Mineral Research, 1997 12: p.
864 - 1872.
{6 1] Hogstrom, l\'1., Nordstr1im, P., and Nordstri\m, A., n-3 Fatty acids are positively
associated with peak bone mineral density and bone accrual in healthy men: the N02
study. The American Journal ofClinical Nutrition, 2007. 85: p. 803-807.
{62] Moselhy, S.S., A l-tvtalki, A. L., Kumosani, T.A., and Jalal, J.A., Modulatory effect of
cod liver oil on bone mineralization in overiectomized f emale Sprague Dawley rats.
(63]
[64]
[65]
{66]
{67]
(68]
(69]
2011.07 482337 11 4 12428.

Lage, S., Bueno, M., Andrade, F., Prieto, J.A., Delgado, C., Legarda, M., Sanj urj o, P.,
and A ldamiz-Echevarria, L.J., Fatty acid profi le in pat ients w ith phenylketonuria and
its relationship w ith bone mineral density. Journal of Inherited Metabolic Disease,
20 I 0.
\Veiler, H. A. and Fitzpatr ick-Wong, S. C ., Modulat ion of essent ial (n-6):(n-3) fatty
acid ratios alters fatty acid status but not bone mass in piglets. Journal of Nutrition,
2002. 132(9): p. 2667-2672.
Evans, D. B., Bunning, R. A. D., and Russe U, R. G. G., The Effects of Recombinant
Human lnterleukin- 1-Beta on Cell ular Proliferation and the Production of
Prostaglandin-E2, Plasminogen-Activator, Osteocalcin and A lkaline-Phosphatase by
Osteob last-L ike Cell s Derived from Human-Bone. Biochemical and Biophysical
Research Communications, 1990. 166( I): p. 208-216.
Atkinson, T. G., Barker, H. J., and Meckli ngG ill, K. A., Incorporat ion of long-chain n3 fatty acids in t issues and enhanced bone marrow cell ularity with docosahexaenoic
acid feeding in post-wean ling Fischer 344 rats. Lipids, 1997. 32(3): p. 293-302.
Kokkinos, P. P., Shaye, R., A lam, B. S., and A lam, S. Q., Dietary Lipids,
Prostaglandin-E(2) Levels, and Tooth Movement in A lveolar Bone of Rats. Calcified
Tissue International, !993. 53(5): p. 333-33 7.
Claassen, N., Coetzer, H., Steinmann, C. M. L., and Kruger, M. C., The EfTect of
Different N-6 N-3 Essential Fatty-Acid Ratios on Calcium Balance and Bone in Rats.
Prostaglandins Leukotrienes and Essential Fatty Acids, 1995. 53( I): p. 13-19.
Pou lsen, R. C.; Moughan, P. J.; Kruger, M. C., Long-Chain Polyunsaturated Fatty
Acids and the Regu lat ion of Bone Metabol ism. Experimental Biology and Medicine,
2007.232: p. 1275- 1288.
70
Gerard G. Dumancas, Rangika S. Hikkaduwa Koralege, Patrisha J. Pham et al.
[70] Watk ins, B. A., Lippman, H. E., Le Bouteiller, L., Li, Y., and Seifert, M. F., Bioact ive
fatty acids: role in bone biology and bone cell function. Progress in Lipid Research,
2001. 40(1 -2): p. 125- 148.
[71 ] Van Papendorp, D. ; Coetzer, H, M.C., K., Biochemical profi le or ost eoporotic patients
on essent ial fatty acid supp lementat ion. Nutr. Res., 1995. 15: p. 325-334.
{72] Macdonald, H. M., New, S. A., Golden, M. H., Campbel l, M. K., and Reid, D. M. ,
Nutrit ional associat ions w ith bone loss during the menopausal transition: evidence of a
beneficial effect of calcium, alcohol , and fruit and vegetable nutrients and of a
detrimental effect of fatty acids . American Journal of Clinical Nutrition, 2004 . 79( 1):
p. 155- 165.
[73] Claassen, N., Potgieter, H. C., Seppa, lVI., Vem1aak, \V. J. H., Coetzer, H.,
Vanpapendorp, D. H., and Kruger, M. C., Supp lemented Gamma-Li no lenic Acid and
Eicosapentaenoic Acid Influence Bone Status in Young Male-Rats - Effects o n Free
Urinary Collagen Cross-Links, Total Urinary HydroxyproJjne, and Bone Calcium
Content. Bone, 1995. 16(4 ): p. S385-S392.
{74] Kruger, !vi. C., Coetzer, H., Dewinter, R., and Claassen, N., Eicosapentaenoic Acid and
Docosahexaenoic Ac id Supp lementat ion Increases Calcium Balance. Nutrition
Research, 1995. 15(2): p. 211 -2 19.
[75] Valentine, R. C. and Va lentine, D. L., Omega-3 fatty acids in cell ular membranes: a
un ified concept. Progress in Lipid Research, 2004. 43(5): p. 383-402.
[76] Bone. 2012 [cited 2012 Apri l I]; Available from: hilp://www.britannica.com/
EBchecked/topic/72869/ bone.
{77] V igor ita, V. J .; Ghelman, B.; M intz, D., Bone stntcture: cortical and trabecular hone
in Orthopaedic Pathology 2nd edition. 2008, Li ppincoli Willi ams and W ilkins:
Phi lade lph ia. p. 44.
[78] Judex, S., Woh l, G. R., Wolff, R. B., Leng, W., Gillis, A. lvl., and Zern icke, R. F. ,
Dietary Fi sh Oil Supplementation Adversel y Affects Cortical Bone lvl orphology and
Biomechanics in Growing Rabbits. Calcified Tissue International, 2000. 66(6): p.
443-448.
{79] Kruger, lvl. C. and Horrob in, D. F., Calcium metabolism, osteoporosis and essential
faily acids: A review. Progress in Lipid Research, 199 7. 36(2-3): p. 13 1- 15 1.
{80] Bonnet, N .; Ferrari , S. L., Effects of long-term supplementation w ith omega-3 fatty
acids on longitudi nal changes in bone mass and microstructure in mice . .f. Nutr.
Biochem ., 20 11. 22(7): p. 665-72.
{8 1] Matsushita, H., Barrios, .f. A., Shea, J. E., and Mill er, S. C., Dietary fish oil results in a
greater bone mass and bone fom1at ion indices in aged ovariectomized rats. Journal of
Bone and Mineral Metabolism, 2008. 26(3): p. 24 1-247.
(82] Lukas, R., Gigliotti, .f. C., Smith, B. J., Altman, S., and Tou, J. C., Consumpt ion of
di fferent sources of omega-3 pol yunsaturated fatty acids by growing female rats affects
long bone mass and microarchitecture. Bone, 2011 . 49 (3): p. 455-462.
[83] Liu, D., The effects of di etary lipids on bone chemical , mechanjcal, and histological
properties in Japanese quai l (Cotum ix C. Japonica), in Animal and Poultly Sciences.
2000, V irginia Pol ytechn ic Inst itute and State Un iversity: Blacksburg.
[84] Malawer MM, Helman LJ, bone., O'Sullivan B. Sarcomas of, DeVita, In: V incent T.,
Hell man, Samuel, and eds, Steven A. Rosenberg, Cancer, Principles and Practice of
Oncology. 7th ed. Philadelph ia, PA: Lipp incou W illiams and W ilkins; 2005.
Eicosapentaenoic Acid (EPA} and Bone Metaboli sm
71
(85] Jude, Sebast ien, Roger, Sebastien, lv!artel, Eric, Besson, Pierre, Richard, Serge,
Bougnoux, Philippe, Champeroux, Pascal, and Le Guennec, Jean-Yves, Dietary longchain omega-3 fatty acid s of marine origin: A compar ison of their protect ive eftects on
coronary heart disease and breast cancers. Progress in Biophysics and Molecular
Biology, 2006. 90(1 - 3): p . 299-325.
{86] Riediger, Natali e D., Othman, Rgia A., Suh, lv!iyoung, and Moghadasian, Mohammed
H., A Systemic Review of the Roles of n-3 Fatty Acids in Health and Disease. Journal
of the American Dietetic Association, 2009. I 09( 4): p. 668-679.
[87] Roynette, Catherine E., Calder, Philip C., Dupertuis, Yves M., and Pi chard, C laude, n-3
Polyunsaturated fauy acids and co lon cancer prevent ion. Clinical Nutrition, 2004.
23(2): p. 139- 15 1.
(88] Tang, Feng-Yao, Cho, Hsin-Ju, Pai, Man-Hui, and Chen, Y e-Hsin, Concomitant
supplementat ion of lycopene and eicosapentaeno ic acid inh ibits the proli ferat ion of
human colon cancer cell s. The Journal of Nutritional Biochemistly, 2009. 20(6): p.
426-434.
[89] Hegde, S., Kaushal, N., Ravindra, K. C., Chiaro, C., Hafer, K. T ., Gandhi, U . H.,
Thompson, J. T., van den Heuvel, J. P., Kennett, M. J., Hankey, P., Pau lson, R. F., an d
Prabhu, K. S., Delia( 12)-prostaglandin .1(3), an omega-3 fatty acid-derived metaboli te,
select ively ab lates leukemia stem cell s in mice. Blood, 2011 . 11 8(26) : p. 6909-69 19.
[90] Ch iu, Lawrence C. lvf. and Wan, Jenn ifer lvf. F., Induction of apoptosis in HL-60 cells
by eicosapentaenoic acid (EPA) is associated with downregu lation of bcl-2 expression.
Cancer Letters, 1999. 145(1 - 2): p. 17-27.
[9 1] S lagsvold, .lens, Pettersen, Caroli ne, Foll estad, Turid, Krokan, Hans, and Sch0nberg,
Svanhi ld, The Ant iproli ferat ive Effect of EPA in HL60 Cell s is Mediated by
Alterati ons in Calcium Homeostasis. Lipids, 2009. 44(2): p. 103 - 11 3.
(92] Mu sumeci, G., Loreto, C., C lement i, G., Fi ore, C . E., and Mart inez, G., An in vivo
experimental study on osteopen ia in diabet ic rats. Acta Histochemica, 2011. 11 3(6): p.
619-625.
(93] Chambers, T. J., McSheehy, P. M . .1., Thomson, B. M., and Full er, K., The EfTect of
Calcium-ReguJat ing Hom10nes an d Prostaglandins on Bone-Resorpt ion by Osteoclasis
Disaggregated from Neonatal Rabb it Bones. Endocrinology, 1985. 11 6( 1): p. 234-239.
[94] Jee, W. S. S., Ueno, K., Kimme l, D. B., Woodbury, D. M., Price, P., and Woodbury, L.
A., The Role of Bone-Cell s in Increasing 1\,letaphyseal Hard Tissue in Rap idl y
Growing-Rats Treated w ith Prostaglandi n-E2. Bone, 1987. 8(3): p. 171 - 178.
[95] Marks, S. C. and Mill er, S., Local Infusion of Prostaglandin- I Stimu lates Mandi bu lar
Bone-Fom1at ion Jnvivo. Journal of Oral Pathology and Medicine, 1988. 17(9- 10): p.
500-505.
[96] Yamada, Yuya, Fush imi, Hisako, lnoue, Toru, Matsuyama, Yukiko, Kameyama,
Masakun i, M inami, Takesb i, Okazaki, Yuko, Noguc.h i, Yasuh isa, and Kasama, Tosh io,
Effect of eicosapentaenoic acid and docosahexaenoic ac id on di abetic osteopen ia.
Diabetes Research and Clinical Practice, 1995. 30( 1): p. 3 7-42 .
{97] Linn, T., Noke, M., Woehrle, M., K loer, H. U., Hammes, H. P., Litzlbauer, D., Bretzel,
R. G., and Federlin, K., Fish Oil Enriched Diet and Reducti on of Low-Dose
Streptozocin Induced Hyperglycemia - Inhibition of Macrophage Activat ion. Diabetes,
1989.38 (1l):p. 1402- 14 11 .
72
{98] Popp-Snijders, C., Schouten, J. A., Heine, R. J., van der Meer, J., and van der Veen, E.
A., Dietary supp lementation o f omega-3 po lyunsaturated fally acids improves insulin
sensitivity in non-i nsuli n-dependent diabetes. Diabetes Res., 1987. 4(3): p. 14 1-7.
{99] Axelrod, L., Omega-3 fatty acids in diabetes mellitus. G ift from th e sea? Diabetes,
1989. 38(5): p. 539-43.
{l OO] Kasim, S. E., Dietary marine fish oi ls and insu lin act ion in type 2 diabetes. Ann. N. Y.
Acad. Sci., 1993. 683: p. 250-7.
{101 ] Malasanos, T. H. and Stacpoole, P. W., Biological effects of omega-3 fatly acids in
diabetes mell itus. Diabetes Care, 199 1. 14( 12): p. 11 60-79.
[ 102] Nestel, P. J., EfTectsofN-3 fau yacidson lipid metabol ism. Annu. Rev. Nutr., 1990. 10:
p. 149-67.
[ I 03] Sorisky, A. and Robb ins, D. C., Fish oil and diabetes. The net effect. Diabetes Care,
1989. 12(4): p. 302-4.
[ 104] Vessby, B., Dietary supplementation w ith N-3 polyunsaturated fatty acids in type 2
diabetes. Effects on glucose homeostasis. Ann. N. Y. Acad. Sci., 1993.683: p. 244-9.
{105] /v!ori, T. A., Fish oil s, dysl ipidaem ia and glycaemic control in diabetes . Practical
Diabetes lnternarional, 1999. 16(7): p. 223-226.
[ I 06] Zwart, S. R., Pierson, D., Mehta, S., Gonda, S., and Smith, S. M., Capacity of Omega-3
Fatly Acids or Eicosapentaeno ic Acid to Counteract Weight lessness-I nduced Bone
Loss by Inh ibiting N F-kappa B Activation: From Cell s to Bed Rest to Astronauts.
Journal of Bone and Mineral Research, 2010. 25(5): p. I 049- 1057.
[ I 07] Cheng, Shi, Bai, Jing, Shang, Yu, and Peng, Liang, The Simu lat ion of Bone Loss Due
to Weight lessness and Bone Recovery Using Finite E lement lv!ethod. 20 I 0.
{I 08] Sakaguchi, K., Morita, 1., and lvlurota, S., Eicosapentaenoic acid inhibits bone loss due
to ovar iectomy in rats. Prostaglandin.~. Leukotrienes and Essential Fatty Acids, 1994.
50(2): p. 8 1-84.
{I 09] Sun, Dongxu, Krishnan, Apama, Zaman, Khal iquz, Lawrence, Richard, Bhattacharya,
Arunabh, and Fernandes, Gabriel, D ietary n-3 Fatty Acids Decrease Osteoclastogenesis
and Loss of Bone Mass in Ovariectomized lvlice. Journal of Bone and Mineral
Research, 2003. 18(7): p. 1206- 121 6.
{ 11 0] Robinson, D. R., Tateno, S., Pate!, B., and Hirai, A., Lipid Mediators of Inflammatory
and Immune-React ions. Journal of Parenteral and Enteral Nutrition, 1988. 12(6): p.
S3 7-S42.
{Ill ] Fi restein, G. S., Budd, R. C., Harr is, Jr. E. D., lvlc lnnes, I. B., and Ruddy, S., Kelley:5
Textbook of Rheumatology. 8th ed. Phil adelph ia, W.B. Saunders Company. 2008.
[ 112] Kremer, J. M., Lawrence, D. A., Jub iz, W., DiGiacomo, R ., Rynes, R., Bartholomew,
L. E., and Shem1an, M., Dietary fish oil and olive oil supplementat ion in pat ients w ith
rheumatoid arthri tis. Clinical and immunologic effects . Arthritis Rheum, 1990. 33(6): p.
8 10-20.
[ 11 3] Kremer, J. M., Effects of modulation of inflammatory and immune parameters in
patients w ith rheumatic and inflammatory disease receiving di etary supp lementat ion of
n-3 and n-6 fatly acids. Lipids, 1996.3 1: p. S243-S247.
[ 11 4] Alexander, J. W., lmmunonutrition: The role o f omega-3 tally acids. Nutrition, 1998.
14(7-8): p. 627-633.
Eicosapentaeno ic Acid (EPA} and Bone Metaboli sm
73
{11 5] Raisz, L. G., A lander, C. B., and S immons, H. A., Efrects of prostaglandin E3 and
eicosapentaenoic acid on rat bone in organ culture. Prostaglandins, 1989. 37(5): p.
6 15-625.
[ I I 6] Pou lsen, Raewyn C. and Kruger, Mar lena C., Detrimental effect of eicosapentaenoic
acid supplementat ion on bone fo llow ing ovariectomy in rats. Prostaglandins,
Leukotrienes and Essential Fatty Acids, 2006. 7 5(6): p. 4 19-427.

Editors: T.G. Bradley and F.P. Vargas, pp. 75-98
ISBN: 978- 1-62257-480-3

2012 Nova Science Publishers, Inc.
Chapter 3
EPAAND DHAANTIDEPRESSANT EFFECT:

ALONE OR TOGETHER?
Car/os Horacio Laino 1' , Martin Gabriel Codagnoni,
Maria Fernanda Podestcf and Analia Reinb/
1
lnstituto de lnvest igaciones en Ciencias de la Salud Humana (IICS HUM),

Departarnento de Ciencias Exactas, Fisicas y Natural es,
Univers idad Nacional de La Rioja, Provincia La Riqja, Argentina
2
1nstituto de lnvestigaciones Farrnacol6gicas ( IN INFA, CONI CET-UBA),
and Catedra de Farmacologia, Facultad de Farmacia y Bioquim ica,
Universidad de Buenos Aires, Buenos Aires, Argent ina
Abstract
Even with che new generation of amidepressams, che creaunem of depression scill shows
mulciple needs. Treaunem issues usually relace eo lack of efficacy, prolong onsec of
amidepressam accion and a plechora of side effeccs (such as sexual dysfunccion,
gascroimescinal evems, weighc gain). Several scudies have shown chac cercain biological
consciruems, when ingesced wich food or adminiscraced in pure forms, can funccion as
amidepressam agems. Epidemiological findings derived in clinical and preclinical swdies
from which several compounds emerged as pocemial new creaunems for depression either as
accive agems or as adjuncc therapy wich advamages over convemional creaunems in cerms of
side effects and drug imeractions. Omega-3 polyunsamraced fany acids (PUFA) are key
nucricional componems chac exerc imporcam physiological and biochemical effeccs on
cardiovascular, immune and cemral nervous syscem (CNS) funccion. Indeed, diec
supplememacion wich omega-3 PUFA has shown to be bene ficial in che creacmem of
depression. However, noc all cypes of omega-3 PUFA are equally efficacious. While eh ere is
more informacion on docosahexaenoic acid (DHA), linle is known abouc eicosapemaenoic
acid (EPA)'s amidepressam effeccs and che mechan ism of accion of chese fany acids. This
review explores clinical and preclinical evidences relaced eo che amidepressam effeccs of
DHA, EPA and fish oil (DHA plus EPA). An overview of DHA and EPA effeccs on CNS
funccion and ulcimacely che hypochesis of che cemral role of omega-3 PUFA on depression are
E-mail address: carloslaino25@gmail.com, Tel: +54-3822-457000 ex~738 1, Fax: +54-3822-457000, Address:
Av Luis M de la Fuente SN (5300) La Rioja, Argentina. (Co tTesponding author)
76
Carlos Horacio Lai no, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
also provided. Undoubtedly, furrher studies are needed to shed light on the mechanisms
involved in the potemial clinical ben efit of omega-3 supplememation for the treannem of
depression.
Omega-3 and Depression: Studies Using Omega-3 PUFA

Monotherapy and Combined Treatment with Antidepressants
Omega-3 PUFA Monotherapy: Cl in ical Studies
Over the past decade, the incidence o f depression has been raising sharp ly in western
countries (Murray and Lopez, 199 7). Epidemiological studi es suggest genetic and
environmen tal components in the et iology of depression. In 1995, it was hypothesized that
low dietary intakes of omega-3 PU FA (pol yunsaturated fatty acids) could predispose to a
higher risk of suffering from depression and committ ing suicide (H ibbeln and Salem, 1995) .
Converging evidence suggests that low levels of omega-3 PU FA (eicosapentaenoic acid, EPA
plus docosahexaenoic acid, DHA.) in cholesteryl esters in serum and phospholipids in red
blood cells are associated w ith depression (Adams et al., 1996; Peet et al., 1998; lvlaes et al.,
1999). A low preval ence of bipo lar disorder in di fferent countri es is strongly associated with
a high consumption o f fish (Noaghuil and Hibbeln, 2003). Indeed, regu lar consumpt ion of
fish was associated w ith a reduction in suicidal tendencies (Tanskanen et al., 200 1) and better
mental health status (Si lvers et al., 2002).
Cl inical studies have shown some bene fits for the treatment of bipolar depression, major
depressive disorder, ch ildhood depression and res istant depression not only w ith DHA plus
EPA therapy but w ith EPA alone as monotherapy. One of the fi rst studies that evaluated the
effect iveness of omega-3 PU FA monotherapy in patients with bipolar disorder demonstrated
that these fatty acids were well tol erated and that improved the outcome of th e illness when
compared to placebo (Stoll et al., 1999). Other trials focused on bipolar pat ients showed a
reduct ion in their depressive symptoms after a 6-month or a 12-week treatment w ith EPA as
monotherapy (Osher et al., 2005, Frangou et al., 2006). A new meta-anal ysis provides strong
evidence that supports the use of omega-3 PUF A therapy to overcome bipolar depressive
sympt oms (Sarris et al., 20 12). On the other hand, Keck et al. (2006) eval uated a 4-month
treatment w ith EPA in patients w ith bipolar depression but did not find any evidence of
emcacy over placebo.
The rol e of omega-3 PUFA has also been investigated in maj or depressive disorder.
Individuals that were supp lemented w ith omega-3 P UFA (Fish oil ) had a significant
improvement in depressed mood (Su et al., 2003; App leton et al., 20 I 0; Lesperance et al.,
2011 ). Similar results were obtained w ith EPA in patients with depression (Peet et al., 2006;
Li n and Su, 2007; Richardson, 2008). On the contrary, there is a study in which patients with
depression were treated over a per iod of 12 weeks w ith omega-3 PUFA showing ml
si gn ificant efrect compared to placebo (S il vers et al., 2005). Interest ingly, other clinical
studi es have been conducted to detem1ine whether omega-3 P UFA treatment is effect ive in
children (6 to 12 years of age) w ith depression. Nemets et al. (2006) observed benefi cial
effects atl er 16 weeks of treatment w ith the flu ty acid therapy.
it should be noticed that potent ial ant id epressant effects of omega-3 PUFA have been
examined in many clinical trials using a comb inat ion of omega-3 PUFA such as fi sh oil. T his
EPA and DHA An tidepressant Effect: A lone or Together?
77
approach makes it diilicuh to distingu ish specific roles of EPA and DHA. In th is regard, it
has been shown that depressive pat ients that were resistant to the standard ant idepressant
treatment had low serum levels of EPA and exhibited an improvement when treated for 12
weeks with EPA; th ereby, EPA monotherapy has proven significant antidepressant efficacy
(Peel and Horrobin, 2002). Another study that examined patients with treatment-resistant
depression found depressive symptom ameliorat ion during the first month in pat ients treated
"i th EPA (Puri et al., 2001). A recen t meta-analysis demonstrated that EPA supp lementat ion
in excess to DHA (at least 60% EPA) was an effect ive treatment for depression (Sublette et
al., 20 11 ). Consequently, no large clinical studi es have been done to date to assess the
efficacy ofDHA in the treatment of depression disorder. it is worth ment ioning that a trial in
wh ich patients w ith depress ion received a 6-week DHA treatment fail ed to show a significant
efTect over p lacebo (Marangell et al., 2003).
it can be concluded that even though cl inical trials show di flerem outcomes, most of th e
results derived from meta-analysis have demonstrated the effect iveness of omega-3 PUFA
(E PA plus DHA) in the treatment of bipo lar disorder, major depression, treatment-resist ant
and childhood depression.
Possi bly, the di fferences seen in cli nical outcome are due t o methodological issues
including the type of placebo, diagnosis, di fferences in the dose and composit ion, as well as
the durat ion of the omega-3 PU FA diet ary supplementat ion.
Omega-3 l'UFA in Combined Treatment: Clinical Studies

Nemets et al (2002) studied the effect of EPA in combinat ion with ant idepressants in
pati ents w ith recurrent unipolar depressive disorder. Interest ingly, data showed reduction of
depressive symptoms after 2 weeks of treatment, an onset of ant idepressant action
comparable to that seen w ith antidepressant monotherapy. S imilar results were also obtained
for the treatment of bipolar disorder (Sarr is et al., 20 11 ) .
Omega-3 l'UF A Monotherapy: Preclinical Stud,ies

Based on clinical and epidemiological evidence, studies in rodent models of depression
have been carried out.
A grow ing number of preclinical studies suggest that chronic supplementat ion with
omega-3 PUFA (EPA plus DHA as fish oil) disp lays antidepressant effects (Car lezon et al.
2005; Lakhwan i et al., 2007; Huang et al., 2008; Venna et al., 2009, Laino et al., 20 10).
However, contrary to the expectations deri ved from clinical evidence, Shaldub ina et al.
(2002) fail ed to show antidepressant effects after chronic treatment w ith EPA. Therefore, it is
still unknown whether DHA, EPA or the ir comb inat ion is responsible for the antidepressant
efTects observed in exper imental parad igms.
78
Carlos Horac io Laino, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
Omega-3 1'UFA in Combined Treatm ent with Antidepressan ts: l'recli nicaJ
Studies
Benefi cial effects have been observed after combined treatment w ith omega-3 PUFA and
antidepressants in experimental paradigms. For instance, chronic fl uoxetine treatment
di splays addi tive etfects when comb ined w ith omega-3 PUFA (Lakhwani et al., 2007; Laino
et al., 2010). S imil ar results have been shown w ith imipramine (Venna et al., 2008).
Surprisingly, subeffect ive doses of ant idepressants (fl uoxet ine and mirtazap ine) have been
shown to potent iate omega-3 PUF A antidepressant effects (Laino et al ., 20 I 0).
Brain Effects of DHA and EPA

1. DHA in the Bra in
The nervous system is a t issue with high content of polyunsaturated fatly ac ids,
part icularly, arachi donic acid (20:4, omega-6, AA) and docosahexaenoic acid (22:6, omega-3,
DH A). W ith in neurons, DH A is high ly concentrated in brain membrane phospholipids in the
posit io n sn-2 of the amino group of glycerophospholipids, phosphatidylethanolamine,
phosphat idy lser ine and plasmenylethanolantine ( Breckenridge et al., 1972). DH A may be
di rectly obtained from the di et, as prefom1ed DH A or synthesized in vivo from other commo n
dietary omega-3 PUFA such as a-linolen ic ac id (LNA), E PA or docosapentaenoic acid (DPA)
(S inclair, 1975). A series of elongat ion and desaturat ion steps in the endoplasmic ret iculum
tbll owed by a fi nal single step I)-ox idation react ion biotransforms these omega-3 PUF A to
DH A (Sprecher, 20 00).
Studies carried out in humans indicate that only a small proportion (< I%) of dietaryingested LNA (the metabolic precursor of omega-3 PU FA family) is metabolized to DHA
(Paw losky et al., 2003; Goyens et al., 2005) and that the rest is rapidly catabolized Q! 1535%) to carbon dioxi de for energy purpose (Vem1unt et al., 2000; Burdge and Woouon,
2002; Burdge et al., 2003; Burdge et al., 2002). This data indi cates that there is a low rate of
LNA conversion to DHA and that the most useful way to increase DHA body levels is to
ingest prefom1ed DH A . However, in healthy indi viduals, hepat ic conversion of LNA to DH A
is enough to cover ret ina and brain demands (Rapoport et al., 20 I 0; Scott and Bazan, 1989).
Although the brain captures DH A from p lasma (Sheaff et al., 1996; Paw losky et al.,
1996; Su et al., 1999; Nouvelot et al., 1986; Scott and Bazan, 1989; S inclair, 1975; Su et al.,
2001), astrocytes can a lso provide neurons w ith their own synthesized DHA , but th is process
is quantitat ively less important (W iJiard et a l., 2001). DH A synthesis depends on its
availability in the brain. However, astrocytes cont inue to synthesize DH A even in excess
condit ions, suggest ing a crucia l role ofastrocytes in this process (Willard et a l., 2001).
Several mechanisms have been re lated to DH A in the CNS, which in summary describe
the crucial DH A role in the brain:
79
l.l. Effects at Membrane Level
Alteration of Membrane Biophysical Proper ties

DHA possesses a tremendously flexible structu re that it is retlected in an unusual degree
of molecular confom1ations and rapid inter-conversions highly affect ing membrane order
C'fl uidity") (Holte et al., 1995; Salem and N iebylsk i, 1995). Several studies in art ificial
membranes demonstrate high fluidity in DHA-containing membranes (Straume and Litman,
198 7; Stubbs, 1992; Mitchell and Litman, 1998). Simil ar resu lts were obtained from
experiments involving cell s cultured in DH.A-rich medi a (Brown and Subbaiah, 1994; Calder
et al., 1994; Sobaj ima et al., J 986; Y orek et al., 1989).
Furthem1ore, since ion permeability appears to direct ly depend on the fatly acid
unsaturat ion degree, DHA-containing phosphol ipids affect membrane permeabili ty (Huster et
al., 1997 ; Stillwe ll an d Wassa il , 2003). Ehringer et al (1 990) showed that DHA has a greater
effect on membrane permeabili ty to ions than LNA (its precur sor), although both fatly acids
have simi lar effects on membrane iluidi ty. On th e other hand, it has also been demonstrated
that high DHA content may increase the eff iciency of membrane fus ion events wh ich is
important for neurotransmission (league et al., 2002) .
Effects on Lipid Rafts

Lipid rafis are regions of the plasma membrane that contain high levels of sphingolipids
and cholesterol. These dynamic membrane microdomains are enriched with a variety of proteins
and act as signaling platfom1s directly influenced by the cholesterol content (Calder and
Yaqoob, 2007) . The incorporation of PUFA and DHA into microdomains reduces cholesterol
content and changes membrane order. T he relatively low afTinity between the OH A-containing
phospholipids and cholesterol may promote phase separat ions in the membrane (Shaikh et al.,
2004; Shaikh et al., 2003; Wassail et al.,2004; Pitman et al., 2004; Shaikh et al., 2006; Harroun
et al., 2006; Marrink et al., 2008). Thus, DH A induces lateral phase separations into DHArich/cholesterol-poor and DHA-poor/cholesterol-rich lipid m icrodomains. Consistent w ith these
findings, in vivo studies showed that dietary DHA modulate lipid raft composition, decreasing
the amount of cholesterol (Fan et al., 2003; l'vta et al., 2004).
Regulation of Dopaminergic, Serotonergic, Cholinergic and Glutamatergic

Neuro transmissions
Several studi es demonstrate that chronic ingest ion of a LNA-deficient di et modifi es man y
propert ies of dopaminergic and serotoninergic neurotransmission, such as receptor density,
vesicular monoamine transporters and endogenous dopamine and seroton in levels. These
effects were associated with a significant reduction in neuronal DHA (Zimmer et al., 2000a;
Zi mmer et al., 1998; Zi mmer et al., 1999; Zi mmer et al., 2000b; Del ion et al., 1994; Del ion et
al., 1996; Chalon et al., 200 I; Kodas et al., 2002; Zimmer et al., 2002; Kodas et al., 2004; du
Bois et al., 2006; Cha Ion et al., 1998).
Oth er studies have also shown that a chron ic LNA-defi cient diet causes changes in
several cholinergic parameters such as release and binding of Acetylcho line to muscarinic
receptors, and loss of neuronal DHA (Ai'd et al., 2003) . Recent studies in hippocampal
80
Carlos Horacio Laino, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
membranes demonstrate that DHA reduces glutamate-mediated neurotoxicity, suggest ing

neuroprotecti ve effects of DHA (Wang et al., 2003; Menard et a l., 2009 ). Indeed, DHA is
able to modulate glutamate transporter activity (Berry and McBean, 2003; Berry et al., 2005).
Effects on the Acth'ity of Membrane Bound Enzy mes
The increment of synaptic membrane DHA cont ent has been described to modulate
Na K-A T Pase act ivity (Bowen and C landinin, 2002) wh ich is li nked to energy consumption
in the brain. Th is may represent a fundamental role of DHA in brain metaboli c act ivity. On
the other hand, chronic LNA-deficient diets modify Na K--ATPase act ivity due to neuronal
DHA reduct ion (Bourre et al., 1989).
Effects on Ion Channels
By cbanging membrane Iipid compOS JtJon, DH A modifies tensions exerted by
phospholipids, wh ich in turn cause conftlmlat ional changes to the ion channels.
Consequent ly, ion nux through voltage-gated K- and Na- channels is altered (Seebungkert
and Lynch, 2002; Leaf et al., 2002).
Effects on Cell Signaling Pathways
DHA has been shown to act ivate some kinases in the dentate gyrus (Vaidyanathan et al.,
1994), inh ibit cAMP-dependent protein kinase act ivity (Speizer et al., 199 1) and modulate Gprotein-coupled signal ing (M itchell et al., 2003; Wood, 1990). Some studies demonstrate that
a rich DHA diet can increase the DHA content of cell phosphol ipids, and as a consequence of
the part ial disorganizat ion of membrane, phospho lipase D can be excluded and act ivated
(D iaz et al., 2002).
1.2. Effect s at Cell ular Level
Regulation of AA -Metabolizing Enzy mes

Studies indicate that a LNA-defkient diet reduces DHA concentration in the brain and
consequently increases the expression of enzymes that regulate AA metabolism (Rao et al.,
2007).
Effect on Lipid Mediators Derived From AA
DHA decreases the product ion of inflammatory eicosanoids deri ved from AA, such as to
the 2-series prostaglandins and thromboxanes. DHA also inh ibits the release of
proinflammatory cytokines, such as inter leuk in- 1 beta, interleuk in-2, inter leuk in-6, interferongamma, and tumor necrosis factor al pha (Si mopolous, 2003; Wall et al., 20 I 0).
EfTects in OH A-Derived Lipid Mediators
DHA is the precursor of resol vins and protect ins. These compoun ds possess potent ant iinflammatory and neuroprotective propert ies (H ong et al., 2003; Marcheselli et al. 2003;
81
Serhan , 2005). For instance, it has been demonstrated a key neuroprotective role of
neuroprotect in D I (NPD I) in brain isch emia-reperfusion (Bazan, 2005; Hong et al., 2003) .
Effect on Neu rite Growth
it has been shown that DH A modifi es neuron size (Ahmad et al., 2002) aND promotes
neurite growth ( lkemoto et al., 1997; lvlartin, 1998; Ca lderon and Kim, 2004) .
Antiap o ptot ic. Effect
DHA is also able to prot ect neurons from apoptot ic cell death (Salem et al 200 l ; Akbar et
al., 2005)
Synaptic Fu nctions
lt has been shown that changes in DHA membrane composi t ion aiTect neurotransmiller
release by altering membrane fluidity (lVIcGahon et al., 1999 ).
Effect s on Glucose as Energy Source
DHA deficiency induces funct ional a lterations in glucose transporters causing changes in
brain energy metabolism (Ximenes da Sil va et al., 2002; Pi fferi et al., 2005).
1.3. Effects on Gene Activity
Many studies have demonstrated that DHA regu lates the express ion of di fTereni genes in
the brain (De Urquiza et al 2000, Rojas et al 2002, Kitaj ka et al 2002, Barcelo-Coblij n et al
2003a, b; Puskas et al 2003; Ki taj ka et al., 2004). DHA increment afler fish oil and LNA
supp lementat ion has been shown to modify th e expression of more than I 00 genes (approx.
equal number over- and under-expressed). M icroarray anal yses revealed that affected genes
include those related to cytoskeleton, synaptic plast icity, signal transduction, ion channel ,
energy metabolism and membrane trafticking (Kitaj ka et al., 2002; Puskas et al., 2003 ).
2. El' A in the Brain

2.1. Effects at Membrane Level
Alteration of Membrane Biophysical P ro perties
Compared w ith I 0-20% of DHA, EPA on ly comprises 0. 1 % of total brain fatty acids
(lvlcNamara et al., 2006). The spat ial con format ion of DHA is di fferent from that ofEPA as a
resu lt of its carbon backbone length and degree of unsaturat ion. EPA is a long-chain PUFA
that has 20 carbon atoms and 5 doub le bonds (20:5). However, DHA has a longer chain, 22
carbon atoms, and 6 double bonds (22:6). EPA possesses a relat ively lower effect than DHA
on membrane structure o rgan ization (Gorjao et al., 2009) .
82
Carlos Horacio Lai no, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
On the other hand, studies in planar lipid bilayer preparat ions demonstrated di llerent effects
of E PA and DHA on proton conductance and pem1eability. Indeed, EPA and DHA increase
proton conductance but the extent of the increment is di~ferent (60% versus I 00%,
respectively). it has been postulated that the different unsaturat ion degree of DHA and E PA
molecules could impact di fferential ly on membrane organization, and, thus result ing in di fferent
proton permeabi lily through proton passive pathways (Haines, 200 I; Decoursey, 2003).
Ion Channel Mod ulat ion
Studies have demonstrated that E PA possesses a lower inhibitory effect than DHA on
sodium and calcium currents (Vreugdenhil et al., 1996).
Effects on Lipid Rafts
EPA, as well as DHA, is able to remodel the lipid rafis by decreasi ng the cholesterol
amount in these microdomains (Fan et al., 2003; Ma et al., 2004 ).
Effects on Synaptic Vesicle Fusio n
Neurotransmitters are released fr om synaptic terminals via exocytosis afier synapt ic
vesicle fusion w ith the plasma membrane. EPA plays an important role in th is process by
increasing fusion phenomena (Ong et al., 2006).
Effects o n Neuronal Memb rane Excitability
Studies have shown that EPA and DHA di splay di fferent effects on neuronal membrane
excitability under control and drug-stimulated condit ions in di fferent hippocampal reg ions
(Xiao et al., 1999).
Effects on Cell Signaling Pathways
As menti oned above, changes in membrane lip ids modi fy enzyme act ivHy. Several
studi es have demo nstrated that EPA as DHA inhibits cA1viP-dependent protein kinase
pathways (Speizer et al., 199!).
2.2 E ffects at Cellula r Level
Antioxidant and Anti-Inflam mat ory Effects
EPA has shown simil ar effects to DHA on oxidative stress and inflammation induced by
cytokines and LPS (Kawash ima et al., 2009; Lynch et al., 2007; lvloon and Pestka, 2003).
EPA reduces the expression of tumor necrosis factor-a lpha, interleukin-6, nitric oxide
synth ase and upregu lates the heme oxygenase- I (Lu et al., 20 10).
EPA and DHA An tidepressant Effect: A lone or To gether?
83
EPA-Derived Proinflammatory Eicosanoids

The AA by act ion of the 5- li poxygenase (5-LOX) and cycloox ygenase (COX) is
transfo m1ed to eicosanoides, such as prostag landins, leukotrienes and thromboxanes, which
are si gnaling molecules that regu late a wide range of biolog ical act ions includi ng potent
effects on inilammat ion, vasodi latat ion, vasoconstr iction, apoptosis, and immune responses
(Tassoni et al., 2008).
EPA is a substrate for both 5-LOX and COX giving rise to 3-series prostaglandi ns,
thromboxanes, and 5 -ser ies leukotrienes. The EPA-derived eicosano ids are li pid mediators
with anti-inflammato ry effects while AA-derived eicosanoides have pro- inflammatory
effects . In contrast, DHA is not a substrate for COX ( Babcock et al., 2000; Lagarde, 2008).
EPA has an important role in balanci ng infl ammat ion by antagonizing membrane AA (an
n-6 PU FA) and reducing prostaglandin E2 synthesis (Farooqui et al., 2006).
No Neuroprotective Effect
Recent studies have demonstrated DH A neuroprotective effects, by reducing the toxicity
induced by A 1vfPA receptor in hippocampal membranes (Menard et al., 2009). This etTect is
not exerted by EPA.
2.3. Effects on Gene Activity

EPA and DHA differ in their effects on gene ex press ion. Several studies demonstrated
that EPA downregulates the en zymes involved in DH A synthes is from its precursor, LNA
(Poumes-Ball ihaut et al., 200 I; Langelier et al. , 2005).
Putative Mechanisms Involved in the Antidepressant Action

When analyzing the antidepressant mechanism of EPA and DH A, various mechanisms
can be suggested but none have been conclusive yet. Omega 3 fatty acids are responsible of
many physiological changes in the brain that can be related to their therapeutic effect. Fi rst,
al terat ions in membrane function seem to be a potent ial target (membrane fl u idity, receptor
function, neurotran smission, membrane-related enzyme and ion channel act ivity, glucose
transport and si gnal transduct ion). lvlood stabilization has been proposed to invol ve the AA
cascade and synthesis ofeicosanoids and to target inilammat ion and expression of many CNS
genes. EPA a lso increases oxygen and glucose supply to the brain (Katsumata et al., 1999)
and pro tects against oxidat ive stress (Young et al., 2005).
DHA and EPA can modulate neuronal function by modulat ing neurotransmiller
production such as serotonin and dopamine (du Bois et al., 2006, Femon et al., 2000).
Changes in Dopa minergic 1\eurotransm ission

DH A and EPA modulate brain function by chang ing dopaminergic and serotonergic
neurotransmission. Major depression is characteri zed by altered neurotransmission and
changes in production and function of neurotransmillers turn relevant, for example, the
84
downregulation of dopamine receptors is thought to play a role in the pathophysiology of

depression. The precise mechanisms li nk ing the supp ly of omega-3 PUFA and dopaminergic
system are probab ly complex and mult i factorial. Unbalanced supp ly (deficiency or excess) of
n-3 PUFA changed several aspects of the dopaminergic neurotransmission in the frontal
cortex of rats.
Chron ic deficiency of n- 3 PUF A strongly acts on dopaminergic neurotransmission in the
nucleus accumbens, affecting both dopamine (DA) metabolism and several molecu lar targets
of DA such as receptors and transporters (Z immer et al., 2000b). Chron ic n- 3 PUFA-defi cient
rats show higher basal levelsofDA and lower basal levels ofmetabolites (DOPAC, HVA) in
the shell part of the nucleus accumbens than animals receiving a dietary supp ly o fn- 3 PUFA
corresponding to need. lvloreover, the vesicular storage pool of DA was markedl y decreased,
whereas the cytoplasmic pool was unchanged. Different studies on the vesicu lar monoamine
transporter (VMA T2) reinforce th is hypothesis, as the number of transporters was found
decreased both in the frontal cortex and accumbens nucleus of deficient rats (Z immer et al.,
2000a).
Modulation of Gene Express ion

DH A can also influence gene express ion in the brain. Omega-3 PUFA are able to modi fy
gene expression in di fferent brain areas (Grimaldi et al., 200 I, Jump et al., 2002, Lapillonne
et al., 2004). it has been shown that omega-3 fatty acids modulate transcription of several
genes involved not on ly in li pid metabolism but also in th e response to oxidat ive stress, the
antiox idant capacity, cell growth, proli feration, apoptosis and signali ng. The fact that many of
the omega-3 effects are speci fi e of brain t issue (Kitaj ka et al., 2004) supports its part ic ipat ion
in the neurocogn it ive and behavioral effects o bserved in hu mans.
Brain-Derived l\eurotro phic Factor (BDI\ F)

Several pieces of evidence suggest a crucial role for BDN F in the pathophysiology of
depression (Shimizu et al., 2003; Hash imoto et al., 2004). Serum levels ofBDN F have been
fhund reduced in depressed patients and negatively correlated to the length of the disease
(Shimizu et al., 2003). Ant idepressant medi cation improves BDN F signaling by increasing the
activity of transcript ion factor CREB (cA!viP response element-binding) through aden ilate
cyclase. Omega-3 fatty acids have been proposed to act via a s imilar mechan ism that involves
BDN F action. Indeed, in a rat model of depression, DH A deprivation decreases BDNF, CREB
and p38 MA PK (Mitogen-act ivated protein) levels in the prefrontal con ex and the addition of
DH A to cultured rat cort ical astrocytes induced BDNF protein expression (Rao et al., 2007).
This data suggests that reversing BDNF/ CREB alterat ions may be involved in the
beneficial effect of omega-3 fatty acids, as well as in the increased neurogenesis descr ibed
'"ith these compoun ds (Kawak ita et al., 2006, Beltz et al., 2007, Dyall 20 I 0).
85
Targeting AA Cascade and Eicosanoid Synthesis

There is another possible ant idepressant mechanism on which focus must be made. EPA
and DHA may have therapeut ics effects on mood stabilization by target ing the A.A. cascade
(Farooqui et al., 2006 and 2007). Arachidonic acid, EPA and DHA are all released from
neuronal membrane phospholipids by members o fphosfoli pase A2 famil y. Arachidonic acid
is re leased by act ion of cPLA2 (Farooqui et al., 2000), and EPA and DHA are released by
act ion of PlsEtn-P LA2 (Plasmalogen -Selective Phospholi pase-A2) (Hirashima et al., 2003).
Arach idonic acid is metaboli zed to prostaglandi ns, leukotrienes, and thromboxanes by
cyclooxygenases and li poxygenases. These molecules have proinflammatory act ions and
infl uence mult iple physio logical and pathological mechanisms in the organism. The
proinflammatory eicosanoids prostaglandin E2 (PG E2) and leukotriene 84 (LT B4) are
derived from AA, whereas anti-inflammatory leukotrienes, prost aglandin D2, prostaglandin
El and prostaglandi ns are der ived from EPA and DHA. EPA competes w ith AA at the
cyclooxygenase level to produce the 3-series prostaglandins (PGE3, PG13, and TXA3) or the
5-series leukotrienes (LT B5, LTC5, and LT D5). These metabolites are less active than th e
corresponding AA-d erived compounds. DHA is not a substrate for cyclooxygen ase but
inhibits cyclooxygenase activity and is metaboli zed to docosanoids. Docosahexaenoi c acid
and its li pid mediators prevent neuroin nammation by inhibiting transcription factor
NFkappaB, preventing cytokine secret ion, blocking the synthesis o f prostaglandins,
leukotrienes, and thromboxanes, and modulating leukocyte traffi cking. They not only
antagonize the e~Yects of AA-derived metabolites but also act potent ly on leukocyte
tra fficking as well as downregu lating the expression of cytokines in glial cell s. T he generation
of docosanoids may be an internal protect ive mechanism to prevent brain damage.
Mod ulation of l nterleukine and i nterferon Production

Depression is character ized by an inmlUne-i nflammatory response (Maes et al., 1995)
\\ith increased cytokine production such as interleukin- 1 ( fL- 1) (Maes et al., 1993; Owen et
al ., 200 I), interleuk in-2 (IL-2) (Maes et al. 1993), interleukin-6 (IL-6) (Maes et al., 1995;
Frommberger, 199 7) and inter feron-g ( IN F-g) (Maes et al. 1994), and increased number of
solub le IL-2 (si L-2R) and l l -6 (sll-6R) receptors (lvlaes et al., 1995). Acute phases of
depression have been associated with I L-6 increases, whil e remi ssion is associated with
decreases (Frommberger et al., 199 7). Indeed, the severity o f depression has been reported to
correlate w ith ll-6 levels (Dentino et al., 1999). In accordance to these observations, IL-2 and
IN F-al pha therapies have been associated with suicidal au empts and successful suicide
(Baron et al., 1993; Janssen et al., 1994; Windemuth et al., 1999; Jonasch et al., 2000).
In th is context, it has been reported that omega-3 P UFA can suppress some of the
pathophysiological features of depression, such as infl ammat ion and immune react ivity
markers. Specifi cally, in vitro studies have shown that EPA and DHA suppress LL-6
production by human endothelial cell s (De Caterina et al., 1994; Khalfound et a l., 199 7). EPA
and DHA have been reported to suppress the in vitro production of ll-1, ll-2, IL-6, tumor
necrosis factor alpha (TN F-a) and IN F-g by human lymphocytes (Purasiri et al., 199 7).
Human studies have indicated that dietary supplementat ion w ith EPA and D HA results in
86
suppression of IL- l, IL-2, fL-6 and TNF-a product ion by monocytes (lvleydani et al., 199 1;
Calder, 1997; Kell ey et a l., 1997).
Therefore, modulat ion of cytok ines, inflammat ion, and ox idat ive stress makes EPA and
DH A neuroprotective compounds, and may explain the a ll eviat ion of depressive symptoms
consideri ng that depression is associated w ith excessive product ion of pro-innammatory
cytokines, including interleuk in (ll )- I beta, IL- 12, IL-6, IN F-g and TN F-rL
Omega-3 Neuronal and Synaptic Effects

N euronal and synapt ic remodeling has been shown in depressed patients and in animal
models of depression (Duman, 2002; Fuchs et al., 2004; Haj szan et al., 2009; lwata et al.,
2006; Reines et al., 2008). Antidepressants have proven beneficial in recoveri ng nom1al
morphology and functi on of neurons, part icularly in the hippocampus (Margar iiios et al. ,
1999; Sheli ne et al, 2000 y 2003; Reines et al., 2008; Haj szan et al., 2009). In th is regard,
omega-3 fatty acids have been reported to share some properties w ith ant idepressants.
DH A promotes neur ite growth and synaptogen esis, and increases glutamatergic syn apse
function in culture neurons (Cao et al., 2009). A lthough the exact mechanism is st ill
unknown, it can be speculated the part icipat ion of troph ic factors. For instance, hippocampal
nerve growth factor (NG F), known to maintain neuronal survival and stimulate
di fferentiation, has been shown to decrease after DH A-deficient di ets d uring pregnancy and
lactation ( lkemoto et al., 2000).
Final Remarks
Even though omega-3 PU FA are suggested to act as ant idepressants, we have provided
evidence indicating that EPA and DH A exert di stinct act ions and effects when adm inistered
indi vidua ll y or in combinat ion (fish oil ). Most reports show ing omega-3 effectiveness to treat
depressive symptoms are epidemiological and clinical studi es that employed omega-3enriched di ets either alone or combined with classical antidepressants. These diets consisted
of supp lementat ion w ith Cish oil that is pri ncipally composed by DH A and EPA. Individual
administrat ion of EPA has rendered ant idepressant eiTects in depressed patients. However, no
data is availab le about DH A to treat depressive symptoms. On the contrary, DH A has sbown
antidepressant act ion in experimental paradigms, either alone or in combinat ion w ith E PA
(as fish oil ).
it is worth noti cing that r eports from li terature point out DH A as th e key fatty acid in fish
oil supplementat ion. Despite th is, EPA seems to be the act ive compound in humans. Possibly,
DH A and EPA doses, length of treatment, class and severity of evaluated symptom are some
variables th at may contribute to these apparent discrepancies. On the other hand, EPA and
DH A di splay different molecular e ~rects o n membrane pem1eabi lity and ion conductance,
di fterences attributed to th eir molecular structures. For instance, DHA carbon chain is longer
than EPA s and consequently, DHA strongly impacts on membrane organization. Therefore,
di st inctive pham1acological effects between EPA and DHA would not be surprising and new
experiments are needed to understand DHA and EPA ant idepressant actions. it should also be
considered that when EPA and DH A are combined w ith antidepressants, the overall ba lance
EPA and DHA Antidepressant Effect: A lone or Together?
87
in omega-3 fauy acid concentration and/ or invo lved mechan isms could vary as a
consequence of the medi cation.
References
Adams, P.B., Lawson, S., Sanigorski, A., S inclair, A.J. Arach idon ic acid to e icosapentaenoic
ac id rat io in blood correlates posit ively with cli nical symptoms of depression. Lipids.
1996; 3 1: S l 57-6 1.
Ahmad, A., Moriguch i, T., Salem, N. Decrease in neuron size in docosahexaenoic acidde6cient brain. Pediatr. Neurol. 2002; 26: 210-8.
Aid, S., Vancassel, S., Poumes-Balli haut, C., Cha lon, S., Guesnet, P., Lavialle, M. Effect of a
di et-induced n-3 PU FA deplet ion on cholinergic parameters in the rat hippocampus. ./.
Lipid Res. 2003; 44: 1545-5 1.
Akbar, M., Calderon, F., Wen, Z., Kim, H.Y . Docosahexaenoic acid: a posit ive modulator of
Akt signaling in neuronal survival. Proc. Natl. A cad. Sci. US A. 2005; 102: 10858-63.
Appleton, K.M., Rogers, P.J., Ness, A.R. Updated systematic review and meta-analysis of the
effects of n-3 long-chain po lyunsaturated fatty acids on depressed mood. Am. .!. Clin.
Nutr.20 JO; 9 1:757-70.
Babcock, T., Helton, W.S., Espat, N.J. Eicosapentaenoic acid (E PA): an anti -innammatory
omega-3 fat. Nutrition. 2000; 16: 1116-8.
Barcei6-Coblij n, G., Kitaj ka, K., Pusk<1s, L.G., Hogyes, E., Zvara, A., Hackler, L. Jr., Farkas,
T. Gene expression and molecular composit ion of phospholipids in rat brain in relation to
dietary n-6 to n-3 fatty acid ratio. Biochim. Biophys. Acta. 2003a; 1632: 72-9.
Barcei6-Coblij n, G., Hogyes, E., Kitajka, K., Puskas, L.G ., Zvara, A., Hack ler, L. Jr., Nyakas,
C., Penke, Z., Farkas, T. lvlodificat ion by docosahexaenoic acid of age-induced
al terat ions in gene expression and mo lecular composit ion of rat brain phospholi pids.
Proc. Natl. A cad. Sci. U S A. 2003b; 100: 11 32 1-6.
Baron, D.A., Hardje, T., Baron, S.H. Possible associat ion of inter leukin-2 treatment w ith
depression and su icide. .f. Am. Osteopath. Assoc. 1993; 93: 799- 800.
Bazan, N.G. Neuroprotectin D1 (NPD I): a DHA-derived mediator that protects brain and
ret ina against cell injury-i nduced ox idat ive stress. Brain Pathol. 2005; 15: 159-66.
Beltz, B.S., T lusty, lvi. F., Benton, J. L., Sandeman, D.C. Omega-3 fatty acids upregu late adult
neurogenesis. Neurosci. Lett. 2007; 4 15: 154-8.
Berry, C.B., McBean, G .J. An invest igation into the role of calcium in the modulat ion of rat
synaptosomal o -f H]aspartate transport by docosahexaeno ic acid . Brain Res. 2003; 973:
107- 11 4.
Berry, C.B., Hayes, D., lvlurphy, A., Wiessner, M., Rauen, T., McBean, G.J. Differential
modulation of the glutamate transporters GLT I, G LAST and EAAC I by
docosahexaenoic acid. Brain Res . 2005; 1037: 123- 133.
Bou rre, J.lvl., Youyou, A., Dumont, 0 ., Piciott i, M., Pascal, G., Durand, G. The effects of
di etary a-linolenic acid on the composition of nerve membranes, enzymat ic activity,
amp litude of electrophysiologica l parameters, resistance to poisons and perfom1ance of
learning tasks in rats . .f. Nutr. 1989; 11 9: 1880- 92.
88
Carlos Horac io Lai no, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
Bowen, R.A., C landinin, lvi.T. D ietary low linolenic ac id compared w ith docosahexaenoic
ac id alter synaptic plasma membrane phospholi pid fatty acid composi ti on and sodi umpotassium ATPase kinet ics in developing rats . .f. Neurochem. 2002; 83: 764- 74.
Breckenr idge, W.C., Gombos, G., Morgan, I.G . The lipid composit ion of adu.lt rat brain
synaptosomal plasma membranes. Biochim. Biophys. Acta. 1972; 266: 695-707.
Brown, E. R,. Subbaiah, P.V. Ditferential effects of eicosapentaenoic acid and
docosahexaenoico acid on human sk in fibroblasts. Lipid.~. 1994; 29: 825- 29.
Burdge, G.C., Wootton, S.A. Conversion of alpha-linolen ic acid to eicosapentaenoic,
docosapentaeno ic and docosahexaenoic acids in young women. Br. .f. Nutr. 2002; 88:
4 11 -20.
Burdge, G .C ., Fi nnegan, Y.E., Minihane, A.M., Williams, C.M., Wootton, S.A. Effect of
al tered di etary n_3 fatty acid intake upon plasma lip id fatty acid composit ion, conversion
of ( 13C]alpha-linol en ic acid to longerchain fatly acids and partit ion ing towards betaoxidat ion in older men. Br. .f. Nutr. 2003; 90: 3 11- 21 .
Burdge, G.C., Jones, A.E., Wootton, S.A. Eicosapentaenoic and docosapentaenoico acids are
the principal products of alpha-linolenic acid metaboli sm in young men. Br. .f. Nutr,
2002; 88: 355- 64.
Ca lder, P.C., Yaqoob, P., Harvey, D.J., Waus, A., Newsholme, E.A. Incorporation of fatty
ac ids by concanavalin A-st imulated lymphocytes and the effect on fatty acid composit ion
an d membrane fluidity. Biochem . .!. 1994; 300: 509- 18.
Calder, P.C. n-3 po lyunsaturated fatly acids and cytokine production in health and disease.
Ann. Nutr. Metah. 1997; 4 1: 203- 34.
Calder, P.C., Yaqoob, P. Lipid rafis--composit ion, characterizat ion, and controversies. J.
Nutr. 2007; 137: 545-7.
Calderon, F., Kim, H.Y. Docosahexaenoic acid promotes neurite growth in hippocampal
neurons . .!. Neurochem. 2004; 90: 979-88.
Cao, D., Kevala, K., Kim, J., lvloon, H.S ., Jun, S.B., Lovinger, D., Kim, H.Y .
Docosahexaenoic acid promotes h ippocampal neuronal deve lopment and synapt ic
functi on . .!. Neurochem. 2009; Ill : 5 10-21.
Carlezon, W.A. Jr., Mague, S.D., Parow, A.M., Stoll , A.L., Cohen, B.M., Renshaw, P.F.
Antidepressant- li ke effects of uridine and omega-3 fatty acids are potent iated by
comb ined treatment in rats. Bioi. Psy chiatly. 2005; 57, 343-50.
Cbalon, S., Delion -Vancassel, S., Belzung, C., Guill oteau, D., Leguisquei, A.M., Besnard,
.I.C., Durand, G . Dietary fish oil affects monoaminergic neurotransmission and behavior
in rats . .f. Nutr. 1998; 128: 25 12- 9.
Chalon, S., Vancassel, S., Zi mmer, L., Gu ill oteau, D., Durand, G. Polyunsaturated fatly
acids and cerebral funct ion: focus on monoam inergic neurotransmissi on. Lipids. 200 I;
36: 937-44.
De Cater ina, R., Cybu lsky, M.l., C linton. S.K., G imbrone. M.A. Jr., Libby, P. The omega-3
fatly acid docosahexaenoate reduces cytok ine-induced expression of proatherogen ic an d
proinnammatory proteins in human endothelial cell s. Arterioscler Thromh. 1994; 14:
1829- 36.
Decoursey, T.E. Voltage-gated proton channels and other proton transfer pathways. Physiol.
Rev. 2003; 83:475-579.
89
Del ion, S., Chalon, S., Herault, J., Guilloteau, D., Besnard, J.C., Durand, G. Chronic dietary
al pha-linolen ic acid deficiency alters dopaminergic and serotoninergic neurotransmission
in rats . ./. Nutr. 1994; 124:2466-76.
Del ion, S., Chalon, S., Guilloteau, D., Besnard, J.C., Durand, G. alpha-Linolenic acid di etary
deficiency alters age-related changes of dopaminergic and serotoninergic
neurotransmission in the rat frontal cortex . .f. Neurochem. 1996; 66: ! 582-9 1.
Dent ino, A.N., Pieper, C.F., Rao, M.K., Currie, lvi.S., Harris, T., Blazer, D.G., Cohen H. J.
Association of interleuk in-6 and other biologic variables with depression in older peop le
living in the commun ity. .f. Am. Geriatr. Soc. 1999; 47: 6-11.
De Urquiza, A.lvl., Liu, S., Sj oberg, tvl., Zeuerstrom, R .H., Griffiths, W., Sjovall , J.,
Perlmann, T. Docosahexaenoic acid, a li gand for the retinoid X receptor in mouse brain.
Science. 2000; 290: 21 40-4.
Diaz, 0., Berquand, A., Dubois, M., Di Agostino, S., Sette, C., Bourgoin, S., Lagarde, M.,
Nemoz, G., Prigent, A. F. The mechan ism of docosahexaenoic acid-induced
phospholi pase D act ivat ion in human lymphocytes involves exclusion of the enzyme
from lipid rafts . .f. Bioi. Chem. 2002; 277: 39368-78.
du Bois, T.M., Deng, C., Bell, W., Huang, X .F. Fatty acids dilrerent ially affect serotonin
receptor and transporter b inding in the rat brain. Neuroscience. 2006; 139: 1397-403.
Duman, R.S . Pathophysiology of depression: the concept of synapt ic plast icity. Eur.
Psy chiatry. 2002; 17: 306- 10.
Dyall , S.C., Michael, G .J., Michaei -T itus, A.T. Omega-3 faily acids reverse age-related
decreases in nuclear receptors and increase neurogenesis in old rats . ./. Neurosci. Res.
2010;88: 209 1- 102.
Edwards, R., Peel, lvl., Shay, J., Horrobin, D. Omega-3 polyunsaturated !ally acid levels in
the di et and in red blood cell membranes of depressed patients. .f. Affe ct Disord. 1998;
48: 149-55.
Ehringer, W., Belcher, D., Wassail , S.R., Sti ll well , W. A comparison of the effects of
linolenic (18:3 omega 3} and docosahexaenoic (22:6 omega 3} acids on ph ospholipid
bilayers. Chem. Phys. Lipids. 1990; 54: 79-88.
Fan, Y.Y., McMurray, D.N., Ly, L H., Chapkin, R.S. Dietary (n-3} polyunsaturated !htty
acids remodel mouse T-cell lipid rafts . .f. Nutr. 2003; 133: 19 13-20.
Farooqui, A.A., Ong, W.Y., Horrocks, L A., Farooqui, T. Brain cytosolic phospholi pase A2:
localization, role, and invol vement in neurological diseases. Neuroscientist. 2000; 6:
169- 180.
Farooqui, A.A., Ong, W.Y., Horrocks, L A. Inh ibitors of brain phospho li pase A2 act ivity:
their neuropharmaco logical effects and therapeutic importance f(lr the treatment of
neuro logic disorders. Phannacoi. Rev. 2006; 58: 59 1- 620.
Farooqui, A.A., Horrocks, L.A., Farooqui, T. Modulat ion of inflammat ion in brain: a maller
of fat../. Neurochem. 2007; 10 1: 577-99.
Fenton, W.S., Hibh len, J., Knable, M. Essential futty acids, lipid membrane abnom1alities and
the diagnosis and treatment of schizophrenia. Bioi. Psychiatry. 2000; 4 7: 8- 21 .
Frangou, S., Lew is, lvl., McCrone, P. Efficacy of ethyleicosapemaeno ic acid in bipolar
depression: randomized doub le-blind placebo-controll ed study. Br. .f. Psy chiatry. 2006;
188: 46-50.
90
Frommberger, U.H., Bauer, J., Haselbauer, P., Fraulin, A., Riemann, D., Berger, M. lnterleuk in6-(LL-6) plasma levels in depression and schizophrenia:comparison between the acute state
and after remission. Eur. Arch. Psychiatry C/in. Neurosci. 1997; 247:228- 233.
Fuchs, E., Czeh, 8 ., Kole, M.H., M.ichaelis, T., Lucassen, P.J. Alterat ions of neurop lasticity
in depression: the hippocampus and beyond. Eur. Neuropsy chopharmacol. 2004; 14:
48 1-90.
Gorjao, R., Azevedo-tvlart ins, A.K ., Rodrigues, H.G ., Abdulkader, F., Arcisio-Miranda, l'vl.,
Procopio, J., Cur i, R. Comparative effects of DHA and EPA on cell funct ion. Phnrmacoi.
Ther. 2009; 122: 56-64.
Goyens, P.L., Sp ilker, M. E., Zock, P. L., Katan, M.B., Mensink, R.P. Compartmental
modeling to quanti fy alpha-lino lenic acid conversion atier longer tem1 intake of mult iple
tracer boluses . ./.Lipid Res. 200 5; 46: 1474-83.
Grimaldi , P.A. Fatty acid regulat ion of gene expression. Curr. Opin. C/in. Nutr. Metah. Care.
2001;4: 433- 37.
Haines, T.H. Do sterols reduce proton and sodium leaks through li pid bilayers? Prog. Lipid
Res. 200 1; 40:299-324.
Hajszan, T., Dow, A., Warner-Schmidt, J. L., Szigeti-Buck, K., Sall am, N .L., Parducz, A.
Remodelling of h ippocampal spine synapses in the rat learned helplessness model of
depression. Bioi. Psy chiatry. 2009; 65: 392-400.
Harroun, T.A ., Katsaras, .1., Wassail , S.R. Cholesterol hydroxyl group is found to reside in the
center of a polyunsaturated lipid membrane. Biochemistry. 2006; 45: 1227-33.
Hashimoto, K., Shimizu, E., lyo, M. Critical role of brain-derived neurotrophic factor in
mood disorders. Brain Res. Brain Res. Rev. 2004 May; 45(2): 104- 14.
Hibbeln, .I. R., Salem, N. Jr. Dietary pol yunsaturated fatty acids and depression: when
cholesterol does not sat isfy. Am . .f. Clin. Nutr. 1995; 62: 1-9.
Hirashima, Y ., Farooqui, A.A., lvlill s, J.S., Horrocks, L.A . Identificat ion and puri ficat ion of
calci um-independent phospholipase A2 from bovine brain cytosol. ./. Neurochem. 2003;
59: 708- 714.
Holte, L.L , Peter, S.A., S innwell , T.lVI., Gawrisch, K. 2H nuclear magnet ic resonance order
parameter profi les suggest a c.hange of mol ecular shape for phosphatidylcholi nes
containing a polyunsaturated acyl chain. Biophy s . .f. 1995; 68: 2396-403.
Hong, S., Gronert, K., Devchand, P.R., Moussignac, R.L., Serhan, C.N. Novel docosatrienes
and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood,
and gli al cells. Autacoids in anti -inflammat ion. ./. Bioi. Chem . 2003; 278: 14677- 87.
Huang, S.Y., Yang, H.T., Chiu, C.C., Par iante, C.M., Su, K.P. Omega-3 fatty ac ids on the
forced-swimming test../. Psy chiatr. Res. 2008; 42: 58-63.
Huster, D., A lbert, J.J., Amold, K., Gawrisch, K. Waterpem1eab ili ty of polyunsaturated lipid
membranes measured by 170 NlVfR. Biophys . .f. 1997; 73: 856- 864.
lkemoto, A., Kobayashi, T., Watanabe, S., Okuyama, H. Membrane fatly acid modi ficat ions
of PC 12 cell s by arachidonate or docosahexaenoate affect neurite outgrowth but not
norepinephrine release. Neurochem. Res. 1997; 22: 67 1-8.
lkemoto, A., N itta, A., Furukawa, S., Ohishi, lvl., Nakamura, A., Fuj ii , Y., Okuyama, H.
Di etary n-3 fatty acid deficiency decreases nerve growth factor content in rat
hippocampus. Neu rosci. Lett. 2000; 285: 99- 102.
Janssen, H.L., Brouwer, J.T., van der Mast, R.C., Schalm, S.W. Suicide associated with a!fainter feron therapy for chronic viral hepatit is . .! Hepatol. 1994; 2 1: 241- 3.
91
Jonasch, E., Kumar, U.N., Linelle, G.P., Hodi, F.S., Soiffer, R .J., Ryan, B.F., Sober, A.J.,
Mihm, M.C ., Tsao, H., Langley, R.G., Cosimi, B.A., Gadd, i'v!.A., Tanabe, K.K., Souba,
W., Haynes, H.A ., Bamh ill, R., Osteen, R., Haluska, F.G. Adj uvani high-dose interferon
alfa-2b in pat ients w ith high-risk melanoma. Cancer ./. Sci. Am. 2000; 6: 132- 134.
Jump, D.B. Dietary polyunsaturated fatty acids and regu lat ion of gene transcr ipt ion. Curr.
Opin. Lipidoi. 2002; 13(2): 155- 64.
Kamada, T., Yamashita, T ., Baba, Y., Kai, M., Setoyama, S., Chuman, Y., Otsuj i, S. Di etary
sardi ne oi I increases erythrocyte membrane fluidity in diabet ic patients. Diabetes. 1986;
35: 604-6 11.
Katsumata, T., Katayama, Y., Obo, R., Muramatsu, H., Ohtori, T., Terash i, A . Delayed
administrat ion of ethyl eicosapentate improves loca I cerebral b lood flow and metaboli sm
"~thou! atrect ing infarct volumes in the rat focal ischemic model. Eur. .f.
Pharmaco/.1 999; 372(2): 167- 74.
Kawaki ta, E., Hash imoto, M., Sh ido, 0. Docosahexaenoic acid promotes neurogenesis m
'itro and in vivo. Neuroscience. 2006; 1393:99 1- 7.
Kawash ima, A ., Harada, T., Kami, H., Yano, T., lmada, K., Mizuguch i, K. Effects of
eicosapentaenoic acid on synaptic plast ici ty, fatly acid profil e and phospho inositid e 3kinase signali ng in rat hippocampus and di fTerentiated PC I2 cell s . .J Nutr. Biochem.
2009;21 : 268-2 77.
Keck, P.E. Jr., lvl intz, J., lvlcE lroy, S.L., Freeman, M. P., Suppes, T., Frye, M.A., Altshuler,
L.L., Kupka, R., Nol en, W.A., Leverich, G.S., Denicoft~ K.D., Grunze, H., Duan, N.,
Post, R.M. Double-bli nd, randomized, placebo-controll ed trials of ethyl-eicosapentanoate
in the treatment of bipolar depress ion and rapid cycling bipolar disorder. Bioi. Psychiatry.
2006; 60: 1020-22 .
Kel ley, D.S., Taylor, P.C., Nelson, G .J., Schmid~ P.C., Ferretti, A., Er ickson, K.L., Yu, R.,
Chandra, R.K., /\lackey, B.E. Docosahexaenoic acid ingest ion inhibits natural kill er cel l
act ivity and product ion of inflammatory mediators in young healthy men. Lipid.~. 1999;
34: 3 17- 24.
Khal foun, B., Th ibault, F., Wat ier, H., Bar dos, P ., Lebranchu, Y. Docosahexaenoic and
eicosapentaenoic acids inh ibit in vitro human endothelial cell production of interleukin-6.
Adv. Exp. Med. Bioi. 1997; 400: 589- 97.
Kitaj ka, K., Puskas, L.G ., Zvara, A ., Hackler, L. Jr., Barcei6-Coblijn, G., Yeo, Y .K., Farkas,
T. The role of n-3 polyunsaturated fatly acids in brain: modulation of rat brain gene
expression by dietary n-3 fatty acids. Proc. Nati. A cad. Sci. US A . 2002; 99: 26 19-24.
Kitajka, K., S inclair, A..l., Weisinger, R.S., Weisinger, H.S., Mathai, M., Jayasooriya, A. P.,
Halver, .I. E., Pusk<ls, L.G. Effects of dietary omega-3 polyunsaturated tally acids on brai n
gene expression. Proc. Nati. A cad. Sci. U S A. 2004; 10 1: 1093 1-6.
Kodas, E., Page, G., Zi nm1er, L., Vancassel, S., Gui lloteau, D., Durand, G., Chalon, S. Neither
the density nor function of striatal dopamine transporters were influenced by chronic n-3
polyunsaturated fatly acid deficiency in rodents. Neurosci. Lett. 2002; 32: 95-9.
Kodas, E., Gali neau, L., Bodard, S., Vancassel, S., Gu ill oteau, D., Besnard, J.C., Cha lon, S.
Serotoninergic neurotransmission is affected by n-3 polyunsaturated fally acids in the rat.
.f. Neurochem. 2004; 89: 695-702.
Lagarde, M. Docosabexaenoic acid: Nutrient and precursor of bioact ive lipids. Eur. .f. Lipid
Sci. Technoi. 2008; 11 0: 673- 678.
92
Laino, C.H., Fonseca, C., Sterin-Speziale, N., Slobodian ik, N., Reines, A. Potentiation of
omega-3 fatty acid ant idepressant-like effects w ith low non-antidepressant doses of
fluoxetine and mirtazap ine. Eur. J. Pharmacol. 20 I 0; 648: 11 7-26.
Lakhwan i, L., Tongia, S.K., Pal , V.S., Agrawal, R.P., Nyat i, P., Phadnis, P. Omega-3 fatly
ac ids have antidepressant act ivity in forced sw imming test in Wistar rats. Acta. Pol.
Pharm. 2007; 64: 27 1-6.
Langeli er, B., A lessandri, J.M., Perruchot, M.H., Guesnet, P., Laviall e, l'vl. Changes of the
transcriptional and fatly acid profi les in response to n-3 fatly ac ids in S H-SY5Y
neurob lastoma cell s. Lipids. 2005; 40: 7 19- 728.
Lapillonne, A., C larke, S.D., Heird, W.C. Pol yunsaturated fatly acids and gene expression.
Curr. Opin. C/in. Nutr. Metab. Care. 2004; 7(2): 15 1-56.
Leaf, A., Xiao, Y. F., Kang, J.X. Interact ions ofn-3 fatty acids w ith ion channels in excitable
tissues. Prostaglandins Leukot. Essent. Fatty Acids. 2002; 67: 11 3-20.
Lesperance, F., Frasure-Smith, N., St-Andre, E., Tureck i, G., Lesperance, P., W isn iewski,
S.R. The efficacy of omega-3 supplementation for major depress ion: a randomized
controll ed tr ial. .f. C/in. Psychiatry. 20 11; 72: I 054-62.
Li n, P.Y., Su, K.P. A meta-analytic review of doub le-blind, placebo-controlled trials of
antidepressant ef1:1cacy of omega-3 fatly acids . .f. Clin. Psychiatry. 2007; 68: I 056-6 1.
Lu, D.Y., Tsao, Y.Y ., Leung, Y.M., Su, K.P. Docosahexaenoic acid suppresses
neurointlanm1atory responses and induces heme oxygenase- ! expression in BV-2
microglia: impli cations of ant idepressant e!Tects
for ro-3
fatty ac ids.
Neuropsychophannacology. 20 I 0; 35: 2238-48.
Lynch, A.M., Loane, D.J., l'vl inogue, A .M., C larke, R.lvl., Kil roy, D., Nall y, R.E., Roche,
O.J., O'Connel l, F., Lynch, l\1f.A. Eicosapentaenoic acid confers neuroprotection in the
amy loid-beta chall enged aged hippocampus. Neurobiol. Aging. 2007; 28: 845-55 .
Ma, D.W., Seo, J., Switzer, K.C., Fan, Y .Y., McMurray, D.N., Lupton, J.R., Chapkin, R.S. n3 PUFA and membrane microdomains: a new frontier in bioactive lipid research . .f. Nutr.
Biochem. 2004; 15: 700 -6.
Maes, M., Bosmans, E., Meltzer, H.Y., Scharpe, S., Suy, E. lnterleuk in-1 beta: a putat ive
mediator ofH PA ax is hyperact ivity in major depression? Am . .f. Psychiatry. 1993; 150:
11 89- 93.
Maes, M., Vandoolaeghe, E., Ranjan, R., Bosmans, E., Bergmans, R., Desnyder, R. Increased
serum interleuk in-1-receptor-antagon ist concentrations in major depression . .f. Affect.
Disord. 1995; 36: 29- 36.
Maes, M., Cbristophe, A., Delanghe, .1., Altamura, C., Nee ls, H., Me ltzer, H.Y. Lowered
omega3 po lyunsaturated fatty acids in serum phospholipids and cholesteryl esters of
depressed pat ients. Psych in fly Res. 1999; 85: 275-9 1.
Magarii\os, A.M., Deslandes, A., McEwen, B.S. Effects o f ant idepressant and benzodiazepine
treatments on the dendrit ic structure of CA3 pyramidal neurons after chron ic stress. Eur .
.f. Pharmacol. 1999; 3 71: 11 3- 22.
Marangell , L.B., Mart inez, J.M., Zboyan, H.A., Kertz, 8 ., Kim, H. F., Puryear, L.J. A doub leblind, placebo-controll ed study of the omega-3 fatly acid docosahexaeno ic acid in the
treatment of major depression. Am . .f. Psy chiatry. 2003; 160: 996-8.
Marcheselli, V. L., Hong, S., Luk iw, W.J., T ian, X.H., Gronert, K., Musto, A., Hardy . M.,
Gimenez, J.M., Chiang, N., Serhan, C.N., Bazan. N.G. Novel docosanoids inh ibit brain
93
ischemia-reperfusion-mediated leukocyte infi ltrat ion and pro-inflammatory gene

expression . ./. Bioi. Chem. 2003; 278: 43807- 17.
Marrink, S.J., de Vries, A.H., Harroun, T.A., Katsaras, J., Wassail, S.R. Cholestero l shows
preference for the interior of polyunsaturated li pid membranes . ./. Am. Chem. Soc. 2008;
130: I 0- 1.
lvlartin, R.E. Docosahexaenoic acid decreases phospholipase A2 activity in the neurites!nerve
growth cones ofPC I2 cell s . ./. Neurosci. Res. 1998; 54:805-13.
McGahon, B.M., lvlartin, D.S.D., Horrobin, D.F., Lynch, M.A. Age-related changes in
synaptic function: Analysis of the effect of dietary supplementat ion w ith omega-3 fatty
ac id . Neurocsience. 1999; 94: 305- 3 14.
i'vlcNamara, R.K., Carlson, S.E. Role of omega-3 fatty acids in brain development and
function: potent ial impli cat ions for the pathogenesis and preventi on of psychopathology.
Prostaglandins Leukot. Essent. Fatty Acids. 2006; 75: 329- 349.
Menard, C., Patenaude, C., Gagne, A.lvl., Massicotte, G. A1v1PA receptor-mediated cell death
is reduced by docosahexaenoi c acid but not by eicosapentaenoic acid in area CA I of
hippocampal slice cu ltures . ./. Neurosci. Res. 2009; 87: 876-86.
Meydan i, S.N., Endres, S., Woods, M.M., Goldin, B.R., Soo, C., Morriii-Labrode, A.,
Dinarello, C.A., Gorbach, S.L. Oral (n-3) fatty ac id supp lementat ion suppresses cytokine
production and lymphocyte proli ferat ion: comparison between young and older women .
.f. Nutr. 199 1; 121: 547- 55.
lvlitchell, D.C., Litman, B.J. Molecular order and dynamics in bil ayers consist ing of high ly
polyunsaturated phospholipids. Biophys. J. 1998; i4: 879- 9 1.
1vlitchell , D.C., N iu, S. L., Li tman, B .J. DHA-rich phosphol ipids optimize G-Prote in-coupled
s ignaling . ./. Pediatr. 2003; 14: 80-6.
Moon, Y ., Pestka, J.J. Deoxyn ivalenol-i nduced mitogen-act ivated protein kinase
phosphorylat ion and IL -6 expression in mice suppressed by fish oi l. ./. Nutr. Biochem.
2003; 14: 717- 26.
Murray, C .J., Lopez, A.D. Alternat ive project ions of mortali ty and disability by cause 19902020: Globa l Burden of Disease Study. Lancet. 1997; 349: 1498-504.
Nemets, B., Stahl, Z., Belmaker, R.H. Addi ti on of omega-3 fatty acid to maintenance
medication treatment for recurrent unipolar depressive disorder. Am . .f. Psychiatry. 2002;
159: 477-9.
Nemets, H., Nemets, B., Apter, A., Bracha, Z., Belmaker, R.H. Omega-3 treatment of
childhood depression: a controlled, double-b.lind pilot study. Am . .f. Psy chiatry. 2006;
163: I 098- l 00.
Noaghiul, S., Hibbeln, J. R. Cross-national comparisons of seafood consumpt ion and rates of
bipolar disorders. Am . .f. Psychiafly. 2003; 160: 2222-27.
Nouvelot, A., Delbart, C., Bourre, J.M. Hepat ic metaboli sm of di etary al pha-linolen ic acid in
suck li ng rats, and its possible importance in pol yunsaturated fatly acid uptake hy the
brain. Ann. Nutr. Metab. 1986; 30: 3 16-23.
Ong, L.W., Jiang, B., Tang, N., Yeo, J.F., Wei, S., Farooqui, A.A., Ong, W.Y. Di fferent ial
effects of polyunsaturated fatty acids on exocytosis in rat pheochromocytoma- 12 cell s.
Neurochem. Res. 2006; 3 1: 4 1- 8.
Osher, Y., Bersudsky, Y., Belmaker, R.H. Omega-3 eicosapentaenoic acid in bipo lar
depression: report o f a small open-label study. ./. Clin. Psy chiatry. 2005; 66: 726-9.
94
Owen, B.M., Eccleston, D., Ferrier, I. N., Young, A.H . Raised levels of plasma interleuk inlbeta in maj or and postviral depression .Acta. Psychiatr. Scan d. 2001; 103: 226- 228.
Paw losky, R.J., Ward, G., Salem, N. Jr. Essentia l fatty acid uptake and metabolism in th e
developing rodent brain. Lipid.~. 1996; 3 1: 103-7.
Pawlosky, R.J., Hibbeln, .J. R., Lin, Y ., Goodson, S., Riggs, P., Sebring, N., Brown, G .L.,
Sal em, N. Jr. Effects of beef- and fi sh-based diets on the kinet ics of n-3 fatly acid
metabolism in human subjects. Am . .f. Clin. Nutr. 2003; 7i: 565- 72.
Peel, M., lvlurphy, B., Shay, .1., Horrob in, D. Dep letion of omega-3 fatly acid levels in red
blood cell membranes of depressive pat ients . Bioi. Psychiatry. 1998; 43: 3 15-9.
Peet, M., Horrob in, D.F. A dose-ranging study of the efTects of ethyl-eicosapentaenoate in
patients w ith ongoing depression desp ite apparent ly adequate treatment w ith standard
drugs. Arch. Gen . Psy chiatry. 2002; 59: 9 13-9.
Peet, M., Keck, P.E. Jr, lvlarangell , L.B., Richardson, A.J., Lake, J., Stoll , A. L. Omega-3 fatly
ac ids: evidence basis for treatment and future research in psychiatry . .J C/in. Psychiatry.
2006; 67: 1954-67.
Pi fTeri, F., Roux, F., Langelier, B., A lessandr i, .Uvl., Vancassel, S., Jouin, !vi., Laviall e, lvl.,
Guesnet, P.(n-3) polyunsaturated fatty acid de ficiency reduces the expression of both
isofom1s of the brain glucose t.ansporter GLUT ! in rats . .J Nutr. 2005; 135: 224 1-6.
Pi tman, lvi.C., Suits, F., lvlackerell , A .D., Fell er, S.E. Molecular-level organ izat ion of
saturated and polyunsaturated fatty acids in a phosphatidylchol ine bilayer containing
cholestero l. Biochemistry. 2004; 43: 153 18-28.
Poumes-Ballihaut, C., Langeli er, B., Hau lier, F., A lessandri, J.M ., Durand, G., Latge, C.,
Guesnet, P. Comparat ive bioavailability of di etary al pha-linolen ic and docosahexaenoic
ac ids in the grow ing rat. Lipid.~. 200 I; 36: 793.
Purasiri , P., i'vlckechn ie, A ., Heys, S.D., Eremin, 0. i'vlodulat ion in vitro of human natural
cytotoxicity, lymphocyte proli ferat ive response to mitogens and cytokine product ion by
essenti al fatty acids. hnmunology. 199 7; 92: 166-72.
Puri, B.K., Coun sell, S.J., Hami lton, G., Richardson, A.J., Horrob in, D.F. Ei cosapentaenoic
ac id in treatment-resistant depress ion associated w ith symptom remission, structural brai n
changes and reduced neuronal phospholipid turnover. hu. .f. Clin . Pract. 2001; 55: 560-3.
Puskas, L.G., Kitajka, K ., Nyakas, C., Barcelo-Coblijn, G., Farkas, T. Short-term
administrat ion of omega 3 fatly acids from fi sh oi l results in increased transthyretin
transcription in old rat hippocampus. Proc. .11/at/. A cad. Sci. US A. 2003; I 00: 1580-5.
Reines, A., Cereseto, M., Ferrero, A., Sifon ios, L., Podesta, lvi.F, Wikinski, S. lvlaintenance
treatment w ith fl uoxet ine is necessary to sustain nom1al levels ofsynapti c markers in an
experimental model of depression: correlat ion w ith behavioral response.
Neuropsychopharmacology. 2008; 33: 1896-908.
Rao, J.S., Ert ley, R.N., Delvl ar, .I.C. Jr., Rapoport, S.l., Bazi net, R.P., Lee, H.J. Dietary n-3
PUFA depri vat ion alters expression of enzymes of the arach idonic and docosahexaenoic
ac id cascades in rat frontal cortex. Mol. Psychiatry. 2007; 12: 15 1-7.
Rapoport, S.l., lgarashi, M., Gao, F. Quant itative contribut ions of diet and liver synthesis to
docosahexaenoi c acid homeostasis. Prostaglandins Leukot. Essent. Fatty Acids. 20 I 0; 82:
273-6.
Richardson, A.J. n-3 Fatty acids and mood: the devil is in the detai l. Br. .f. Nutr. 2008; 99:
221 -3.
95
Rojas, C.V ., Greiner, R.S., Martinez, J. l., Sa lem, N ., Jr., Uauy, R. Long-term n-3 fatty acid
deficiency modi fies peroxisome prol iferator-activated receptor beta mRNA abundance in
rat ocular t issues. Lipids. 2002; 37: 367-3 74.
Salem, N. Jr., N iebylski, C.D. The nervous system has an absolute molecular species
requ irement for proper funct ion. Mol. Membr. Bioi. 1995; 12: 13 1-4.
Salem, N. Jr., Litman, B., Kim, H.Y., Gawrisch, K. Mechan isms of act ion of
docosahexaenoic acid in the nervous system . Lipid.~. 200 I; 36: 945-59.
Sarris, J., lv!ischou lon, D., Schweitzer, I. Adjunct ive nutraceuticals w ith standard
pharmacotherapies in bipolar disorder: a systemat ic review of clinical trials. Bipolar
Disord. 201 1; ( 3: 454-465.
Sarris, J., Mischoulon, D., Schweitzer, I. Omega-3 for bipolar disorder: meta-analyses of use
in man ia and bipolar depression . .1. C/in. Psychiatry. 2012; 73: 8 1-6.
Scott, B.L., Bazan, N .G. Membrane docosahexaenoate is supplied to the developing bra in and
retina by the liver. Proc. Natl. A cad. Sci. US A. 1989; 86: 290 3-7.
Seebungkert, B., Lynch, J. W. E fl'ects of polyunsaturated fatty acids on voltage-gated K + and
Na+ channels in rat olfactory receptor neurons. Eur. ./. Neurosci. 2002; 16: 2085-94.
Serhan, C.N. Novel w-3-derived local mediato rs in anti-i nnammat ion and resolut io n.
Pharmacol. Ther. 2005; I 05: 7- 2 1.
Shaikh, S.R., Dumaual, A.C., LoCassio, D., S iddiqui, R.A., Sti ll well , W. Acyl chain
unsaturation in PEs modulates phase separation from lipid raft molecules. Biochem.
Biophys. Res. Commun . 2003; 3 11 : 793- 6.
Shaikh, S.R., Dumaual, A.C., Castillo, A., LoCascio, D ., S iddiqui, R .A., Stillwe ll, W.,
Wassail , S.R. Oleic and docosahexaenoic acid differentiall y phase separate fr om lipid ra il
molecu les: a comparat ive NM R, DSC, A FM, and detergent extract ion study. Biophys. .f.
2004; 87: 1752- 66.
Shaikh, S.R., Cherezov, V., Callrey, M., Soni, S.P., LoCascio, D., Still well, W., Wassail,
S.R. Molecu lar organization of chol esterol in unsaturated phospbatidylethanolami nes: Xray di nract ion and solid state 2H NM R reveal di fferences with phosphatidylcholines . .f.
Am. Chem. Soc. 2006; 128: 53 75-83.
Shaldubina, A., Nemets, B., Bersudsky, Y. Lack of effect of eicosapentaenoic acid in the
Porsolt forced sw imming test model of depression. Acta. Neumpsychiatrica. 2002; 14:
203- 20.
Sheline, Y., Sanghavi, M., Mintun, M.A., Gado M.H. lv!RI studies of neuroanatomic changes
in un ipo lar major depression: the role of stress an d medical comorb idity. Bioi.
Psy chiatry. 2000; 48: 79 1-800.
Sheli ne, Y. l., Gado, M.H., Kraemer, H.C. Untreated depression and hippocampal vol umeloss.
Am . .f. Psychiatry. 2003; 160: 15 16-8.
Sheaft'Greiner, R.C., Zhang, Q., Goodman, K.J., Gi ussan i, D.A., Nathan ielsz, P.W., Brenna,
J.T. Linoleate, alpba-li nolenate, and docosahexaenoate recycling into saturated and
monounsaturated fatly acids is a major pathway in pregnant or lactat ing adu lts and fetal
or infant rhesus monkeys. Lipid Res. 1996; 37: 2675-86.
Shim izu, E., Hash imoto, K., Okamura, N., Koike, K., Komatsu, N., Kumakiri, C., Nakazato,
M., Watanabe, H., Shinoda, N., Okada, S., .lyo, M. Alterat ions of serum levels of
brainderived neurotrophic factor (B DN F) in depressed patients w ith or w ithout
antidepressants. Bioi. Psychiatry. 2003; 54: 70-5.
96
Silvers, K.M., Scoll, K.lvl. Fish consumption and self reported physical and mental health
status. Puhlic Health Nutr. 2002; 5: 427-3 1.
Sil vers, K.lvl., Woolley, C.C., Hamilton, F.C., Watts, P.M., Watson, R.A. Randomized
doub le-blind placebo-contro lled trial of fish oil in the treatment of depression.
Prostaglandins Leukot. Essent. Fatty Acids. 2005; 72: 211 -8.
Simopou los, A .P. Essent ial fatty acids in health and chron ic diseases. Forum Nutr. 2003; 56:
67- 70.
Si nclair, A ..I. Incorporation of radioactive po lyunsaturated fatty acids into li ver and brain of
developing rat. Lipid.~. 1975;10:175-84.
Sobajima, T., Tamiya-Koizumi, K., Ishihara, H., Kojima, K. Efrects of fatty acid modi fication
of ascites tu m or cells on pulmonary metastasis in rat. Jpn . .f. Cancer Res. !986; 77: 657- 63.
Speizer, L.A., Watson, M.J., Brunton, L.L. Di fferent ial effects of omega-3 fish oils on protein
kinase activit ies in vitro. Am . .!. Physiol. 199 1; 26 ! : 109- 11 4 .
Sprecher, H. Metaboli sm of high ly un saturated n-3 and n-6 fatty acids. Biochim. Biophys.
Acta. 2000; 1486:2 19-3 1.
Still well , W., Wassail. S.R. Docosahexaenoic acid: membrane propert ies of a un ique fatty
ac id. Chem. Phys. Lipids. 2003; 126: 1- 27.
Stoll, A. L., Severu s, W.E., Freeman, M.P., Rueter, S., Zboyan, H.A., Diamond, E., Cress,
K.K., Marangell, L.B. Omega 3 faHy acids in bipolar disorder. a preliminary doub leblind, placebo-controlled tri al. Arch. Gen. Psychiatry. 1999; 56: 407- 12.
Straume, M., Litman, B.J. Equi librium and dynamic structure of large, un ilamell ar,
unsaturated acyl chain phosphatidylcholi ne vesicles. Higher order analysis of 1,6diphenyl- I ,3,5-hexatriene and 1-[ 4-(trimethylammonio) pbenyl]-6-phenyl-1,3,5hexatriene an isotropy decay. Biochemistry. 1987; 26: 5 11 3-20.
Stubbs, C.D. The structure and funct ion of docosahexaenoic acid in membranes. ln: Sinclair
A, Gibson R (Eds), Essential Fatty Acids and Eicosanoids, AOCS, Champaign, Illinois,
1992 , p 11 6- 121.
Su, H.lvl., Bemardo, L., Mirmiran, M., Ma, X.H ., Corso, T.N ., Nathanielsz, P.W., Brenna,
J.T. Bioequivalence of dietary alpha-lino len ic and docosahexaenoic acids as sources of
docosahexaenoate accretion in brain and associated organs of neonatal baboons. Pediatr.
Res. 1999; 45:87-93.
Su, H.lvl., Huang, M.C., Saad, N.lvl., Nathan ielsz, P.W., Brenna, J.T. Fetal baboons convert
18:3n-3 to 22:6n-3 in vivo. A stable isotope tracer study . .f. Lipid Res. 2001;42:58 1-6.
Su, K.P., Huang, S.Y., Chiu, C.C., Shen, W.W. Omega-3 fally acids in major depressive
disorder.
A
pre li minary
double-blind,
placebo-controlled
trial.
Eur.
Neuropsychopharmacol. 2003; 13: 267-71.
SubleHe, rvi.E ., E ll is, S.P ., Geant, A. L., Mann, J.J. Meta-analysis of the effects of
eicosapentaenoic acid (E PA) in clinical trials in depression . ./. Clin. Psychiatry. 2011 ; 72:
1577 -84.
Tanskanen, A., Hibbeln, J. R., Hintikka, J., Haatainen, K., Honkalampi, K., Viinamaki , H.
Fish consumpt ion, depression, and suicidality in a general popu lat ion. Arch. Gen.
Psy chiatry. 200 I; 58: 5 12-3.
Tasson i, D., Kaur, G., Weisinger, R.S., Sinclair, A.J. The role of eicosanoids in the brain.
Asia Pac. .f. C/in. Nutr. 2008; 17: 220-8.
Teague, W.E., Full er, N.L., Rand, R.P., Gawrisch, K. 2002. Pol yunsaturated li pids in
membrane fusion events. Cell Mol. Bioi. Lett. 2002; 7: 262-4.
97
Vaidyanathan, V .V., Rao, K.V., Sastry, P.S. Regulat ion of diacylglycerol kinase in rat brain
membranes by docosahexaenoic acid. Neurosci. Lett. 1994; 179: 171-4.
Venna, V .R., Deplanque, D., A llet, C., Belarb~ K., Hamdane, M., Bordet, R. PU FA induce
antidepressant-li ke effects in parall el to structural and mol ecular changes in the
hippocampus. Psychoneuroendocrinology. 2009; 34: 199-2 11.
VemlUnt, S.H., l\1fensink, R.P., Simonis, M.M ., Homstra, G. Effects of dietary alpha-l inolen ic
acid on the conversion and oxidation of 13C-alpha- linolen ic acid. Lipids . 2000; 35: 13 7-42.
Vreugdenhil, M., Bruehl, C., Voskuyl, R .A., Kang, J.X ., Leaf, A., Wadman, W..l.
Polyunsaturated fatty acids modulate sod ium and calcium currents in CA I neurons. Proc.
Not/. A cad. Sci. U S A. 1996; 93: 12559- 63.
Wall , R., Ross, R.P., Fitzgerald, G .F., Stanton, C. Fatly acids from fish: the anti-inflammatory
potential of long-ch ain omega-3 fatly acids . Nutr. Rev. 2010; 68: 280 -9.
Wang, X ., Zhao, X ., Mao, Z.Y., Wang, X .M., Liu, Z.L. Neuroprotective effect of
docosahexaenoic acid on glutamate-induced cytotoxicity in rat hippocampal cultures.
Neuro repon. 2003; 14: 2457-6 1.
Wassail , S.R., Brzustowicz, M.R., Shaikh, S.R., Cherezov, V., Caffrey, M., Stillwell , W.
Order from disorder, corralling chol esterol with chaotic lipids - the role of
pol yunsaturated lipids in membrane raft fbm1at ion. Chem. Phys. Lipid.~. 2004; 132:
79- 88.
Willi ard, D .E., Harmon, S.D., Kaduce, T.L., Preuss, M., Moore, S.A., Robb ins, M.E.,
Spector, A .A. Docosahexaenoic acid synthesis from n-3 polyun saturated fatty acids in
di fferent iated rat brain astrocytes . ./.Lipid. Res. 200 !; 42: 1368-76.
Windemuth, D., Bacharach-Buhles, M., Hoffmann, K., A ltmeyer, P. Depression and suicidal
intentions as a side effect of high dosage interferon-alpha therapy- two cases. Hautarzt.
1999; 50: 266-9.
Wood, J.N. Essential fatly acids and their metabolites in signal transduction. Biochem. Soc.
Trm1s. 1990; 18: 785-786.
Xiao, Y. F., Li, X .Y. Polyunsaturated fatly ac ids modi fy mouse hipocampal neuronal
excitabili ty during excitotox ic or convulsant stimulat ion. Brain Res. l 999; 846: 11 2- 2 1.
Xi menes da S ilva, A., Lavialle, F., Gendrot, G., Guesnet, P., A lessandri, J.M., Lavial le, M.
Glucose transport and ut ili zation are altered in the brain of rats deficient in n-3
pol yunsaturated fatty acids . .f. Neurochem. 2002; 8 1: 1328-3 7.
Yorek, M., Leeney, E., Dun lap, J., Ginsberg, B. Effect of fatty acid composition on insulin and
IG F-I binding in retinoblastoma cells. Invest. Ophthalmol. Vis. Sci. 1989; 30: 2087- 92.
Young, G. , Conquer, J. Omega-3 fatly acids and neuropsychiatric disorders . Reprod. Nutr.
Dev. 2005; 45: 1- 28.
Zimmer, L., Hembert, S., Durand, G ., Breton, P., Guilloteau, D., Besnard, J.C ., Chalon, S.
Chronic n-3 pol yunsaturated fatty acid diet-defic iency acts on dopamine metaboli sm in
the rat frontal cortex: a microdial ysis study. Neurosci. Let/. 1998; 240: 177-8 1.
Zimmer, L., Durand, G ., Guill oteau, D., Chalon, S. n-3 pol yunsaturated fatty acid deiici ency
and dopamine metaboli sm in the rat frontal cortex. Lipids. 1999; 34: 25 1.
Zi mmer, L., Delpal , S ., Guill oteau, D., Aloun, J., Durand, G., Cha lon, S. Chronic n-3
pol yunsaturated fatty ac id deficiency a lters dopami ne ves icl e density in the rat frontal
cortex. Neurosci. Lett. 2000a; 284: 25-8.
98
Zimmer, L., Delion-Vancassel, S., Durand, G., Gui ll oteau, D., Bodard, S., Besnard, J.C.,
Chalon, S. Mod ificat ion of dopamine neurotransmission in the nucleus accumbens of rats
deficient in n-3 polyunsaturated fatty acids . ./. Lipid Res. 2000b; 4 1: 32-40.
Zimmer, L., Vancassel, S., Cantagrel, S., Breton , P., Delamanche, S., Gu ill oteau, D., Durand,
G., Chalon, S. The dopamine mesocorticoli mbic pathway is affected by defici ency in n-3
pol yunsaturated fatly acids. Am . .!. Clin. Nutr. 2002; 75: 662- 7.

Editors: T.G. Bradley and F.P. Vargas, pp. 99- 11 6
ISBN: 978- 1-62257-480-3

20 12 Nova Science Publishers, Inc.
Chapter 4
EICOSAPENTAENOIC ACID:
THE ROLE IN MALIGNANT DISEASES
Vesna VuCic'" and Danijela Ristic-Medic
Centre of Research Exce llence in l\utrition and Metabolism,
Institute for Medical Research, University of Belgrade, Belgrade, Serbia
Abstract
A number of epidemiological and experimental evi dences have linked polyunsarurmed fatty
acids (PUFA) of n-3 series to reduced cancer risk. Particularly the long chain n-3 PUFA
eicosapemaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to have
potent antinunor effects. Increased consumption of EPA and DH A, found namrally in sea
food, may lower the risk of cancer development. Both EPA and DH A also exert amiangiogenic effects, inhibiting production of many important angiogenic mediators.
Furthermore, nmrirional intervention with EPA decreases weight loss, promotes weight gain
and increases survival rimes in pmiems a ffecred with cancer cachexia.
Namre of aminunor effects of EPA is nor clearly understood, but one of the mechanisms
is competitive inhibition of the use of arachidonic acid, an n-6 fatty acid, for the production of
eicosanoids. Eioosanoids derived from arachidonic acid have been associated with both nunor
promotion and progression. EPA is also a porem angiogenesis inhibitor, which suppresses
production of crucial angiogenic mediators namely: Vascular Endothelial Growth Factor
(V EGF), Platelet-Derived Growth Factor (PDG F), cyclo-oxygenase 2 (COX-2), Nuclear
Factor Kappa Beta (N FKB) and nitric oxide.
Proportion of EPA in plasma or ery1hrocyre phospholi pids in health y individuals depends
on the dietary intake and endogenous metabolism. However, clinical evidence suggests that
EPA status in newly diagnosed cancer pmiems and patients undergoing chemotherapy is
usually markedly lower in comparison with healthy population. Low EPA srams was found in
patients with cancer on different sires, including pancreatic, lung, prostate cancer, and nonHodgkin lymphoma. In addition, EPA and the other n-3 PUFA were shown to be particularly
depleted in advanced cancer pariems, during chemotherapy and in cancer pariems close to
death. Therefore, both the disease itself and therapeutic rreaunems may be contributing factors
in the decline of EPA status in patients with cancer. The present review will focus on the
E-mail address: vesna.vucic.imr@gmait.com; Tet: +38 11 1 303- 1 997~ Fax: +381 11 2030- 169; Address: Institute
for Medical Research, Centre of Research Exce.Hence in Nutrition and r..letabolism, University of Belgrade-,
Tadeu!\3 Koscuska I, I 11 29 Belgrade, Serbia. (CotTesponding author)
Vesna Vucic and Dan ijela Ristic-lv!edic
100
currem knowledge related m the anticancer effects of EPA on differem stages of disease, from
initiation and promotion, m progression and neoplastic transformation.
Introduction
Chronic noncommun icable diseases are rapidl y becoming epidemic worldwide, with
about 60% of all causes of deaths [ 1]. The reasons for increased chron ic disease preva lence
include genetic factors, but also modi fiable factors such as smoking, li festyle and nutrit ion
[2]. Among nutrit ional factors, dietary po lyunsaturated fally ac ids (PUFA) are often
associated to develo pment and progression of chron ic di seases, includi ng cancer.
Although adults in Western countries consume on average 30 to 45 % of the ir total
energy from fat, a small proport ion of fats consists of long-chain PUFA [3]. Dietary PU FA
are involved in many biological processes and part icularly two fatty acids (FA) must be
obtained from the diet: linoleic acid (LA, 18:2n-6) and a -lino lenic acid (A LA, 18:3n-3).
These FA are essential, since they cannot be synthesized in humans, and they are precursors
iOr two di fferent series ofPUFA: n-6 PlJFA are deri ved from LA, wh ile ALA is a precursor
for n-3 P UFA [4]. ALA and LA are both important for cell structure and are in compet it ion
for the same enzyme systems [5] . l.n addition, their metabolites, most ly arachidon ic acid (AA,
20:4n-6) and eicosapentaenoic acid (E PA, 20:5n-3) are involved in product ion ofeicosano ids,
wh ich inter alia, modulate the immune functions (6, 7]. AA is a precursor for the syntheses of
leukotriene B4, thromboxane A2, and prostaglandin E2 (PGE2), wh ich are potent ial
mediators of inflammat ion, wh ile EPA decreases their product ion and acts as an antiinfl ammatory agent [8]. Eicosapentaenoic acid competes w ith AA for prostaglandin and
leukotriene synthesis at the cyclooxygenase and lipoxygenase level [9]. A ll these eicosanoids
are strong mediators of a number of biochemical processes, play important roles in regulati on
of cell funct ions, but are also included in cancer development and promoti on [ 10]. In
part icular, PGE2 has been li nked to angiogenesis and carcinogenes is in studies of hornlOnerelated cancers {11, 12], and its role in these processes will be di scussed later in th is review.
Therefo re, n-6 an d n-3 PU FA have di flerent funct ions in the body and di etary intake of
these FA could markedly infl uence human health. However, n-6 fatty ac ids are the
predominant PU FA in most diets. When the diet is enriched with n-3 P UFA, they partiall y
replace n-6 PlJFA in the vast maj ority of cell membranes (e.g. erythrocytes, platelets,
monocytes, lymphocytes, granulocytes, and endotheli al neuronal, co lon, and hepatic cell s)
[ 13]. Main dietary sources and competit ion between n-6 PU FA and n-3 PUFA in
prostaglandi n forn1at ion are outli ned in Figure I .
Increasing body of epidemiological and experimental evidences has li nked n-3 PUF A to
reduced cancer r isk. Part icu lar ly the long chain n-3 PU FA EPA and docosahexaenoic acid
(DH A) have been shown to have potent ant itumor influence. The potent ial ofE PA and DHA
to inh ibit cancer growth at early stages of cancer development have been addressed in several
reviews [9, 14- 17]. Furthermore, E PA and DHA supplementation have been shown to
improve the effect of chemotherapy and to reduce the tox ic effects of the therapy [ 18, 19].
101
Eicosapeniaenoic Acid: The Role in Malignant Diseases

Soybt~n. ~ o1'n,
sunOowtr- oil
Linolric at"id (18:2n.$1
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c~
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~~
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TXR,
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(lO:Sn-3)
L.ipoxygenn<>t
nzrs
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(18: ~013)
alpha~llnoltnlc
arid
Colcl ~'"altJ' fish
LOX
Leuco tritnt
LTA,
LTB,
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L TD;
LT,.
(18:3 n-3)
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oil
Figure I. Dietary sources and simplified metabolism of n-3 and n-6 PUFA. COX- cyclooxygenase,
LOX-Iipoxygenase.
Finall y, in patients with advanced cancer EPA has allenuated the weight and Jean body
mass loss {20-23 ]. The present review wil l focus on the current know ledge related to th e
status and ant i-cancer et-reels of EPA on di ff'erent stages of disease, from init iat ion and
promotion, to angiogenesis and metastasis.
Dietary EPA: The Role in Cancer Risk Reduction

The evidence of an inverse association between cancer risk and fish intake has been
found for many cancer types, although these data are inconsistent and stat istical ly
102
Vesna Vu cic and Danije la Ristic-lv!edic
insign ifi cant in some reports. Prospecti ve and case-control studi es have shown that high
consumption of 11sh, wh ich is the major djetary source of EPA, was inversely associated w ith
colorectal cancer [24-27], oesophagus and stomach cancer [28], ovarian cancer [28-30] and
breast cancer [3 1]. In a large case-control study, Fritschi et al. [32] have found a strong
protective effect of fresh llsh intake for leukemia, myeloma and non-Hodgkin lymphoma.
However, epidemiological evidence on the relation between fish or n-3 PU FA consumpt ion
and cancer risk are often conflict ing. For instance, several cohort studies have been examined
the associat ion of fish, n-3 PUFA, EPA, and DHA intake with incident breast cancer (33-37],
and in most of th em no association was found. On the contrary, a prospect ive stud y of women
in Singapore, who consume fish much more than people in the Western countries, showed a
sign ificant inverse association between intake of n-3 PU FA from sea food and breast cancer
risk (33]. In addjt ion, Saadatian-E iahi et al. [38] conducted a meta-anal ysis o f studi es that
anal yzed blood biomarkers of PUFA in associat ion w ith breast cancer risk and found inverse
associat ions fo r both EPA and DHA . The si milar situation has been observed in seven
prospective studies on EPA and/or DHA intake and prostate cancer r isk. One of these studies
reported a posi t ive associat ion between dietary intake of n-3 PUFA an risk of prostate cancer
(39], three papers showed an inverse association [40-42] and in the rest 3 studies [43-45] no
associat ion was found. Jn a case-control study, Fradet et al. {46] showed that increased
consumpt ion of dietary long chain n-3 P UFA appeared protective for aggressi ve prostate
cancer, but th is effect was modj li ed by th e gene variant (rs46483 10) single nucleot ide
pol ymorphism in cyclo-oxygenase 2 (COX-2). This enzyme is involved in FA metaboJjsm
and is a well known risk factor for prostate cancer. In the li ght of these ti ndings, only men
carrying the variant allele maintained a strong inverse association between llsh intake and
prostate cancer [47]. Therefore, further high quali ty studies seem 10 be required in a large
cohort, w ith minjmal confounding factors to establish a convincing link between n-3 PU FA
intake and cancer risk.
EPA Status of the Cancer Patients

Lipid metabolism is frequently altered in patients sufl:'ering from malignant diseases, but
in spite of th is fact, only few studies have detem1i ned the status of FA in these patients. In
part icular there is very li!!le data on changes of FA during the cancer progression or in the
course of treatment. Most of these studies have demonstrated a low EPA status in cancer
patients.
EPA Status at Diagnosis

FA composit ion of plasma phospholipids refl ect overall metaboli sm of endogenous and
dietary FA [48] . Thus at ear ly stages of cancer FA status depends on the previous intake, in
addit ion to an impaired metabolism specific to the disease. Popu lation studi es have shown
di fferent status of EPA and the oth er FA in blood lipids in subj ects from di fferent regions [ 12,
49, 50]. lv!oreo ver, a cross-sect ional study conducted in more than 3000 individuals from 16
centers in 10 European countries revealed that the most important detem1inam of plasma FA
was region [5 1]. The correlation between consumpt ion and biomarker status in blood
103
Eicosapentaenoic Acid: The Role in Malignant Diseases
phospholipids is especiall y strong for EPA (5 1]. Accordingly, EPA status in cancer pat ients
must be interpreted in comparison with healthy populat ion from th e same region.
Several studi es have found impaired n-3 PU FA level in blood and cell lipids in patients
with di fferent cancer types [52-54]. Abnom1al plasma (or serum) EPA status has been shown
in pat ients with a new diagnosis of pancreat ic, non-small -cell lung o r oesophageal cancer
(55], b ladder cancer [53], uterine cervical cancer [56], and non-Hodgkin lymphoma (NH L)
that has been recently published hy our group {57]. Furthermore, Agatha et al. [52] found
lower EPA in Jymphocytes phospholipids in ch ildren with acute Jeukemia when compared
"ith healthy ch ildren. EPA status at diagnosis in plasma/serum phospholipids, lymphocytes
and erythrocytes phospho lipids of patients with cancer is presented in Table I. On ly papers
wh ich also reported EPA biomarker status in healthy controls are included. In all papers
lower proport ion of EPA has been reported in pat ients w ith cancer than in the con trol group,
except in patients w ith oesophageal cancer {55]. lvlarkedl y di ft'erent values of EPA in both the
patient and control groups ori ginate not only from di fferent regions, but from di fferent un its
(mol%, weight% etc.) used in papers.
Table 1. EPA biomarker status in patients with different

cancers compare.d to healthy subj ects
Author, year (ref)
pop ulation grou p
EPA status in patients

(source of FA, units)
Cancer type
Agatha, 200 I [52]

children
I. Acute lymphobla-stic
leukemia (A LL)
2 Acute m ye! oid
leukemia(AM L)
:VIcCiimon, 1991 [53]

adults
Bladder
Zuijdgeest-van
Leeuwen, 2002 [55]
adults
I. Pancreatic
2. Lung
3. Oesophagial
Lis boa, 2008 [56]

adult women
Cvetkovic, 20 10 [57]
adults
\Virfalt, 2004 [58]
adult women
"'orrish, I 999 [59]
adult men
Godley, 1996 [60]
adult men
..
Urerine cervix
"'on-H odgkin
lymphoma
Breast
Prostate
Prostate
I. A LL 0.52 = 0.1 2
(Ly membrane PL; % of total
FA)
2. AM L 0.38 = 0.1 3
(Ly membrane PL; % of total
FA)
1.1 0.8*
(Plasma PL;% of total FA)
1. o.68,!, o.os
2. 0.85 = 0.09
3. 1.29 = 0.1 5
(Plasma PL, weight % of total
FA)
0.88 0.3 4
(%of total FA)
0.20. 1*
(Plasma PL, mol %)
Er I. 70,!, 0.57
(Er PL, mol%)
0.67= 0.02*
( Er Pl, % of total FA)
0.54 (mean only)
(Er PL, mol%)
PL - phosphoh p1ds, Ly-h mphocytcs, FA - Jatty ac1d, Er-ctythrocytes.

P<0.05.
EPA stat us
in healthy
subject
0.60,!, 0.44
1.4 1. 1
1.24
=0. 14
=0.73
0.3 =0.2
1.20
(mol%)
1.7 1 = 0.58
0.62
=0.02
0.59
(mean only)
104
Vesna Vu cic and Danije la Ristic-lv!edic
Erythrocyte El'A Status and Cancer Risk

The fatty acid compos ition of erythrocyte membranes depends both on the fatty acids
present at the time of erythropoesis and on the current plasma concentration [58]. Thus, the
erythrocyte membrane levels of EPA can be used as biomarkers for long-tem1 EPA dietary
intake [6 1]. However, an Italian study examining flllty acids of erythrocyte membranes in
relation to breast cancer r isk, indicated that tissue levels of FA were not simple reflections of
diet, but the result of a mu lt itude of metabolic act ivities [62]. The relationship between n-3
PUFA status in erythrocytes and cancer risk was addressed in several studies. A sign ificant
inverse association between EPA and DHA content in erythrocytes and the r isk of prostate
cancer was observed in a case control study by Norrish et al. [59]. The authors found that
decreased prostate cancer risk was associated with high erythrocyte phosphalidylcholine levels
of EPA (muJtivar iate relative r isk = 0.59; 95 % conli dence interval 0.37-0.95, upper vs lowest
quartile) and DHA (multivar iate relative risk = 0.62; 95% confidence interval 0.39-0.98, upper
vs lowest quartile). Reduced prostate cancer risk associated w ith the highest quart ile of EPA
(OR 1.36, 95 % Ctln fidence interval 0.47-3.96; OR 0.74, 95 % confidence interval 0.23-2.33;
lowest vs upper quartil e) and DHA levels in erythrocytes (OR 1.22, 95 % confidence interval
0.47-3.96; OR 0.36, 95 % confidence interval 0. 10- 1.27; lowest vs upper quart ile) has also been
reported in an outpatient cli nic-based study of89 cases and 38 controls [60].
Studies related to breast cancer risk showed di fferent results. The study conducted by
Wir fat et al. (58] has not found any associat ions between the proport ions of fatty acids in
erythrocyte membranes and breast cancer risk. However, a large European mult icentre study
(EU RAM IC Study) in adipose t issue samples and found consistent inverse associations of the
n-3/n-6 PUFA rat ios w ith breast cancer risk across most study centers [63]. One case-control
study that exami ned FA composit ion from breast adi pose t issue in breast cancer cases
compared to controls w ith ben ign breast di sease f(lund a protective effect of n-3 fluty acids
[64]. T his indi cated that preferred t issue as a biomarker of di etary fally acids in relat ion to
breast cancer risk wou ld be breast adipose t issue.
El'A Status in Advanced Cancer Patients

The data on EPA status in pat ients w ith advanced cancers are very sparse. In one of the
fi rst studi es on ad vanced cancer pat ients, Prali et al. [65] found 50 % lower amount ofE PA in
plasma phospholipids in pat ients w ith advanced cancer at di fferent sites (prostate, pancreas,
breast, rectum etc.) than in healthy individuals: I 0.2 and 2 0.4 ~tg/mL plasma,
respectively. A recent study by Cvetkovic et al. [57] was the first to show that the proport ion
of individual FA varied according to the stage and aggressiveness of the cancer. However,
proport ion of EPA in total phospholipids did not di ffer significantly among patients w ith
di fferent clinical stages. One of the possible reasons is th at th is study assessed the percentage
of E PA in total phospholi pids and not the amo unt of FA. Considering that concentration of
phospholipids is usuall y much lower in cancer pat ients [66, 67], and that the autho rs reported
lower total phospholip ids in the pat ients group, it can be concluded that amount of EPA was
sign ificantly reduced in patients with non-Hodgkin lymphoma. Furthem1ore, EPA has been
observed to be particu larly low in advanced stage lung cancer [54] and its status has been
assoc iated w ith low muscle mass in these patients [68]. A ll these studies suggest a potent ia ll y
Ei cosapentaenoic Acid: The Role in Malignant Diseases
105
important deplet ion of n-3 and n-6 PU FA in advanced cancer, and a tendency towards
increased n-6/ n-3 ratio .
E.PA Status during Cancer Trea tment

Metaboli sm and status of FA duri ng treatment for cancer are not well character ized. ln a
pi Jot study MacDonald et al. [69] foun d a select ive deplet ion of long-chai n PUF A, most ly
EPA and DHA, in plasma and membrane p hospholipids by a high-dose combination of
cyclophosphamide, taxotere and 5-fl uorouraci l. lvlarra et al. [70] repo rted that cytotox ic
agents may li mit the endogenous production of long chain P UFA; however, li ttle is known
about the activity o f desaturase/elongase system in cancer patients. New data from our
laboratory on patients w ith non-Hodking lymphoma have reveal ed an associat ion between
EPA status and the response to chemotherapy. In pat ients who achieved remission proport ion
of EPA in plasma phospholipids increased al most double aft er the treatment, pat ients w ith
stable disease kept unchanged proportion of EPA, but in pat ients with cancer progression
percentage of EPA further decreased (manuscript in preparat io n).
Depletion of EPA in cancer pat ients is o f particu lar interest because of its ant itumor
effects. Although the causes o f the aberrant EPA status in p lasma PL are unclear, it has been
shown that both chemotherapy and disease itself may contribute to PUFA dep letion {71 ] .
Low EPA status together with the data show ing an associat ion between EPA and the
mai ntenance of lean mass, give a rat ionale for supp lementat ion of EPA in cancer patients.
Supplementation Studies
Supp lementat ion with EPA in pat ients suffering form cancer was the obj ective in many
trials. At fi rst, studi es on animal models have consistently been shown to improve the effects
of chemotherapy and to protect from the side effects caused by the treatment [72, 73]. T he
first clinical tri als suggested that similar effects co uld be expected in humans. The rationale
for these studi es included the effects o n tumor-associated weight loss, nutrit ional and
inilammatory status o r respo nse to therapy [23, 74-77]. The obtai ned results suggested
beneficial effects of supp lementation in compari son w ith p lacebo (or rarely no effects of
supp lementat ion) on these parameters. Tolerance of EPA even at h igh doses was very good in
al l trials and side effects were rare and relat ively mild [78].
A recent meta anal ysis have addressed the role of fish oi l/n-3P UFAIE PA
supp lementation for the treatment of cachex ia in advanced cancer patients, included 38
pub Ii cation, and concluded that there was not enough evid ence to support a net benefi 1 of n-3
PU FA intervent ion on cachexia in advanced cancer [79]. However, most of the trials included
in th is meta analysis used fish oil (rich in both EPA and DHA) for supp lementation, and it
was di fficultto attribute th e obtained results only to EPA or DHA. it rema ins unclear whether
EPA and DHA exert the same eifects and do they use the similar pathways Jo r the ir act ion.
Th e doses for supp lementat ion wh ich have some beneficial effects should also be betler
defi ned in the future trials. A lso, we did not ident ify any studies that assessed infl uence of
EPA (or fish oil) on clinical outcomes after chemoth erapy or radiat ion surgery. For example,
si gn ifi cantly improved plasma FA profil e was found afler only 7 days of supplementat ion
106
Vesna Vu cic and Dan ije la Ristic-lv!edic
with 5 g o f fish o il daily (0.9g EPA + 0.6g DHA) in a pilot study o n pat ients with obstruct ive
jaund ice [80]. Th is study included patients with hepa tocellu lar carcinoma but the authors d id
not report the effects o f supp lementat ion on clin ical outcomes.
The potential role of n-3 PUFA for supplementat ion in cancer patients has rec.ently been
rev iewed by lv!urphy et al. {8 1]. Review ing recent trials, the y have found a n effect o f n-3
supp lementat ion on weight, lean body mass an d treatmen t ou tcomes throughout
antineoplastic therapy and concluded that n-3 PU FA supp lementat ion had beneficial effect s at
early stages o f cancer, during anti cancer therapy for preservation o f muscles and o n better
to lerance o f the treatment. T he influence of n-3 P UFA on the p hospho li pids fraction o f the
immune cell popu lat ion, alte ration in membrane structure, and changes in cellular signal
transduct ion that in fl uence growth and proli feration may be possible mechan isms of tumor
growth inh ibition by fi sh o il. Nevertheless, a longer survival was not foun d in pat ients treated
" i th EPA [82].
Cancer Cachexia and EPA

Malnutrition is o fien present in cancer pat ients. it contributes to reduced qua lity o f li fe and
shorter survival ti me. Many factors in fluence the weight loss in pati ents w ith cancer, some o f
them can be related to reduced d ietary nutrient intake, wh ile others are associated w ith
metabo lic changes induced by system ic inflammatory responses. These nutrition modulation
processes activate protein degradat ion and lipo lytic pathways, resulting in tissue loss (83].
Loss o f skeletal muscle appears to relate to poor n-3 PUFA status in advanced cancer
patients (8 1]. A systemat ic rev iew by Dewey et al. [84] selected on ly fi ve randomized contro l
trials on pa tients w ith advanced cancer and a c li nica l d iagnos is o f cachexia (or self-reported
\\e ight loss >5 %) w ith oral EPA supp lementation. Authors concluded that there were
insu ffi cient data to define the opt imal dose o f EPA su pp lement in re li eving symptoms
assoc iated w ith the cachexia synd rome in ca ncer pat ients.
Prev ious stud ies in cachect ic pancreatic cancer pat ients sh owed that fish o il or h igh purity
EPA capsules (2-6 g EPA/ day) supply was assoc iated w ith weight stability and an incr ease in
total energy expend iture and physical activity level [85, 86] . Murphy et al. {74] showed that
nutrit ional treatment w ith 2.2 g EPA/ day pro vides a bene fit over standard o f care, allowing
patients w ith non-small ce ll lung cancer to maintain their weight and muscle mass during
chemotherapy. T hese ben efits may be related to the effect o f EPA on certain tumor specifi c
factors such as proteo lysis inducing factor, but might also be related to downregu lat ion o f
proinflanm1ato ry cytokine production. Gogos et al. [87 ] no ted that immunoresto rat ive effect
o f fish o il in malnourished patients w ith advanced cancer was through a sign ificant increase
o fT-he lperIT -suppressors ce ll s ratio and TN F-a p roduct ion, wh ich were sign i f:icam ly lower in
malnourished patients. Howeve r, both consumpt ion of n-3 PU FA and biomarker status seam
to be independent prognostic factors for survives in patients w ith cancer. High doses of
di etary n-3 PUFA (1 8 g/day), in combinat ion with antiox idant supplementation, may prolong
the survival o f patients with generalized ma lignancy [87] .
107
EPA and A ngiogenesis

Angiogenesis is the fommt ion of new blood vessels induced by physiological or
pathological reasons [88]. This is one of the most important processes in tumor growth and a
fundamental step in transit ion of tumor from a dom1ant state into mali gnant one. Namely, for
tumor growth o ver a size of 1-2 mm>, a new blood supply must develop to support the
increasing metabol ic needs [89]. Clinical st udies have shown that angiogenesis in solid
tumors relates to a poor prognosis and, in premali gnant lesions, indicates potential for
cancerous transfom1ation.
There are a number of molecu les involved in stimu lation or inhibition of angiogenesi s,
including growth factors and cytokines, but the most investigated compound is Vascular
Endothelial Growth Factor (VEGF) [90]. VEGF is a potent stimulator of angiogenesis which is
up-regulated in various cancers [9 1-95]. Several in vitro studies have shown that EPA
suppressed VEGF-stimulated endothelial cell proliferation, migration and tube format ion (96,
97] and inhibited increased VEGF expression in colon cancer cell lines [98]. The antiangiogenic potential of EPA was also contim1ed in vivo: Fischer 344 mice with EPA
supp lemented di et showed sign ificantly decreased tumor volume and V EGF-mRNA than the
control mice, afier flank implantation of fibrosarcomas {99]. A lthough human supplementation
studies in cancer patients investigating the effects of EPA on VEGF and angiogenesis have not
been conducted so far, Am bring et al. [ I 00] have shown that n-3 PUF A rich diet decreased
levels of ci rculating V EGF in healthy volunteers. it should be confim1ed in further studies
whether the same eflects of n-3 PUF A would be found in patients with cancer.
Anoth er growth factor wh ich plays an important role in angiogenesis is Platelet-Derived
Growth Factor (PDGF). PDGF has mitogen ic and chemo-attractant propert ies for vascular
smooth musc le cells. However, it has been revealed that EPA could inhibit vascular smooth
muscle cell proli feration as well as PDGF-induced mi grat ion of these cells in vivo through
modulating various steps of the si gnal transduction by PDGF. Namely, Terano et al. [ I 0 I]
revealed that EPA inhibited PDGF binding on its receptor, activat ion of protein kinase C and
suppressed c-fos mRNA expression, o ne of immediate early genes, through partly inhibit ing
c-fos transcription. Hypothetical scheme show ing the potential mechanisms of EPA m
inhibit ing carcinogenesis at di fferent stages is presented in Figure 2.
The above mentioned compet ition between EPA and AA for the product ion of
eicosanoids is o f part icuJar interest in angiogenesi s. T he conversion of AA into
prostaglandi ns catalyzed by enzyme COX-2 leads to production of PGE2, which has been
associated w ith increased intlanm1at ion and tumor angiogenesis [73]. Prostaglandin E2 is a
part icularly potent regu lator of the synthesis and release of pro-inflammatory cytokines, such
a~ interleukin 6 and tumo r necrosis factor [8]. Both COX-2 and PGE2 are elevated in many
cancer types and are beli eved to actively part icipate in colorectal cancer development [ 102,
103]. Treatment with EPA inhibited COX-2 expression and PGE2 product ion in animal
models as well as in cell s cultured in vitro suggest ing possible chemo prevent ive effects of
EPA in humans [ I 04, 105].
In addit ion, PGE2 has an important role in hormone-related cancers. it is known that
estrogen has proliferative effects on estrogen -sensi t ive t issues and that its high concentrat ions
may increase the r isk of hom10ne-dependent cancers, most ly of breast cancer.
Vesna Vu cic and Dan ijela Ristic-lv!edic
108
Di~ta~ iur:-.kt.
endog~uou s
syathesi.>
- -- -.-- - -- -- -- -. -- -- -. -- -.
E l'A
EP.-\ -dnived
ticosa_noids
..
---~~-
Inflammation
ROSIR"S
-.-
\_
... . ... ...

,._
''
-- ...-...
- --
COX-2, PGE2,
\ 'EGF, PDGF
METASTASIS
CA.'iCER
Figure 2. Potemial mechanisms whereby EPA may suppress carcinogenesis at different stages. COX-2
- cyclo-oxygenase 2, PG E2 - prostaglandin E2, V EGF - Vascular Endothelial Growth Factor, PDGF Platelet-Derived Growth Factor, ROS - Reactive Oxygen Species, R.~S - Reactive "'itrogen Species.
stimulation,
--- - - "' inhibition.
PGE2 has been found to stimu late the act ivity of aromatase P450, wh ich converts 19carbon steroids to estrogens. In contrast, EPA-derived eicosanoid PGE3 does not activate this
enzyme. lv!oreover, n-3 PU FA are potent inhibitors of various human P450 enzymes [ 107].
Hence, an increased consumption of E PA, which leads to increased product ion of PGE3 and
decreased product ion of PGE2, di minishes the estrogen fom1at ion and consequently estrogen
109
induced growth of cell s involved in breast cancer. The modulat ion of breast cancer
proli ferat ion by EPA and DHA has been confi m1ed in cell line experiments [ 108]. Studies in
vitro have also shown that n-3 FA induced apoptot ic cell death in human breast cancer cell s,
probably as an effect of altered phosphorylation and membrane raft localizat ion of epidermal
growth factor molecules {109]. In spite of these results, no studies have yet di rect ly examined
th is issue in humans.
Nitric oxide and Nuclear factor Kappa Beta (N FKB) are also important factors in process
of angiogenesis, and n-3 PUFA have been found to down-regulated these fact ors in human
cancer cell lines [ I 06]. However, th ese e ffects have most ly been allr ibuted to DHA and A LA,
\\h i le the role of EPA in th is pathway has not been well establi shed so far.
Conclusion
In conclusion, cancer remains a significant public health burden worldwide. PU FA ofn-3
series, pr imarily EPA, may have signi ficant bene fits for a var iety of cancer-related
compli cations. T he role of EPA in di fferent points in cancer trajectory is beginn ing to be
exp lo red, but several questions are st ill unanswered. In spite of a number of ep idemiological
studi es, the associat ion between EPA consumpt ion and cancer incidence is still unclear. Thus,
prospective studi es to assess the role of EPA in the growth and development of a part icular
type o f cancer might be warranted.
The data on the mechanism of the effects of EPA are limited and th is area is st ill at its
early stages. Further investigat ions are required to explore the act ion of EPA at the cell ular
levels in cancers. Nutriti onal and EPA status in cancer patients is identi fied in some cancer
types, although the data, part icularly in advanced cancer pat ients, should be extended. Future
supp lementat ion studi es in larger, randomized trials are needed to reveal the fu ll potential of
EPA as an adjuvant to ant ineoplastic therapy. All these research efforts w ill show the possible
ut ility of EPA in the fi ght against cancer and hence are awaited w ith interest.
Acknowledgement
This work was supported by the Project 11 14 1030 financed by the Ministry of Science of
the Republic of Serbia.
References
( I]
[2]
{3]
W ardhana, Surachmanto ES, Datau EA: The ro le of omega-3 fatty acids contained in
olive oil on chronic infl ammat ion. Acta Med. lndones. 20 11, 43: !38- 143.
WHO : G lo bal status report on noncommun icab le diseases. World Health Organization
20 I 0.
Baracos VE , lv!azurak VC, Ma DW: n-3 Polyunsaturated fatty acids throughout the
cancer traj ectory: influence on di sease incidence, progression, response to therapy and
cancer-associated cachexia. Nutr. Res . Rev. 2004, 17: 177- 192.
11 0
(4]
{5]
{6]
{7]
(8]
Vesna Vu c ic and Dan ije la Ristic-lv!edic

Gibson RA, Muh lhausler B, Ma krides lv!: Conversion o f lino le ic acid and a lphali no len ic acid to long-cha in po lyunsaturate d fatty acids (LC PUF As), with a focus on
pregnanc y, lacta tion and the 6 rst 2 years o f li fe. Matern. Child Nutr. 2011, 7 Suppl
2: 17-26.
Simopo u los AP: Human requ irement fur N-3 po lyunsaturated fatty acids . Poult. Sci.
2000, 79 :96 1-9 70.
Kogteva GS, Bezuglov VV : Unsaturated fatly acids as endo genous b ioregu lators.
Biochemisfly (Mos c). 1998, 63 :4- 12.
A rsic A, Vuc ic V, Tepsic J, et a l. : A lte red p lasma and eryth rocyte p hosp ho li p id fatty
acid profil e in e li te fema le water po lo and footba ll p layers. Appl. Physiol. Nutr. Metab.
2012, 37: 40 -47.
Kawakam i Y, lv!urakami Y, Kawaka mi T, e t al .: Abnom1a l fally acid profi le o f b lood
ce ll p hospho lip ids and di etary fally acid intake in patients w ith u lcerat ive colit is . J.
C/in. Biochem. Nutr. 2005, 3 7:95- 102.

(9]
( 10]
( I I]
( 12]
( 13]
{14]
{15]
( 16]
{17]
{18]
{19]
(20]
l\!cEntee MF, W he lan J: Dietary po lyunsaturated fally ac ids and colorecta l neop lasia.
Biomed. Pharmacother. 2002, 5 6:380-387.
Comba A, Lin Y H, Eynard A R, et a l. : Basic aspects o f tumor ce ll fally ac id-regulate d
signa ling and transcription factors . Cancer Metastasis Rev. 2011 , 30: 32 5-342.
Rose DP: Dietary fally acids and prevention o f hormone-resp onsive cancer. Proc. Soc.
Exp. Bioi. Med. 199 7, 2 16:224 -233.
Terry PD, Rohan T E, Wo lk A: Intakes of fi sh and marine fally acids and the risks o f
c ancers o f the breast a nd prosta te and o f other hom10ne-rela ted cancers: a review o f the
epidemio log ic ev id ence. Am. J. C/in. Nutr. 2003, 77:532-543.
Ristic Medic D, Perun icic -Pekovic G, Rasic-M il ut inov ic Z: PU FA status and omega-3
fatty acid supplementat ion in hemodi a lysis pat ients: pathophysio log ical aspects an d
c lin ical outc omes . Nutritional Therapy and Metabolism. 2011 , 29: 178- 187.
Bartsch H, Nair J, O wen RW: Dietary po lyunsaturated fatty acids and canc ers o f th e
breast and co lorectum: emerging ev idence for the ir ro le as ri sk modi fi ers.
Carcinogenesis. 1999, 20:2209-22 18.
Black HS, Thornby Jl, Gerguis J, Lenger W: In fluence of di etary omega-6, -3 fatty ac id
sources on the initiation and promot ion stages o f photocarc inogenesis. Photochem.
Photobiol. 1992, 5 6: 195- 199 .
.f iang WG, Bryce RP, Horrob in DF : Essent ia l fa tty acids: mo lecu lar and cell u la r basis
o f the ir ant i-cancer act ion and c lin ic a l implicatio ns. Crit. Rev. Oncol. Hematol. 1998,
27: 179-2 09 .
Rose DP, Conno ll y JM : O mega-3 fatty acids as ca ncer ch em opreventive agents .
Pharmacal. Ther. 1999, 83 :2 17-244 .
Bougnoux P, Hajjaji N, Ferrasson MN, et a l. : Improv ing outc ome o f c hemotherapy of
metastat ic breast cancer by docosahexaeno ic ac id: a phase 11 trial. Br. ./. Cancer. 2009,
I 0 I : 19 78- 1985.
Murphy RA, Mourtzak is M, Ch u QS, et a l. : Supp lementat ion with fi sh o il incre ases
first-li ne chemotherapy e~1icacy in pat ients w ith advanced nonsmall cell lung cancer.
Cancer. 20 11 , 11 7: 3 774-3 780.
Co lomer R, M oreno -Nogueira JM, Garcia -Luna PP, et a l. : N-3 fatty ac ids, cancer and
cachex ia: a systematic rev iew o f the literature. Br. .! Nutr. 2007, 97 :82 3-83 1.
11 I
(2 1] G iacosa A, Rondanelli M: Fish o il and treatment of cancer cachexia. Genes Nutr. 2008,
3:25-28.
(22] Fearon KC, V on Meyenfeldi MF, Moses AG, et al.: Effect of a prote in and energy
dense N-3 fatly acid enriched oral supplement on loss of weight and Jean tissue in
cancer cachexia: a randomised doub le b lind trial. Gut. 2003, 52: 1479- 1486.
{23] .fato i A, Row la nd K, Lopr inzi CL, et al. : An e icosapemaenoic ac id supp lement versus
megestrol acetate versus both for patients with cancer-associated wast ing: a North
Centra l Cancer Treat ment Group and Nat ional Cancer Inst itute o f Canada coll aborat iv e
e ffort. ./. C/in. Oncol. 2004, 22:2469-2 476.
[24 ] Gaard !vi , Tret li S, Loken EB: Dietary factors and risk of colon cancer: a prospective
stu dy o f 50,535 young Norwegian men and women. Eur. .f. Cancer Prev. 1996, 5 :
{25]
[26]
(27]
{28]
{29]
[30]
{3 1]
{32]
445-454.
Kato I, A khmedkhanov A, Koenig K, et al. : Prospect ive study of di et and female
colorecta l cancer: the New York University Women's Health Study. Nutr. Cancer.
1997, 28:276-28 1.
Norat T, Bingham S, Ferrar i P, et al.: Meat, fish, and colorecta l cancer risk: the
European Prospective Investigat ion into cancer and nutrit ion . .f. Natl. Cancer Inst.
2005, 97:906-9 16.
Tiemersma EW, Kampman E, Bueno de Mesqu ita H B, e t a l. : Meat consumpt ion,
c igarelle smoking, and genet ic suscept ibility in the et io logy o f colorecta l cancer: results
from a Dutch prospective study. Cancer Causes Control. 2002, 13:383-393.
Fernandez E, Chatenoud L, La Vecch ia C, Negri E, Francesch i S : Fish consumpt ion
and cancer risk. Am. J. C/in. Nutr. 1999, 70:85-90.
Bosetti C, Negri E, Francesch i S, et al. : Diet and ovarian cancer r isk: a case-control
study in Ital y. lnt . .f. Cancer. 2 00 I, 93:9 11 -9 15.
Kolahdooz F, van der Pols JC, Bain CJ, et al .: Meat, fish, and ovarian cancer risk:
Results from 2 Australian case-control studies, a systematic review, and meta-a nalysis.
Am . .f. Clin. Nutr. 20 I 0, 9 1: 1752- 1763.
Kim J, Lim SY, Sh in A, et al.: Fatty fish and fish omega-3 fa tty acid intakes decrease
the breast cancer risk: a case-control study. BMC Cancer. 2009, 9:2 16.
Fri tsch i L, Ambrosin i G L, Kliewer EV, Johnson KC: Dietary fish intake and risk o f
leukaemia, mult ip le myeloma, and non-Hodgk in lymph oma. Cancer Epidemiol.
Biomarkers Prev. 2004, 13:532-53 7.
(33] Gago-Dominguez !vi, Yuan JM, Sun C L, Lee HP, Yu MC: Opposing effects of dietary
n-3 and n-6 fatty ac ids on mammary carcinogenesis: The Singapore Ch inese Health
Study. Br. .f. Cancer. 2003, 89: 1686- 1692 .
(34] Holmes lv!D, Hunter DJ, Coldi tz GA, e t al.: Associati on o f dietary inta ke of fat and
fatly acids w ith risk o f breast cancer. .lAMA. 1999, 28 1:9 14-920.
(35] Kim EH, W illell WC, Co lditz GA, et al.: Dieta ry fat and risk of postmenopausal breast
cancer in a 20-year follow-up. Am . .f. Epidemiol. 2006, 164:990-997.
(36] VaHen LJ, Sol vo ll K, Loken EB: Frequency o f meat and fis h intake and risk o f breast
cancer in a prospect ive study of 14,500 Norwegian women. lnt . .f. Cancer. 1990, 46:
12- 15.
[37] Voorrips LE, Brants HA, Kard inaal AF, et a l. : Intake of conjugated linole ic acid, fat,
and other fatly acids in re lat ion to postmenopausal breast cancer: the Nether lands
Cohort Study on Diet and Cancer. Am . .f. C/in. Nutr. 2002, 76:873-882.
11 2
(38] Saadat ian-Eiahi lv!, Norat T, Goudable J, Riboli E: Bi omarkers of dietary fatty acid
intake and the risk of breast cancer: a meta-analysis. /nt . .f. Cancer. 2004, Ill :584-59 1.
(39] Wallstrom P, Bj artell A, Gull berg 8 , O lsson H, W irfali E: A prospect ive study o n
dietary fat and incidence of prostate cancer (Malmo, Sweden). Cancer Causes Control.
2007, 18: 11 07- 11 2 1.
(40] Augustsson K, Michaud DS, Rimm EB, et al.: A prospect ive study of intake of fish and
marine fatty acids and prostate cancer. Cancer Epidemiol. Biomarkers Prev. 2003,
12 :64-6 7.
[4 1] Leitzmann MF, Stampfer MJ, Michaud DS, et al .: Dietary intake of n-3 and n-6 tally
ac ids and the risk of prostate cancer. Am . .f. C/in. Nutr. 2004, 80:204-2 16.
[42] Park SY, Murphy S P, Wilkens LR, Henderson BE, Kolonel LN: Fat and meat intake and
prostate cancer risk: the mul!iethnic cohort study. lnt. .f. Cancer. 2007, 12 1:1339- 1345.
[43] Meyer F, Bairati I, Shadman i R, Fradet Y, Moore L: Dietary fat and prost ate cancer
survival. Cancer Causes Control. 1999, I 0:245-25 1.
[44] Schuum1an AG, van den Brandt PA, Dorant E, Brams HA, Goldbohm RA : Association
of energy and fat intake w ith prostate carcinoma risk : results from The Nether lands
Cohort Study. Cancer. 1999, 86: I 0 19- 1027 .
{45] Veierod lv!B, Laake P, Thelle DS: Dietary fat intake and risk of prostate cancer: a
prospecti ve study of25, 708 Norwegian men. lnt. .f. Cancer. 199 7, 73:634-638.
[46] Fradet V, Cheng I, Casey G, W iue JS: Dietary omega-3 fal!y acids, cyclooxygenase-2
genet ic variation, and aggressive prostate cancer risk. Gin. Cancer Res. 2009, !5:
2559-2566.
[47] Hedeli n M, Chang ET, W iklund F, et al.: Association of frequent consumpt ion of fally
fish with prostate cancer risk is modi fied by COX-2 polymorph ism. lnt . .f. Cancer.
2007, 120:398-405.
[48] Teps ic J, Vucic V, Arsic A, et al.: Plasma and erythrocyte phospholipid faHy acid
profi le in professional basketball and football players. Eur . .f. Appl. Physiol. 2009,
I 07: 359-365.
[49] Amiano P, Dorronsoro lv!, Larranaga N, Renobales lvt, Ruiz de Gordoa JC: Very-l ongchain omega-3 fally acids as markers for habitual fi sh intake in Spain. !A RC Sci. Puhl.
2002, 156:201-202.
[50] Sun Q, Ma J, Campos H, Hankinson SE, Hu FB: Compari son between p lasma and
erythrocyte tally acid content as biomarkers of fatty acid intake in US women. Am . .f.
C/in. Nutr. 200 7,86:74-8 1.
(5 1] Saadat ian-Eiahi M, S limani N, Chajes V, et al.: Pl asma phospholipid fatty acid profiles
and their association w ith food intakes: results from a cross-sect ional study within th e
European Prospective Invest igat ion into Cancer and Nutriti on. Am . .f. Clin. Nutr. 2009,
89:33 1-346.
(52] Agatha G, Hafer R, Zint l F: Fatly acid composit ion of lymphocyte membrane
phospholipids in children w ith acute leukemia. Cancer Lett. 200 I, 173:139- 144.
(53] McC ii nton S, Mo !Tat LE, Horrobin DF, Manku MS: Abn om1alit ies of essential fally
acid distribut ion in the plasma phospholi p ids of patients w ith bladder cancer. Br. .f.
Cancer. 199 1,63:3 14-3 16.
(54] Murphy RA, Bureyko T F, Mourtzakis M, et al.: Aberrat io ns in plasma phospholipid
Fatty acids in lung cancer pat ients . Lipid.~. 20 12, 47: 363-369.
1!3
(55] Zuijdgeest-van Leeuwen SD, van der Heij den lviS, Rietveld T, et al. : Fatty acid
composit ion of plasma lipids in pat ients w ith pancreat ic, lung and oesophageal cancer
in compari son w ith heal th y subjects. C/in. Nutr. 2002, 21 :225-230.
(56] Lisboa AQ, Rezende M, Muni z-Junqueira Ml, lto MK: A ltered plasm a phospholipid
fatty acids and nutritional status in pati ent s w ith uterine cervical cancer. Clin. Nutr.
2008,27:37 1-3 77.
{57] Cvetkovic Z, Vucic V, Cvetkovic B, et al.: Abnormal fatty acid distribution of the
serum phospholipids of pati ents with non-Hodgkin lymphoma. Ann Hematol. 2010,
89: 775- 782.
(58] W irfalt E, Vessby B, lvlatt isson I, et al. : No relations between breast cancer risk and
fatty acids of erythrocyte membranes in postmenopausal women of the Malmo Diet
Cancer cohort (Sweden). Eur. .f. Clin. Nutr. 2004, 58:7 6 1-770.
(59] Norrish A E, SkeaffClvl, Arribas GL, Sharpe SJ, Jackson RT: Prostate cancer risk and
consumpt io n of fish oi ls: a di etary biomarker-based case-control study. Br. J. Cancer.
1999,8 1:1 238- 1242.
[60] Godl ey PA, Campbell lv!K, Gall agher P, et al.: Biomarkers of essential fatty acid
consumpt ion and risk of prostatic carcinoma. Cancer Epidemiol. Biomarkers Pre11.
199 6, 5:889-895.
(6 1] Fuhm1an BJ, Barba lvl, Krogh V, et al.: Erythrocyte membrane phosphol ipid
composit ion as a hio marker of dietary fat. Ann. Nutr. Metab. 2006, 50:95-l 02.
(62] Pala V, Krogh V, Muti P, et al.: Erythrocyte membrane fatty acids and subsequent
breast cancer: a prospect ive Italian stud y. .!. Natl. Cancer Inst. 2001, 93: 1088- 1095 .
[63] Simonsen N, van't Veer P, Strain JJ, et al.: Adipose t issue omega-3 and omega-6 fatty
acid content and breast cancer in the EVRAM IC study. European Commun ity
Mult icenter Study on Antioxidants, lvlyocardial Infa rct ion, and Breast Cancer. Am. J.
Epidemiol. 1998, 147:342-352.
[64] Kl ein V, Chaj es V, Germain E, et al. : Low al pha-linolenic acid content of adipose
breast tissue is associated w ith an increased ri sk of breast cancer. Eur. .f. Cancer. 2000,
36:335-340.
[65] Pratt VC, Watanabe S, Bruera E, et al. : Plasma and neutrophil fatly acid composit ion in
advanced cancer patients and response to fish oil supplementat ion. Br. .f. Cancer. 2002 ,
87 :1370- 1378.
(66] Cvetkovic B, Vucic V, Cvetkov ic Z, Popovic T, Gl ibet ic M: Systemic alterations in
concentrations and distribution of plasma phospholi pids in prostate cancer pat ients .
Med. Oncol. 2011.
[67] Cvetkovic Z, Cvetkovic B, Petrovic lvl, et al.: Lip id profi le as a prognost ic factor in
cancer patients . .! BUON. 2009, 14 :50 1-506.
[68] Murphy RA, Mourtzakis lvl , Chu QS, Reiman T, Mazurak VC : Skeletal muscle
depletion is associated w ith reduced plasma (n-3) fatty acids in non-small cell lung
cancer patients . .! Nutr. 20 l 0, 140: 1602- 1606.
[69] MacDonald N, Easson AM, Mazurak VC, Dunn GP, Baracos V E: Understanding and
managing cancer cachexia . .! Am. Call Surg. 2003, 197:143- 16 1.
{70] Marra CA, de A laniz MJ, Brenner RR : Modulation of delta 6 and delta 5 rat liver
microsomal desaturase act ivities by dexamethasone-i nduced facto r. Biochim. Biophys.
Acta. 1986, 879:388-393.
11 4
(71 ] Murphy RA, W il ke MS, Perrine lv!, et al. : Loss of adipose t issue and plasma
phospho lipids: relat ionship to survival in adva nced cancer patients. Clin. Nutr. 20 I 0,
29 :482-487.
(72] Ak inseie JA, Ion G, W itte TR, Hardman W E: Consumpt ion of high omega-3 fally acid
diet suppressed prostate tumorigenesis in C3(1 ) Tag mice. Carcinogenesis. 20 12,
33: 140- 148.
(73] Hardman WE : (n-3) fatty ac ids a nd cancer therapy. ./. Nutr. 2004 , 134:3427 S-3430S.
{74] Murphy RA, Mo urtzakis M, Chu QS, et al. : Nutrit ional inte rvention w ith fish o i I
provides a benefit over sta ndard of care for weight and skeletal muscle mass in pat ients
with nonsmall ce ll lung cancer receiving chemotherapy. Cancer. 20 11 , 11 7: 1775- 1782.
{75] Ryan A lvt, Reyno lds JV, Healy L, et al.: Enteral nutrit ion en riched with
eicosapentaenoic acid (E PA) preserves lean body mass following esophageal cancer
surgery: results o f a double-bli nded randomized controll ed trial. Ann. Surg. 2009, 249:
355-363.
[76 ] Weed HG, Ferguson M L, Gaff RL, et al.: Lean body mass gai n in pat ients w ith head
a nd neck squamo us cell cancer treated per ioperat ively w ith a p rotein- and energyd ense nutrit ional supplement containing eicosapentaeno ic acid . Head Neck. 20 11, 33:
1027- 1033 .
(77] Read JA, Beale PJ, Vo lker DH , et al. : Nutrition intervention using an eicosapentaenoic
ac id (EPA)-containing supple ment in patients w ith advanced colorectal cancer. Effects
on nutri tional and inflammatory status: a phase 11 trial. Support Care Cancer. 2007,
15 :30 1-307.
[78] Barber lv!D, Fearon KC : Tole rance and incorporat ion of a high-dose eicosapentaeno ic
acid d iester emulsion by pat ients w ith pancreat ic cancer cachexia. Lipids. 200 1, 36:
347-35 1.
(79] Ries A, Trol!enberg P, Elsner F, et al. : A systematic review on the ro le of fish o il for
the treatment o f cachexia in advanced cancer: An EPCRC cachex ia guidelines proj ect.
Pallial. Med. 2011.
{80] Popovic T, Ranic M, Bu lajic P, et al. : E ffects of n-3 Fatty Acids Supplementation o n
Pl asma Phospho lipids Fatty Acid Composit ion in Pat ients w ith Obstructive Jaundice- a
Pil ot Stud y. .f. C/in. Biochem. Nutr. 2009, 45:3 70-3 75.
{8 1] Murphy RA, M ourtzakis M, Mazurak VC: n-3 po lyunsaturated fatty acids: the potent ial
ro le for supp lementat ion in cancer. Curr. Opin. Clin. Nun. Metab. Care. 20 12.
{82] Barber M D, Fearon KC, T isdale MJ, Mclvtillan DC, Ross JA: Efteci of a .fish o ilenriched nutrit ional supp lement on metabo li c medi a tors in patients with pancreat ic
cancer cachexia. Nutr. Cancer. 2001, 40: 11 8- 124.
{83] S iddiqui R, Pandya D, Harvey K, Zaloga GP: Nutrit ion modulat ion o f
cachex ia/proteo lysis. Nutr. Clin. Pract. 2006, 2 1: 155- 167 .
{84] Dewey A, Baughan C, Dean T, Higgins B, Johnson !: Eicosa pentaenoic acid (EPA, an
omega-3 fatty acid from fish o ils) for the treatment of cancer cachexia. Cochrane
Database Syst. Rev. 2007:CD00459 7.
{85] W igmo re SJ, Barber MD , Ross JA, T isdal e i'v!J, Fearon KC: Etfect o f o ral
eicosapentaeno ic acid on weight loss in pat ients w ith pancreatic cancer. Nutr. Cancer.
2000, 36: 17 7- 184.
{86] Wigmore SJ, Ross JA, Falconer JS, et al. : The eff ect of po lyunsaturated fau y ac ids on
the progress o f cachexia in patients with pancreatic cancer. Nutrition. 1996, 12:S27-30.
11 5
(87] Gogos CA, G ino poulos P, Salsa B, et al.: Dietary omega-3 polyunsaturated fatty acids
plus vitamin E restore immunodeficiency and prolong survival for severely ill patients
with generali zed mali gnancy: a randomized control trial. Ca ncer. 1998, 82:395-402.
(88] Spencer L, Mann C, Metcalfe M, et al.: The effect of omega-3 FAs on tumour
angiogenesis and their therapeut ic poten tial. Eur. .f. Cancer. 2009, 45:2077-2086.
{89] l\l!cDougall SR, Anderson A R, Chaplain MA: Mathemat ical modelli ng of dynamic
adapt ive tumo ur-i nduced angiogenesis: cli nical impli cat ions and therapeutic targeting
strategies . ./. Theor Bioi. 2006, 24 1:564-589.
{90] Ferrara N : Vascular endotheli al growth factor : basic science and clinical progress.
Endocr. Rev. 2004, 25:58 1-6 11.
(9 1] Baritaki S, S ifakis S, Huerta-Yepez S, et al.: Overexpression of VEGF and TGF-beta l
mRNA in Pap smears correlates with progression of cervical intraepitheli al neoplasia to
cancer: imp licat ion ofYY I in cervical tumorigenesis and H PV infect io n. /nt. .f. Oncol.
2007, 3 1:69- 79.
(92] Giatromanolaki A, Koukourakis Ml, S imopoulos C, Polychron idis A, S i,7idis E:
Vascular endotheli al growth factor (V EGF) expression in operable gall bladder
carcinomas . Eur. .f. Surg. Oncol. 2003, 29:879-883.
[93] Jovino F, Ferraracc io F, Orditura M, et al.: Serum vascular endotheli al growth factor
(V EG F) leve ls correlate w ith tumor V EG F and p53 overexpression in endocrine
posit ive primary breast cancer. Cancer In vest. 2008, 26:250-255 .
(94] Tang RF, ltakura J, A ikawa T, et al.: Overexpression of lymphangiogen ic growth factor
VEG F-C in human pancreat ic cancer. Pancreas . 2001, 22:285-292.
[95] W ong C, Well man T L, Lounsbury KM: VEGF and HrF- Ialpha expression are
increased in advanced stages of ep ithelial ovarian cancer. Gy necol. Oncol. 2003 , 9 1:
5 13-5 17.
[96] T suzuk i T, Shibata A, Kawakami Y, Nakagaya K, Miyazawa T: Anti-angiogen ic e ffects
of conj ugated docosahexaenoic acid in vitro and in vivo. Biosci. Biotechnol. Biochem.
2007, 71: 1902- 19 10.
(97] Yang S P, Morita I, Murota S I: Eicosapentaenoic acid au enuates vascu lar endotheli al
growth factor-i nduced proli feration via inhibiting Flk- 1 receptor expression in bovine
carotid artery endothelial cell s . ./. Cell Physiol. 1998, 176:342-349.
[98] Calviell o G, Di Nicuolo F, Gragnoli S, et al.: n-3 PUFAs reduce VEG F expression in
human co lon cancer cell s modulating the COX-2/PGE2 induced ERK- 1 and -2 and
H rF- 1alpha induction pathway. Carcinogenesis. 2004, 25:2303-23 10.
(99] T evar R, Jho D H, Babcock T, Helton WS, Espat N J: Omega-3 fatty acid
supp lementat ion reduces tumor growth and vascu lar endotheli al growth factor
expression in a model o f progress ive no n-metastasizing mali gnancy . .!PEN .f. Parenter
Enteral Nutr. 2002, 26:285-289.
{l OO] Ambri ng A, Johansson M, Axelsen M, et a l.: Medi terranean-inspired diet lowers the
rat io of serum phospholipid n-6 to n-3 fally acids, th e number of Jeukocytes and
platelets, and vascular endotheli al growth factor in healthy subj ects . Am . .f. C/in. Nutr.
2006, 83:57 5-58 1.
{10 1] Terano T, Shii na T, Tamura Y: Eicosapentaenoic acid suppressed the proli ferat ion of
vascular smooth muscle cell s through modulat ion of vari ous step s of growth signals .
Lipids. 1996, 3 1 Supp l :S30 1-304.
11 6
{!02] Hendrickse CW, Kell y RW, Radl ey S, et al.: Lipid perox idat ion and prostaglandins in
co lorectal cancer. Br. .f. Surg. 1994, 81 : 1219 - 1223.
( 103] Vinogradova Y, Hippisley-Cox J, Coup land C, Logan RF: R isk ofco lorectal cancer in
patients prescribed stat ins, nonsteroidal ant i-inflammatory drugs, and cyclooxygen ase-2
inh ibitors: nested case-control study. Gastroenterology. 2007, 133:393-402.
( 104] Fini L, Piazzi G, Ceccarelli C, et al.: High ly purified eicosapentaenoic acid as free fally
ac ids strongly suppresses polyps in Apc(Min/+) mice. C/in. Cancer Res. 2010, 16:
5703-57 11.
{105] Hawcrofi G, Loadman PM, Belluzzi A, Hull MA: Hrect ofeicosapentaenoic acid onEtype prostaglandin synthesis and EP4 receptor signaling in human colorectal cancer
cell s. Neoplasia. 20!0, 12:6 18-627.
{106] Narayanan BA, Narayanan N K, Simi 8 , Reddy BS: Modulation of induc ible nitric
ox ide synthase and related proinflammatory genes by the omega-3 fally acid
docosahexaenoic acid in human colon cancer cell s. Cancer Res. 2003, 63:972-979.
( 107] Yao HT, Chang YW, Lan SJ, et al.: The inh ibitory effect of polyunsaturated tatty acids
on human CYP enzymes. Life Sci. 2006, 79:2432-2440.
( 108] Hammamieh R, Chakraborty N, M ill er SA, et al.: Differenti al effects of omega-3 and
omega-6 Fauy ac ids on gene expression in breast cancer cell s. Breast Cancer Res Tre<lt.
2007, 10 1:7- 16.
{109] Schley PD, Br indley DN, Fie ld CJ: (n-3) PUFA alter raft lipid composit ion an d
decrease epidermal growth factor receptor levels in lipid rafts of human breast cancer
cell s. J. Nutr. 2007, 137:548-553.

Edi tors: T.G. Bradley and F.P. Vargas, pp. I I 7- 127
ISBN: 978- I -62257-480-3

Chapter 5
R OLE OF EICOSAPENTAENOIC ACID

IN MENTAL H EALTH
.2 t
Kalpana Poudel-Tandukar I' and Krishna C. Poudet'
1
Waseda Institute for Advanced Study, Waseda University,

Shinj uku-ku, Tokyo, Japan
2
Departrnent of Public Health, School of Public Health and Health Sciences, Un iversity
of Massachusetts, Amherst, MA, USA
Abstract
!merest in the role of polyunsamrated fatry acids (PUFAs), particularly long-chain n-3
PUFAs, in memal health is increasing. Epidemiologic and case-control data suggest that
increased dietary imake of n-3 PUFAs may be of benefit in alleviating different memal health
problems. However, the results of randomized controlled trials are inconsistent and
controversy exists as to whether either eicosapemaenoic acid (EPA) or docosahexaenoic acid
(DHA) or both are responsible for the reported benefits. Evidence is growing for the more
intluemial role of EPA in cognition, behavior, and mood. T here are many lines of evidence
indicating why EPA might be more beneficial than DHA in mental health. This chapter
discusses brietly the beneficial roles of EPA in mental health and, where known, the
underlying mechanisms. T his is followed by a review of the emerging literature on the
potemial therapeutic utility of pure EPA in different mental disorders or illnesses.
Introduction
Increasing ev idence suggests that P UFAs are important for brain development and
function (Bourre et al., 199 1; Yehuda et al., 1999). The brain conta ins a high concentrat ion of
PUFAs (approx imately 20 percent of dry weight). One out of every three latty acids belongs
to the PUFA group (Bourre et al., I 99 I; Yehuda et al., I 999). PUFAs are required for the
nom1al development of the brain and the structure of every phospholi pid membrane in the
body. Important ly, neuronal membranes are largely made up of phospholipids (Horrob in et
~ Email
address-es: katpana@aoni. \V~eda.jp; kkpoudel@hotmail.com.

t Email address: kcpoudel@hotmail.com.
118
Kalpana Poudei-T andukar and Krishna C. Poudel
al., 1994). In the brain, the P UFA metabolites, namely EPA and docosahexaeno ic acid
(DH A), are considered to be the most important PU FAs in relat ion to brain funct ion. EPA has
important physiological funct ions that can affect neuronal act ivity; it helps in balancing the
immune function and physical health hy reducing membrane arach idonic acid (AA) and
prostaglandi n E 2 synthesis (Farooqui et al., 2006). DHA is a major structural component of
neuronal membranes and its deficit is associated w ith d ysfunctions of neuronal membrane
stab ility and transmission of serotonin, norepinephrine, and dopamine (H orrobin and Bennett,
1999; Chalon, 2006; Suet al., 2003).
Many lines of evidence suggest that EPA m ight be more beneficial than DH A for several
mental health problems li ke depression, schizophren ia, and mood disorder w ith respect to the
inflammatory hypothesis of disease causation (Bennett and Horrobin, 2000; Lin and Su,
2007; Martins, 2009). The inflanmmtory markers are increased in depression, and
inflammatory cytokines can produce depressive symptoms in humans (M ill er et al., 2009;
Maes and Smi th, 1998; Raison et al., 2006). In th is vein, there are a number of known EPA
effects that may have relevance for the pathophysiology of menta l health problems.
Fi rst, EPA has been considered as an important compet itor w ith arachidon ic acid ( Peet
and Horrob in, 2002a), abnom1alit ies in wh ich can cause mood dysregulat ion (Lee et al., 2007;
Rapo port et al., 2009; Sublette et al., 2004). EPA competes with arach idonic ac id fbr cyclooxygenase, increasing production of anti-inflammatory prostaglandins (Peet and Horrobin,
2002a; Smith, 2005). Second, EPA-der ived eicosanoids, un like the A4/ J4 neuroprostanes
derived from DHA and ox idi zed derivat ives of EPA, have benefici al ant i-inflammatory
effects (Calder, 2006). Th ird, EPA is more effective than DHA at reducing the inflammatory
cytokines tumor necrosis factor-alpha (TN F-al pha), IL-6, and IL- Ib, an act ion that occurs via
the mechan ism of EPA inh ibit ing the acti vity of nuclear !actor kappa-B (N FkB) (Zhao et al.,
2004). Fourth, al though dietary EPA and DHA incorporate into cell membranes w ith equal
facili ty, dietary EPA is more effect ive at reducing inflammat ion in vivo (S ierra et al., 2008).
Finall y, EPA has neuroprotect ive actions on lipopolysaccharide (L PS)-i nd uced hippocampal
dysfun cti on through the prevention of LPS-induced phosphorylat ion of c-Jun N-terminal
kinase, c-Jun, and Bcl-2, wh ich in turn prevents the secretion of interleuk in I B ( Il - l B),
prevents increases in mitochondrial membrane pem1eability, prevents release of cytochrome
C, and prevents neuronal apoptosis ( Lonergan et al., 2004). Based on these stands of
evidence, several investigators have argued that EPA m ight be more effect ive than DHA for
the treatment of mental health problems.
Depression
Depression is characteri zed by feelings of unhapp iness, loss of energy and interest,
fatigue, poor concentration, altered appetite, s leep disturbances, diminished cognit ive
function, weight gain/l oss, anxiety, agitat ion or irritability, chron ic indecisiveness, and o ften,
suicidal ideat ion (Bruinsma and Taren, 2000; Stoll et al., 1999). Depression is the most
common of all mental health disorders and one of the leadi ng causes of disability worldw ide,
''"i th a high li fet ime prevalence rate (The World Health Report, 200 I).
Rol e of Eicosapentaenoic Acid in lvlental Heallh
11 9
Major Depressive Disorders

Maj or depression (also known as clinical depression or un ipolar depression) is a severe and
persistent f()rm of depression. According to the Diagnost ic and Statistical Manual of lvlental
Disorders, founh edition (DSM-IV), pub lished by the American Psych iatric Association, major
depression is defined as two or more weeks of low mood or dimin ished interest in usual
activit ies, combined with four or more of the follow ing symptoms: s leep alteration ( increased or
decreased); inappropr iate gui it or loss of self-esteem, allered appetite (increased or decreased);
diminished energy; di minished concentrat ion; psychomotor symptoms (either agitation or
retardat ion); or suicidal ideat ion (National inst itute of l'vlental Health, 2001).
The World Heallh Organ izati on has estimated that maj or depressive disorder (MDD) will
become the second leading cause of disab ility wor ldwide by 2020, and w ill be the leading
cause in develop ing regions (Murray and Lopez, 1997). A growing prob lem globall y, MDD
has been linked to lower levels of n-3 PUFAs including EPA and DHA in serum an d red
blood cell membranes (Maes et al., 1999; Peel et al., 1998). Abnom1ali t ies in PU FA
composit ion in cell membranes can aller membrane microstructure, cause abnom1al signal
transduct ion and immunologic d ysregu lation, and possibly increase the risk of developing
depression (Horrobin and Bennell, 1999; Logan, 2003). Ecologic and observat ional studi es
suggest that greater intake of n-3 PUFAs is associated with reduced risk of depression
(H ibbeln, 1998; S i Jvers and Scou, 2002), cross-sect ional studies have shown an associat ion
between reduced n-3 PUFA in serum and the occurrence of maj or depressi on (Maes et al.,
1996; 1999; Adams et al., 1996; Edwards et a l., 1998), and randomized controll ed trials
(RCTs) provide evidence of beneficial effects ofn-3 PUFAs compared w ith placebo (Stoll et
al., 1999; Su et al., 2003; Frangou et al., 2006). However, other ecologic and observat ional
Siudi es that suggest no association between n-3 PU FAs and depression can also be idenl i fied
(Hakkarainen et al., 2004; lvliyake et al., 2006; Assies et al., 2004), and RCTs have li kew ise
been pub li shed that suggest no benefits (S il vers et al., 2005; Grenyer et al., 2007). Further,
three meta-analyses that combine the avail able evidence did not show any benefit of n-3
PUF A supplementat ion for depression ( Freeman et al., 2006; Ross et al., 2007; Lin et al.,
2007; lvlartins, 2009; Appleton et al., 2010).
Interest is growing on specific n-3 PUFAs such as EPA because of increasing evidence
on its potent ial role in all eviat ion of depression (Ross et al., 2007; Mart ins JG, 2009). There
are relat ively few stu dies that have assessed the effect of n-3 PUFA mono-therapy, using
either EPA or DHA, for treatment or prevention of depression. The first study that showed the
effectiveness of EPA in a therapeut ic case study of depression was by Puri and coll eagues
(Puri et al., 2001), wherein they added EPA to the conventi onal antidepressant treatment of a
treatment-resistant severely depressed male pat ient. The addition of ethyi-EPA led to
sustained clinical improvement in all the symptoms of depression, including suicidal ideat ion
and social phob ia, w ith in one month. Subsequently, a number of double-bli nd, placebocontroll ed trials have been conducted that have demonstrated the effecti veness of EPA for
treatment of depress ion.
The fi rst doub le-blind placebo-controll ed trial to test the effect iveness of EPA in MDD
was conducted by Peel and Horrobin (Peel and Horrobin, 2002a). They administered I, 2, or
4 g!day ofethyi-EPA or a li qu id paraffin placebo for 12 weeks to 70 pati ents w ith MDD who
had persi sten t symptoms of depression. In th is tria l, the authors reported that the lower dose
120
Kalpan a Poudei-T andukar and Krishna C. Poudel
of ethyi-E PA (I g/day), but not the higher doses (2 or 4 g/day), was sign ificantly more
effect ive than placebo to improve depression symptoms rated using the Hamilton Depression
Rat ing Scale (HDRS), the Montgomery-Asberg Depression Rat ing Scale (MADRS), and th e
Beck Depression Inventory (BD[), w ith the effect of therapy being apparent at the ear liest
measured interval (4 weeks). The ethyl E PA reduced the H DRS score by 3.8 points more than
the placebo. Furthermore, a higher proport ion of pat ients receiving I g/day bad a 50%
improvement in their symptoms compared to the placebo group (9/ 17 vs. 5/ 17). In another
trial among 20 medicated patients with MDD, Nemets and coll eagues provided a 2 g/day dose
of etbyi-EPA or placebo for 4 weeks (Nemets et al., 2002). They found a sign ificant reduction
in depress ion symptoms as measured by the HDRS score in the EPA group compared to the
placebo group afl er 2 and 3 weeks of treatmen t. A sign ificantl y higher proport ion of patients
reported 50% reduction in symptoms in the EPA compared to the placebo group. The
magn itude of the di fference between the EPA and placebo groups was not small ; ethyl EPA
reduced the H DRS score by I 0.8 po ints over th e placebo. The authors used per-protocol
analysis and excluded participants wh o did not complete the tria l, rather than the more w idely
accepted intent-to-treat analysis.
In a latest clinical tri al, .lazayeri and coll eagues administered a dail y dose of either 1000
mg EPA or 20 mg fluoxeline, or a combinat ion thereof, to 60 patients with a diagnosis of
/VIDD (Jazayeri et al., 20 08). The patients were assessed at 2-week intervals for 8 weeks using
DSM -IV criter ia and a cut-off score > 15 on the ! 7-item Hamilton Depression Rat ing Scale
(HDRS). The comb ination of EPA and fluoxetine bad better therapeut ic effects in MDD
compared w ith either treatment alone. The response rates (>50% decrease in baseline HDRS
score) were 50%, 56%, and 81% in the fluoxetine, EPA, and combination groups,
respectively. Recently, l\1lischou lon and co ll eagues carried out a double-blind randomized
controll ed trial of ethy l-eicosapentaenoate in 57 adults with MDD, using a dose o f l g of EPA
or placebo for 8 weeks (M ischoulon et al., 2009). HDRS scores decreased from 21.6 +/ -2.7to
13.9 +/ -8.9 Jor the EPA group and from 20.5+/ -3.6 to 17.5+/ -7.5 for the p lacebo group
(p=O. I23); the etTect size for EPA was 0.55 in the intent ion to treat analysis group (n=35) .
HDRS scores decreased from 21.3+/ -3.0 to 11.1 +/ -8. 1 for the EPA group and from 20.5+/ -3 .8
to 16.3+/ -6.9 for the placebo group (p=0.087); the e!Tect size for EPA was 0.73 in the study
completers group (n=24). The effect iven ess of EPA over placebo did not reach statist ical
sign ificance in th is trial, possibly due to the small sample size and low comp leters rate. In all
of these trials w ith posit ive results, treatment responses were rapid, w ith di flerences observed
in a minimum of two weeks; effect sizes were large; and no sign ificant adverse side effects
were reported.
Bipolar Disorder (Manic Depression)

Bipolar di sorder (or manic depression) is characterized by extreme mood swmgs,
al ternating between per iods of man ia and periods o f depression. Accordi ng to DSM-IV,
man ia is defined as a distinct change in mood and functi on ing, lasting at least one week, and
is characterized by a euphoric or irritable mood accompan ied by symptoms such as increased
energy, decreased need for sleep, rap id th inking and speech, grandiosity, poor j udgement and
impulsivity, an d in some cases, psychosis (i.e., delus ions and/or hall ucinations) (Nat ional
Institute of Mental Health, 2001).
121
Studies in bipolar di sorder have reported an anti-depressant eflect of EPA similar to that
observed in lVfDD. EPA supp lementation in bipolar di sorder has been reported to increase
brain N-acetylaspartate (Frangou et al., 2007), a marker for neuronal heallh. For examp le,
Frangou et al. provided the Hrsl evidence of a probable neurotroph ic role for ethyi-EPA in
treatment of bipolar dep ression (Frangou et al., 2007). They observed a sign ificant rise inNacetyl-aspartate in the ethyi-EPA treatment group compared w ith the placebo group, wh ich is
a marker of neuronal integrity. Frangou et al. conducted a double-blind, randomized placebocontroll ed trial of adjunct ive EPA in 75 patients w ith bipolar depression (Frangou et al.,
2006). They administered either I g or 2 g/day of ethyl EPA or placebo among pat ients
randomly assigned to the three treatment groups for 12 weeks. The results indicated that
neither dose of EPA had any statisticall y sign ificant effect upon mania as measured by the
Young Man ia Rating Sca le (YMRS), but EPA groups did have stat ist ically sign ificant
improvements in HDRS, Ylv!RS, and C linical G lobal Impressions scale scores compared to
the placebo group. In contrast, another previous trial (Keck et al., 2006) did not find any
di fference in benetlt among the intervent ion as compared to the placebo groups. Keck and
coll eagues con ducted a double-bli nd, p lacebo-controlled trial of 4-month treatment w ith
either 6 g/day of ethyi-EPA o r a liquid para!lin placebo among 95 patients w ith bipolar
depression or 86 pat ients w ith rap id-cycli ng bipolar disorder (Keck et al., 2006). The authors
reported no s ign ificant di fferences in man ic or depression symptoms in the E PA group versus
the placebo group, assessed using the YMRS and the Inventory of Depression
Symptomatology, respectively. The no-benefit resu lt in th is trial may be due to us ing too high
a daily dose of the Iipid in the absence of sufficient quantities of DHA or other component of
the crude oil (Ross et al., 2007). Overall , the results of the bipo lar trials are inconsistent
regarding the beneficial effect ofEPA.
Schizophrenia
Schizophren ia is a debil italing condition characterized by perceptual and behavioral
di sturbances, conceptual disturbances, impaired ability to commun icate, and social/occupational
dysfunction. The di sease is ep isodic and is characterized by two classes of symptoms: positive
(wh ich includes hall ucinations, delusions, and disorganized thought and behavior) and negative
(including llattened mood, poverty of speech, and deiicits in goal-directed behavior), the latter
of wh ich account for most of the morbidity associated w ith the illness (Horrobin, 1998).
Generally, diagnost ic criter ia for schizophren ia include at least two of the foll owing "active
phase" symptoms that persist f()r a sign ificant portion of t ime during a one-month period:
delusions, hallucinat ions, disorgan ized speech, disorgan ized or cataton ic behavior, or negative
symptoms (i.e., affective Aauen ing, alogia, or avolit ion). Only one of these symptoms is
required if it is accompan ied with hearing a voice that keeps a runn ing commentary on the
person's behavior or thoughts, or if two or more voices are talking w ith each other.
Mechan ist icall y, it has been suggested that schizophren ia is a pro- infl ammatory
condit ion, which is supported by increased risk of autoimmune disorders in iirst-degree
relatives of schizophren ic patients, and increased levels of pro- inflammatory cytok ines in
schizophren ic pat ients (Gaughran, 2002). EPA may have protective effect in sch izophren ia
through its anti-inflammatory effect. it has also been suggested that EPA is work ing as an
122
Kalpana Poudei-T andukar and Krishna C . Poudel
inhibitor of phospholipase A2, wh ich is known to be e levated in pat ients w ith schizophren ia
(Bennett and Horrobin, 2000).
Studies have suggested a more beneficial effect of EPA compared w ith DHA or placebo
in treati ng schizophren ia. Fi ve o f six doub le-bli nd, placebo-controlled trials have reported the
therapeutic benefit trom E PA in treating sch izophrenia, part icularly in the context of
comorbidi ty w ith an exist ing psychotropic condit ion (Peet et al., 200 I; Emsley et al., 2002;
Peet and Horrobin, 2002b; Fenton et al., 200 1; Berger et al., 2004). Peet and col leagues
randomly assi gned 45 pat ients w ith schizophren ia, hitherto in receipt of conventional ant ipsychot ic medications, to treatment w ith adjunctive EPA, DHA, or p lacebo ( Peet et al.,
200 I). They compared the effect of giving an EPA-enriched oil containing 2 glday ofEPA, a
DHA-enr iched oi l containing 2 glday of DH.A, or a corn-oil placebo fur 3 months. The
Posit ive and Negative Syndrome Scale (PANSS) scores were sign ificantly lower in EPA
groups than in the placebo group, although the di fference was not large. The change from
baseli ne symptom scores for the EPA-group minus the change from baseli ne scores was -4 .1
for the placebo group and +2.2 for D HA group. One further tr ial that administered a higher
dose of ethyi-EPA (3glday) or placebo in 40 medicated pati ents w ith schizophren ia reported
the benefi t of addi ng EPA to the antipsychotic medicat ion in tem1s o f overall improvement in
symptoms (Emsley et al., 2001). They reported improvement after 12 weeks of treatment, not
only in pat ients w ith schizophrenia but also in those with tardive dyskinesia, wh ich is a
movement disorder seen in sch izophrenic pat ients treated long-tem1 w ith older antipsychot ic
drugs (Emsley et al., 2002).
Peet and Horrobin followed up on previous research in trials that showed a dose-ranging
effect of EPA in 11 5 pat ients w ith treatment-resistant schizophrenia (Peet and Horrobin,
2002b). Patients received I, 2, or 4 glday of adjunctive ethyi-EPA or a liquid paratlin placebo
tor 12 weeks. Th is trial reported that treatment w ith 2glday of EPA (but not I or 4 glday)
resulted in improvements from baseline scores among patients who were receiving
ant ipsychotic clozap ine treatment (Peel and Horrobin, 2002b). In a further study by Berger and
colleagues, ethyl EPA or placebo was given to first-episode sch izophren ic patients together w ith
standard treatment with risper idone (Berger et al., 2004). They observed that patients who
received ethyl EPA required sign ificantly lower dosages of ant ipsychotic medicat ion to produce
the same cli nical benefit than those given the placebo (Berger et al., 2004).
In contrast, one study foun d no benefi t trom adding 3glday of ethyl E PA to treatment
\vi th existing antipsychotic medi cation among 87 patients w ith schizophren ia (Fenton et al.,
2001). Th is trial found no sign ifi cant di fference in PANSS scores between the EPA and
placebo groups at any of the reported t ime points.
Other Psychiatric Disorders

Borderli ne Personality Disorder, accordi ng to DS M-!V, is characterized by a pervasive
pattern of unstable interpersonal relat ionships, self image, and behavior. it is a type of mental
illness in wh ich patients fom1 infl exi ble patterns of act ions and thoughts (American
Psychiatric Associat ion, 1994). T o date, only one tr ial has tested the e fli cacy of EPA in
treating personality disorders . Zanarini and Frankenburg randomized 30 femal e outpatients
ident ified as having borderli ne personality disorder (Zanarini and Frankenburg, 2003).
Pat ients received either l glday of ethyl EPA or placebo for 8 weeks and were followed at 2,
Role of Eicosapentaenoic Acid in lvlenlal Heallh
123
3, 4, 6, and 8 weeks to assess for aggression and depression using the lvlodi fied Overt
Aggression Scal e (lv!OAS) and the Montgometry-Asberg Depression Scale (lv!OAS). The
researchers reported that ethyl EPA was more effective in reducing aggression as well as !he
sever ity of dep ression symptoms than the placebo .
On another tronl, obsessi ve--compulsi ve disorder (OC D) is an anxiety disorder
character ized by obsessive thoughts and compulsi ve actions (e.g., cleaning, order ing,
counting) that produce uneasiness, apprehension, fear, or worry, by repetit ive behaviors
ai med at reducing the associated anx iety or distress. A si ngle placebo-controll ed cross-over
trial of adjunct ive EPA was conducted in eleven pat ients w ith cu rrent OCD (Fux et al., 2004).
The patients were randomly all ocated to treatment with either 6 weeks of placebo (2 g liquid
paraftin per day) fullowed by 6 weeks of2 g of EPA, or w ith EPA fullowed by placebo. The
resulls of th is study indicated no clini cal benefit of EPA in treati ng OC D.
Conclusion
The di (ferent studi es oull ined above as well as several meta-analysis studi es (Ross et al.,
2007; lvlart ins, 2009) suggest that EPA may be an etfect ive PUFA component in the
treatment of psychiatric disorders. A meta-anal ysis study by Ross et al. that included I 0
studi es on lv!DD or bipol ar trials reported that the predicted overall standardized mean
di fference (i.e. Slv!D: the mean di fference between P UFA and placebo groups scaled to the
standard deviat ion) for treatment w ith EPA or a predominant ly EPA mixture used wou ld be
1.1 8, whereas for a D HA or predom inantly DHA m ixture, the predicted overall S lviD would
be 0.06 (Wald test for di fference: p=0.007) (Ross et al., 2007). This original observation
made by Ross et al. was further confi rmed by another review study done by Mart ins (lvlartins,
2009). In th is review, the symptoms of depression were si gn ifi cantly reduced in 13 studies
using supp lements containing greater than 50% EPA (Sl\.m = - 0.446, 95% C l = - 0.753 to 0. 138, z = - 2.843, p = 0.005) and in 8 studies using pure ethyi-EPA (S MD = - 0.396, 95% C l
= - 0.650 to - 0.14 1, z = -3.05 1' p = 0.00 2).
In agreement with Ross et al. (Ross et al. , 2007) and Mart ins (Martins, 2009), the rec.ent
meta-analysis by Sub lette et al. (Sub leue et al., 20 11) (which included 15 double-bli nded,
placebo-controlled trials) reported that the effects of EPA were stat isticall y sign ificant when
the concentration of EPA was increased to 10% above the DHA level (Sub lette et al., 2011 ).
Supplements w ith EPA > 60% showed benefit on standardized mean depression scores (effect
size = 0.352, 95% C l, 0.277 to 0.733; t=4 .195; p<O.OO I) versus supp lements with EPA < 60%
(effect size = - 0.026, 95% C l, - 0.200 to 0.148; t= - 0.3 16; p=0.756). Mechanist icall y, E PA
and DHA are structura ll y similar and there might be I :I compet it ion between them for an
unknown biological site (Sub lette et al., 2011). Thus, EPA in excess of DHA may be
considered to be unopposed EPA and to be the benefi cial component for mental heal th. The
unopposed doses of EPA were 2200 mg/d and 2800 mg/d in the latter successfu l studies (Stoll
et al., 1999; Suet al., 2003; Sublelle et al., 20 11 ).
In conclusion, E PA and not D HA may be effective fur treating psychiatric disorders based
on overall available evidence. However, further studi es of strong methodological quality and
us ing a larger samp le size are necessary to evaluate long-term efticacy ofEPA supplementation
and to confim1 the effect of EPA in mental health . Furthermore, a direct comparative
randomized trial of EPA versus DHA is also needed to compare the effects of speci fic PUFAs.
124
Kalpana Poudei-T andukar and Krishna C . Poudel
Competing I nterests
The authors declare that they have no compet ing interests.
References
Adams, PB; Lawson, S. Sanigorski A, Si nclair A.l. Arachidonic acid to eicosapentaenoic acid
rat io in blood correlates positively with clinical symptoms of depression.
Lipid.~. l 996;3 1 :S 157-S 16 1.
American Psychiatric Associat ion: Diagnostic and statistical manual of mental disorders
Fourth edition. Washington, DC: American Psychiatric Association; 1994.
App leton, K1vl; Rogers, PJ; Ness, AR . Updated systematic review and meta-analysis of the
effects of n-3 long-chain pol yunsaturated fatty acids on depressed mood. Am J C/in
Nutr.20 I 0;9 1:757- 770.
Assies, J; Lok, A; Bockting, C L; Weverling, GJ; Lieverse, R ; V isser, I; Abeling, NG; Duran,
M; Schene, AH . Fatly ac ids and homocysteine levels in pat ients w ith recurrent
depression: an exp lorative pil ot study. Proscag/.a ndins Leukot Essent Fatty Acids. 2004;
70:349- 356.
Bennett, CN; Horrobin, DF. Gene targets related to phospholipid and fatty acid metabol ism in
sch izophren ia and other psych iatric disorders: an update. Prostaglan dins Leukot Essent
Fatty Acids. 2000;63:47- 59.
Berger, GE; Proffltt, T; Wood, S; et al. Ethyl-eicosapentaenoic acid (E-EPA) supplementat ion
in early psychosis: a double-bli nd randomised placebo-controlled add on study in 80 drug
naive fi rst episode psychosis patients {abstract]. lnt .I Neuropsychopharmacol 2004; 8
Supp l. I : S422.
Bourre, JM ; Dumont, 0 ; Piciotti, M; C lement, M; Chaudiere, J; Bonneil , M; Nalbone, G;
Lafunt, H; Pascal , G; Durand, G . Essentiali ty of omega 3 fatty acids for brain structure
and funct ion. World Rev Nutr Diet. 199 1;66 : I 03- 117.
Bruinsma, KA; Taren, DL. Di eting, essential fatty acid intake, and depression. Nutr Rev.
2000;58:98- 108.
Calder, PC. n-3 polyunsaturated fatly acids, infl ammation, and inflammatory diseases. Am J
C/in Nutr. 2006;83: 1505S- 15 19S.
Cha lon S. Omega-3 !ally acids and monoamine neurotransmission. Prostaglandins Leukot
Essent Fatty Acids . 2006;75 :259- 269.
Edwards, R; Peel, M; Shay, J; Horrobin, D. Omega-3 pol yunsaturated fatty acid levels in the
di et and in red blood cell membranes of depressed patients . .I Affect Disord.
1998;48: 149- 155.
Emsl ey, R; Myhurgh, C; Oosthuizen, P; van Rensburg, SJ. Randomized, placebo-controll ed
study o f ethyl-eicosapen taenoic acid as supplemental treatment in sch izophren ia. Am .I
Psychially. 2002; 159: 1596-1598.
Farooqui, AA; Ong, WY; Horrocks, LA . Inh ibitors of brain phospholipase A2 act ivity: their
neuropharmacological effects and therapeut ic importance for the treatment of neurologic
disorders. Pharmacol Rev. 2006;58:59 1- 620.
125
Fenton, WS; Dickerson, F; Boronow, J; Hibbeln, JR; Knable, M. A placebo-controlled tria l of

omega-3 fally acid (ethyl eicosapentaen oic ac id) supp lementat ion for residual symptoms
and cogni tive impairment in schizophrenia . Am J Psychiatry. 200 1;158: 207 1- 2074.
Frangou, S; Lewis, M; McCrone, P. Effi cacy of ethyl-eicosapentaenoic acid in bipo lar
depression: randomised double-blind placebo-controlled study. Br .! Psychiatry.
2006; 188:46-50.
Frangou, S; Lew is, !vi; Woll ard, .1 ; Simmons, A . Preli minary in vivo evidence of increased Nacetyl-aspartate fo ll ow ing eicosapentanoic acid treatment in pat ients w ith bipolar
di sorder. .! Psy chopharmacoi. 200 7; 4:43 5-439.
Freeman, MP; H ibbeln, JR; Wisner, KL; Davis, JM; Mischoulon, D; Peet, !vi; Keck, PE Jr;
1\,larangell , LB; Richardson, AJ; Lake, J; Stoll, A L. Omega-3 fally acids: evidence basis
for treatment and future research in psychiatry. J C/in Psy chiatry. 2006;67: 1954-67.
Fux, M; Benjamin, J; Nemets, B. A placebo-controll ed cross-over tr ial of adj unct ive EPA in
OCD. J Psychiatr Res. 2004;38:323- 325.
Gaughran, F. Immun ity and schizophrenia: autoimmun ity, cytokines, and immun e responses.
l nt Rev Neurobioi. 2002;52:275- 302.
Grenyer, BF; Crowe, T; Meyer, B; Owen, AJ; Gr igonis-Deane, EM; Caput i, P; Howe, PR.
Fi sh oil supp lementat ion in the treatment of maj or depression: a random ised doublebli nd placebo-controll ed trial. Prog Neuropsychoph armacol Bioi Psy chiatry. 200 7; 3 1:
1393- ! 396.
Hakkarainen, R; Partonen, T; Haukka, J; V irtamo, J; A lbanes, D; Lonnqvist, J. Is low di etary
intake of omega-3 tally acids assoc iated w ith depression? Am .! Psychiatry. 2004;
16 1:567-569.
Hi bbeln JR. Fish consumption and maj or depression. Lancet. 1998; 35 1: 12 13.
Horrobin, DF; G len, A I; Vaddadi, K. The membrane hypothesis of schizophrenia. Schizophr
Res. 1994; 13 :195- 207.
Horrobin, DF. T he membrane phospholipid hypothesis as a biochemical basis for the
neurodevelopmental concept of sch izophren ia. Schiz ophr Res. 1998;30: 193- 208.
Horrobin, DF; Bennett , CN. Depressi on and bipolar disorder: relationships to impaired fa tty
ac id and phospholip id metabo li sm and to di abetes, card iovascular di sease,
immunological abnormalit ies, cancer, ageing and osteoporosis. Possible candidate genes .
Prostagiandins Leukot Essen! Fatty Acids. 1999;60:2 17- 234.
Jazayeri, S; Tehran i-Doost, M; Keshavarz, SA; Hosseini, M; Djazayery, A; Amini, H; .l alali ,
M; Peel, M . Comparison of therapeutic effects of omega-3 fatty acid eicosapentaenoic
ac id and fluoxetine, separately and in comb inat ion, in major depressive di sorder. Aust N
Z.f Psy chiatry. 2008;42: 192- 198 .
Keck, PE Jr; Mintz, J; McE Jroy, S L; Freeman, MP; Suppes, T; Frye, MA; Altshuler, LL;
Kupka, R; Nolen, WA; Leverich, GS; Den ico ft~ KD; Grunze, H; Duan, N; Post, RM.
Doub le-bli nd, randomized, placebo-controll ed trials of ethyl-eicosapentanoate in the
treatment of bipolar depression and rapid cycling bipolar disorder. Bioi Psychia try.
2006 ;60: I 020- 1022.
Lee, HJ; Rao, JS; Rapoport, S I; Bazinet, RP. Ant imanic therapies target brain arachidonic
ac id signaling: lessons learned about the regu lation of brain fatty acid metabolism.
Prostagiandins Leukot Essent Fatty Acids. 2007; 77:239- 246.
Lin, PY; Su, KP. A meta-analytic review of doub le-blind, placebo-controlled trials of
antidepressant et1icacy of omega-3 fauy acids . .! Clin Psychiatry. 200 7;68: I 056- l 06 1.
126
Kalpan a Poudei-T andukar and Krishna C. Poudel
Logan, AC. Neurobehavioral aspects of omega-3 fatty acids: possible mechan isms and
therapeutic value in maj or depression. A/tern Med Rev. 2003;4:410-425.
Lonergan, PE; Mart in, DS; Horrob in, DF; Lynch, Nlt\.. Neuroprotective act ions of
eicosapentaenoic acid on lipopolysaccharide-induced dys funct ion in rat hippocampus . .I
Neurochem. 2004;9 1:20- 29.
lvlaes, lvl; Smi th, R; Christophe, A; Cosyns, P; Desnyder, R; lvleltzer, H. Fatty acid
composit ion in major depression: decr eased omega 3 fractions in cholesteryl esters and
increased C20: 4 omega 6/ C20:5 omega 3 ratio in cholesteryl esters and phosphol ipids. .I
Affect Diso rd. 1996;38:35- 46.
Maes, M; Smith, RS. Fatty acids, cytok ines, and maj or depression. Bioi Psychiatly .
1998;43:3 13- 3 14.
Maes, M; Christophe, A; Delanghe, J; A ltamura, C; Neels, H; Meltzer, HY. Lowered omega3
polyunsaturated fatty acids in serum phospholipids and cholesteryl esters of depressed
patients. Psychiatry Res. 1999;85: 275-29 1.
Martins, JG. EPA but not DHA appears to be responsible for the efficacy of omega-3 long
chain polyunsaturated fatty ac id supp lementat ion in depression: evidence from a metaanalysis of randomized controlled trials . .I Am Col/ Nutr. 2009;28:525-452.
Mi ll er, AH ; Malet ic, V; Raison, CL. Inflammat ion and its di scon tents: the role ofcytokines in
the pathophysiology ofmajor depress ion. Bioi Psychiatry. 2009;65: 732- 74 1.
lvfischoulon, D; Papakostas, Gl; Dordi ng, ClVI; Farabaugh, AH; Sonawall a, SB; Agoston,
AM; Smith, J; Beaumont, EC; Dahan, LE; Alpert, JE; Nierenberg, AA; Fava, M. A
doub le-blind, randomized controlled trial ofethyl-eicosapentaenoate for maj or depressive
disorder. .I C/in Psychiatly. 2009;70: I 636-1644.
lvfiyake, Y; Sasaki, S; Yokoyama, T; Tanaka, K; Ohya, Y; Fukushima, W; Saito, K; Ohluj i,
S; Kiyohara, C; Hirota, Y. Risk of postpartum depression in relat ion to d ietary fish and
fat intake in Japan: the Osaka lvlatemal and Child Health Study. Psycho/ Med. 2006; 36:
1727- 1735.
Murray, CJ; Lopez, AD. Alternat ive proj ect ions of mortality and disability by cause 19902020: Globa l Burden of Disease Study. Lancet. 1997; 349: 1498-1504.
Nat ional Inst itute of Mental Health. The numbers count: mental disorders in America. A
summary of statistics describing the prevalence of mental disorders in America.
Bethesda, MD: Nat ional Institute oflvlental Health, 200 I . N IH Pub Iication No. 0 1-4584.
Nemets, B; Stahl, Z; Belmaker, RH. Addi tion of omega-3 fatty acid to maintenance
medication treatment for recurrent un ipolar depressive disorder. Am .I Psychiatry. 2002;
159:477-479.
Peet, M; Murphy, B; Shay, J; Horrobin, D. Dep letion of omega-3 fatty acid levels in red
blood cell membranes of depressive pat ients. Bioi Psychiatly . 1998;43:3 15- 3 19.
Peet, M; Brind, J; Ramchand, CN; Shah, S; Vankar, GK. Tw o doub le-bli nd p lacebocontroll ed pilot studies of eicosapentaenoic acid in the treatment of schizophrenia.
Schizophr Res. 200 I ;49: 243- 25 1.
Peet, M; Horrobin, DF. A dose-ranging study of the effects of ethyl-eicosapentaenoate in
patients w ith ongoing depression despite apparentl y adequate treatment w ith standard
drugs. Arch Gen Psychiatry. 2002a;59:9 13- 9 19.
Peet, M; Horrobin, DF; E-E Mu lticentre Study Group. A dose-ranging exploratory study of
the effects ofethyl-eicosapentaenoate in pat ients with persistent schizophren ic symptoms .
.I Psychintr Res. 2002b;36:7- 18.
Role of Eicosapentaenoic Acid in lvlental Heallh
127
Puri, BK; Counsell, SJ; Hami lton, G; Richardson, AJ; Horrobin, DF. Eicosapentaenoic acid
in treatment-resistant depression associated w ith symptom remission, structural brain
ch anges and reduced neuronal phospholipid turnover. bu J C/in Pract. 200 1;55:560- 563.
Raison, CL; Capuron, L; Mill er, A H. Cytokines sing the blues: inflammat ion and the
pathogenesis of depression. Trends lmmunol. 2006;27: 24-3 1.
Rapoport, S I; Basselin, M; Kim, HW; Rao, JS. Bipo lar disorder and mechanisms of action of
mood stabili zers . Brain Res Rev. 2009;6 1:185- 209.
Ross, BM; Segu in, J; S ieswerda, LE . Omega-3 fally acids as treatments for mental illness:
wh ich di sorder and wh ich !ally ac id? Lipid.~ Health Dis. 2007;6:21.
Sierra, S; Lara-V illoslada, F; Comalada, lvl; O livares, M; Xaus, J. Dietary eicosapentaenoic
ac id and docosahexaenoic acid equall y incorporate as decosahexaenoic acid but di ffer in
inflammatory effects. Nutrition . 2008;24 :245- 254.
Sil vers, Klvl; Scott, KM. Fish consumpt ion and self-reponed physical and mental heallb
status. Public Health Nutr. 2002;5:427-43 1.
Sil vers, KM; Woolley, CC; Hamilton, FC; Watts, Plvl; Watson, RA. Randomised doubleblind placebo-controlled tr ial of fish oi l in the treatment of depression. Prostaglandins
Leukor Essent Fatty Acids. 2005; 72:211 - 218.
Smith, WL. Cyclooxygenases, peroxide tone and the all ure of fish oiL Cu rr Opin Cell Bioi.
2005;17: 174-182.
Stoll, A L; Locke, CA; Marangell, LB; Severus, WE. Omega-3 fatty acids and bipolar
disorder: a review. Prostaglandins Leukot Essenr Fatty Acids. 1999;60:329- 37.
Su, KP; Huang, SY; Chiu, CC; Shen, WW. Omega-3 fally acids in maj or depressive di sorder.
A preli minary double-blind, placebo-controll ed tr iaL Eur Neuropsychop harmacol.
2003;! 3:267- 271.
Sub lelle, ME; Russ, MJ; Smith, GS. Evidence for a role of the arach idonic acid cascade in
affect ive di sorders: a review. Bipolar Disord. 2004;6:95- 105.
Sub lette, ME; Ell is, SP; Geant, A L; lvlann, JJ. l\lleta-analysis of the effects of
eicosapentaenoic acid (EPA) in clinical trials in depression . J C/in Psychiatly .
2011 ;72: 1577- 1584.
The World Health Report. Mental Health: News Understanding News Hope. Geneva: World
Heallh Organizat ion, 2001.
Yehuda, S; Rabino,;tz, S; M osto[~ky, DJ. Essent ial fatty acids are mediators of brain
biochemistry and cogn itive functions. J Neurosci Res. 1999;56:565- 570.
Zanarin i, lv!C; Frankenburg, FR. omega-3 Fatty acid treatment of women with borderline
personality di sorder: a double-blind, placebo-controll ed pilot study. Am J Psychiatry.
2003 ;1 60: 167- 169.
Zhao, Y; Joshi- Barve, S; Barve, S; Chen, LH. Eicosapentaenoic acid prevents L PS-induced
TN F-alpha expression by prevent ing N F-kapp aB activation. J Am Coli Nutr. 2004;
23:7 1- 78.

Editors: T.G. Bradley and F.P. Vargas, pp. 129- 142
ISBN: 978- 1-62257-480-3

Chapter 6
COMBINED SUPPLEMENTATION WITH E PA -RICH

FISH OIL A ND PHYTOSTEROLS I MPROVE PLASMA
L IPID P ROFILE AND C - REACTIVE PROTEIN (C R P)
IN INDIVIDUALS WITH H YPERLIPlDEMIA
Michelle A. Micallef and Manohar L. Garg
Nutraceut ica ls Research Group, School of Biomedica l Sc iences and Pharmacy,
Faculty of Hea lth, Univers ity ofl\ewcast le, Cal laghan, NSW, Austral ia
Abstract
The treatmem of hyperlipidemia, particularly reducing LDL-cholesterol and triglyceride
concemration, is established as an efficacious means of reducing both morbidity and mortali ty
from CVD. Dietary supplememation with phy10sterols is known to reduce LDL-cholesterol,
whilst omega-3 PUFA are hypotriglyceridemic and have immuno-modulatory properties. The
obj ective of this study was to examine the effect of combined supplememation with E PA-rich
fish oil and phytosterols on cardiovascular risk factors in individuals with combined
hyperlipidemia. Fifty-eight healthy hyperlipidemic adults, were randomly assigned to
consume 3 x I g EPA-rich (550 mg 20:5n-3 and 120 mg 22:6n-3) fish oil capsules daily or 3 x
I g placebo (sunola oil) capst~es daily, either alone or in combination with 25 g ph}~osterol
enriched spread (2 g phytosterols) daily for 3 weeks. Blood pressure, plasma lipid profi le,
plasma fatty acids and inflammatory status were analysed. Ph}~Osterol supplememation
significamly reduced total-cholesterol and LD L-cholesterol (-8.0 % and -7.5 %, P < 0.01),
whilst fish oil supplememation significamly improved triglycerides and HD L-cholesterol (-8.5
%, P < 0.05; +6.5 %, P < 0.01). Total-cholesterol, LDL-cholesterol, triglycerides and HD Lcholesterol were significamly improved (-9.5 %, P < 0.0 I; -7.5 %, P < 0.01; -22.2 %, P <
0.01; +7. 1 %, P < 0.01) following the combined dietary supplememation. CRP concemration
was reduced by 11 % (P = 0.02) in the combination group. In conclus ion, the combined
supplementation of EPA-rich fish oil capsules and a phytosterol-enriched spread provide
cardiovascular health benefits, by way of optimising plasma lipid profile and improving
inflammatory staws in individuals with combined hyperlipidemia.
E-mail address: manohar.garg@newcastle.edu.au; Tel: +61 02 4921 5647; Fax: +6 1 02 4921 2028; Address:
Nutraceuticals Research Group, School of Biomedical Sciences and Pharmacy, Faculty of Health, University
ofNewc~stle, Callaghan, NSW, 2308 Australia. (Corresponding Author)
130
M ichell e A. M icallef and l\<(anohar L. Garg
Keywords/ P hrases: Omega-3 P UFA, Phytosterols, Hyperlipidemia, CV D.
Introduction
Di et can play a pivotal role in the progression of atherosclerosis and cardiovascular
disease (CV D). For examp le, dietary sources of phytosterols and omega-3 pol yunsaturated
fatty acid (P UFA), such as, nuts, seeds, fru its and vegetables, fi sh and vegetable oils, are
important components of a healthy diet, as they contribute towards lipid metabolism and
excretion pathways, eicosanoid production, cell membrane phospho lipid composit ion and
gene expression [ 1].
There is an emerging need to ident ify treatments which all eviate risk fact ors associated
" i th CV D development and progression. Modifiab le cardiovascular risk factors such as
hyperlipidemia, contribute to the underlying mechanisms of atherosclerot ic disease,
promot ing endothelial dysfunct ion, oxidat ive stress and pro-i nflanm1atory pathways (2-4] .
Phytostero ls are well known for their hypocho lesterol emic properties. The mechanism is
primaril y through the compet itive inh ibit ion ofmicell ular sol ub ilisat ion and hence intest inal
absorpt ion of both di etary and biliary chol esterol [5, 6]. Phytosterol bal ance studies show
marked decreases in intestinal cho lesterol absorption after the administrat ion of phytosterols,
despite increases in Jathosterol, a biomarker of cho lestero l synthesis (7-9]. On average, a
phytosterol consumpt ion of2 g dail y can reduce low-density lipoprotein (LDL) -chol esterol
by I 0 %, and has also been shown to reduce circulat ing concentrat ion of inflammatory
markers, such interleukin-6 (! L-6), tumor necrosis factor-alpha (TN Fcl) and C-react ive protein
(CRP)[ I 0, 11 ].
Alternat ively, fish oi ls rich in omega-3 P UFA have been shown to have
hypotriglycer idemic and immuno-modulatory propert ies [ 12- 14]. Eicosapen taenoi c acid
(E PA), one of the most important long-chain P UFA, can be fo und in large quanti ti es in fish
oil and has been shown to prov ide cardiovascular health benefits. Studies provide strong
eviden ce that an increased consumpt ion of fi sh and/or fi sh oi l is correlated w ith a reduced risk
of CV D, coronary heart disease (CH D), stroke, total mortality and sudden death [ 15] .
Additionall y, inflammatory cytokines, adhesion mol ecu les and vasoconstrictive eicosano ids
have all shown to be beneficiall y reduced with the consumpt ion o f omega-3 PU FA [ 16].
In combination, phytosterols and omega-3 PUFA may off er a co mprehensi ve treatment
for th e optimisat ion of circulat ing plasma lipids, whilst providi ng additional health benefi ts,
such as ant i-inflammato ry, hypotensive and ant i-arrhythmic propert ies in hyperlipidemic
individuals {17]. !t has previousl y been shown that the combination of a phytosterol-enriched
spread and omega-3 P UFA supp lementat ion, provides li pid lower ing and anti-i nflammatory
effects in byperl ipidemic indi viduals ( 18, 19]. Gi ven the heightened interest of both
phytosterols and omega-3 PUF A as food/dietary addit ives o r funct ional foods, it is important
to further invest igate the potent ial cardiovascular health benefits with their concomitant
supp lementat ion. Hence the primary aim of th is study is to examine the effect of combined
supplementat ion with pbytosterols and o mega-3 PU FA o n cardiovascular risk factors in
indi viduals w ith combined hyperlipidemia.
EPA and Phytosterol s in Hyperlipidemia
13 1
Methods and Materials

This was a 3 week randomised, doub le-bli nd, placebo controlled trial. This study was
approved by the Human Research Eth ics Comm ittee from The Un iversity of Newcast le,
NSW, Australia. Study procedures were rev iewed with partic ipants and informed written
consent was obtained pri or to commencement A ll data was coll ected at baseli ne (week 0) an d
post-intervent ion (week 3).
1vli Idly hyper lipidemic, yet otherw ise healthy adults aged 25 to 70 years were recru ited
through a vol unteer database (Hunter Medi cal Research Inst itute, John Hunter Hospital,
NSW, Australi a) and advertisements in local media throughout Newcast le, NSW, Australia.
The primary inclusion criteria included a fasting plasma total-cho lesterol concentrati on> 6.0
mmol/ 1 (231 mg/dL), a fasting plasma triglycer ide concentration> 1.5 mmoVJ (1 32 mgfdL)
and no previous or current cardi ovascu lar events, diabetes mellitus, chron ic inflammatory
di sease, hypenension or liver/ renal disease. Part icipants were excluded if they were tak ing
anti-i nflammatory, ant i-hypertensive or hypolipidemic agents, regularly consumed
phytosterol-enriched foods and/or fi sh oil supplements, bad a usual weekly consumption of
more than 2 faHy fish meals per week, or if they had a strong aversion or known
al lergy/intolerance to the foods involved in the study.
Fi fty-eight part icipants were recru ited and randomly assigned to an intervention group.
Each parti cipant was instructed to consume 3 x I g capsules dai ly with each meal. Each
capsule contained either I g of sunola oil (B1vi -Cogn it ive-S B20-EO, E PAX, Norway), or I g
ofE PA-rich fish oi l (EPAX 55 10 TG/N, EPAX, Norway), containing 550 mg EPA and 120
mg docosahexaenoi c acid (DHA) (marine-der ived) in a triacylglycerol fom1. Part icipants
were randomly assigned to consume the capsu les alone or in combination w ith 25 g (2 g total
phytosterols) of a phytostero l-enriched spread (Logicol, Goodman Fi elder, Australi a) dail y
for 3 weeks . This conm1erciall y avail able spread is canol a oil-based and predominant ly I)si tosterol, campesterol and st igmasterol. The 4 intervent ion group s were identi fied as p lacebo
(n= l 4), fish oi l (n= l 5), phytosterol (n= l 4) and combination (n= J5).
The oil s were contained w ith in an opaque brown capsu le and flavoured with peppem1int
for bli nding. The phytosterol-enriched spread was provided as individuall y weighed tubs (2 5
g each), and was to replace all hab itual margarine/butler consumpt ion during the study.
Participants were instructed to use the spread on bread or crackers, melted over vegetab les or
mixed into mashed potato, pumpkin, rice etc. The spread was not to be used for high
temperature cooking or frying. Un intent ional consumpt ion of phytosterols from other food
sources was not possible to mon itor or restrict.
Compli ance was mon itored by week ly telephone contact w ith participants, weigh ing of
spread tubs and capsu le count-back upon their return, interview ing participants regarding th e
consumpt ion of the spread and an evaluati on of their dietary records. Pan icipants also kept a
dail y record of their consumpt ion of capsules and/or spread.
A general medical quest ionnaire, including medi cal history, current medi cal condition/s
and prescr ibed medications was coll ected from each pan ic ipant at baseline. Weight (kg) and
height (m) were recorded to the nearest 0.1 using a calibrated balance beam scale (PCS
Measurement, NSW, Australi a), waist and hip girth (cm) were measured wh ile dressed m
2
2
light clothing. Body mass index ( BM I) was calculated as weight!height (kgfm ).
132
Michell e A. Micallef and l\<(anohar L. Garg
Single frequency bioelectrical impedance was used to assess body composition (Maltron
International, Essex, UK). Measurements were recorded in the supine posit ion fo llow ing a > I Oh fast and partici pants refrained from strenuous physical activity and alcohol consumption 24-h
pr ior to testing. Fat mass and fat free mass were recorded as a percentage of total body weight.
Part icipants were asked to maintai n their habitual dietary and alcohol intakes during the
intervent ion. A 24-h food recall was collected at baseline and post-intervention and analysed
fbr macronutrient, micronutri ent and fauy acid consumpt ion usi ng a food database program
(FoodWorks, Xyri s, QLD, Australia). Participants did not receive additional di etary
counsell ing pri or to, or d uring the study.
Blood pressure and heart rate were measured usi ng an automated monitor (lv!icrolife BP
3A D 1-A, Heerbrugg, Sw itzerland) (pressure 3 nun Hg; pu lse 5 %) fr om the supported left
am1 of the rested (1 0 minutes), seated participant. Systolic blood pressure (SBP) an d diastolic
blood pressure (DBP) were based on the average of two independent measurements.
A blood sample was obtained by venipuncture, following an overn ight fast at baseline
and post-intervent ion. Plasma was prepared by centri fug ing (Heraeus Bio fuge Straw s) for 10
minutes x 3000 g at 4C. A liquots were coll ected and stored at -80C until analysed.
Plasma total-chol esterol, triglyceride and high-density lipoprote in (H DL) -cholesterol
concentration were measured by automated methods on an auto analyser, using standardized
reagents (Hunter Area Pathology Service, NSW, Australia). LDL-cholesterol concentrat ion
was calculated usi ng the Friedewald equation [20].
Plasma faHy acid concentration was detemu ned using the method establi shed by Lepage
and Roy (2 1]. Fatty acid methyl ester peaks were ident i tied by comparing their retent ion
limes w ith those of a standard and quant ified usi ng a Hewle!! Packard 6890 series gas
chromatograph w ith Chemstat ions version A .04.02 for GC anal ysis.
TNFcl and IL-6 were anal ysed using en zyme-linked immunosorbent assay (EL!SA) kits
(R&D Systen1s, Minneapolis, MN, USA), with a minimum detection concentration of 0. 106
and 0.039 pg/ml , respectively, and an intr a- and interassay CV of < 9 %. CRP analysis was
carr ied out usi ng an immuno-turbidimetric method (Hunter Area Pathology Service,
Newcastle, NSW, Australia). Plasma leukotriene B4 (LT B4) concentrat ion was detem1ined
u.~ing an EL!SA kit based on the compet ition between LT B. and its conj ugate (Cayman
Chemical Company, Ann Arbor, M !, USA). The intra- and inter-assay CV was 8.3 % and 9 .7
%, respect ively, with a detect ion li mit of 13.0 pglml.. A ll analysis was assayed in duplicate
\vi th in th e same series to avoid assay variabi lity.
All data are presented as means SEM. Stat ist ical significance was set at P < 0.05.
Changes from baseline were determ ined using non-parametric analyses (Wilcoxon signed-rank
test). The effect of treatment on the percentage change between groups was detem1ined usi ng
one-way anal ysis of variance w ith post-hoc compar isons (Tukey HSD). The 95 % C l is given
when differences in change between groups is shown. Two-way analysis of var iance was used
to test for interaction effects between the 2 independent var iables (phytosterols and EPA-rich
fish oil) on each of the plasma lipids. All results were analysed using SPSS version 17.0.
Results
Part icipants (mal e n = 2 1 and femal e n = 37) had a mean SEM age of 56.7 2.2 years
and a BM! o f 25.7 + 1.0 kglnl . Anthropometric characteristics of the 4 groups at baseline
EPA and Phytosterols in Hyperlipidemia
133
were well matched and were not signi fi cant ly di fferent (Tab le I) . There were no signi fi cant
di !Terences between groups in fat mass an d fat free mass at baseline (33.4 + 2.0 % and 66.4
2.0 %, respect ively) . Blood pressure and heart rate were also not sign ificantly di fferent
between groups and did not significant ly change over the course of the intervent ion per iod (3
weeks). Participants had a mean baseline SBP of 133.4 4.0 mm Hg, a DBP of 82.2 + 2.6
mm Hg and a heart rate of 66.9 2 .8 bpm.
Evidence of adherence to the study was analysed by 24-h food recall , capsu le counts,
\\e ighing of spread tubs and analysis of plasma fatty acid concentration. The capsules were
"ell tol erated and compli ance as detem1ined by capsu le count-back was 97.3 0.03 %. T he
average amount of spread consumed was 24.2 0.2 g dail y and was most frequent ly
consumed at breakfast and lunch.
Dietary consumpt ion of macronutrient, micronutr ient and fatty acids were analysed using
24-h food recall (Tab le 2). The mean energy consumption at baseline was 7822.4 768.4 kJ
dail y, comprising of44.5 2.5 % carbohydrate, 20 .27 1.4 % protein and 33.5 2.3 % total
fai. Dietary nutritional status was not significantly di fferent between the 4 groups at base line
and did not change th roughout the study. Consumpt ion of alcohol and physical act ivity were
also unchanged.
Participants had a mean SEM plasma total -cho lestero l and triglyceride concentrat ion of
6.4 0. 1 mmoi/L and 1.5 0.2 mmoi/ L, respect ively. Plasma lipid profi le did not di ffer
among the groups at baseline (Table 3). There were no sign ificant changes in plasma lipid
profi le in the placebo group. Supplementat ion with 2 g of phytosterols dail y for 3 weeks,
si gnificantly reduced plasma total -cho lesterol concentration by 8.0 2.2 % (P < 0.01), LDLcho lesterol by 7. 5 1.9 % (P < 0.01) and triglycerides by 5.9 4. 1 % (P < 0.05).
Supplementation w ith EPA-rich fish o il (1 .65 g EPA dail y) d id not affect total -cholesterol or
LDL-cho lestero l, however a significant reduct ion in triglyceride concentration (8.5 5.7 %, P
< 0.05) and a signi ficant increase in HD L-cho lesterol (6.5 1.4 %, P < 0.0 1) was achieved .
The treatment group assigned the combined supplementat ion o f 2 g phytosterols and 3 g
EPA-rich fish oil dail y, ach ieved significant reduct ions in plasma total-cho lestero l, LDLcholesterol and triglyceride concentration of 9.5 1.6 % (P < 0.0 1), 7.5 1.4 % (P < 0.01)
and 22.2 3. 1 % (P < 0.001), respectively. A 7. 1 2.5 % (P < 0.01) increase in HD Lchol estero l concentration was also evident in the comb ination group.
A comparat ive analysis between each of the 4 treatment groups using one-way AN OVA
was perfom1ed. A signifi cant di fference between the placebo and combination group was
found fbr the change in plasma total -cholesterol (P= 0.04, 95 % Cl - 14. 1, -0. 1), triglycerides
(P = 0.02, 95% C l -45 .0, -6.1) and HD L-cho lesterol (P = 0.04, 95 % Cl 0. 1, 18.7) from
baseli ne. A significant di fference in the change in plasma total-cholesterol from baseli ne
between the fish o il and phytostero l and fish oi l and combinat ion groups was found (P =
0.01, 95 % C l 3.2, 17.3; P < 0.001, 95 % C l 4.7, 18.5, respect ivel y). Treatment affect was
assessed for phytosterol and EPA-rich fish oi l supp lementat ion. Phytostero l supplementation
had a sign ificant effect on change in tota l-cholesterol (P < 0.001) and triglyceride (P = 0.04)
concentration, whi lst fish oi l supplementat ion had a sign ifi cant effect on change in
triglyceride (P = 0.01) and HD L-cho lesterol (P = 0.02) concentration. A possible interaction
between phytostero ls and EPA-rich fish oil supplementat ion was assessed, compared to the
placebo group. No signi ficant interactions (phytostero l x EPA) were found.
Table I. rartidpanl characteristics (mean :: SEM) iu each of the inlervculion groups a1 baseline
Placebo
SEM
14
53.9
2.5
7 1.7
2.7
24.9
0.9
0 .8
0.02
65.7
1.7
341
1.7
3.4
127.0
792
2.8
68.1
3.1
39.6
8.9
2.2
0 .2
!.5
0 .3
2.9
0.3
~lean
N=
Age (years)
Body
W~-i hl
(kg)
BMI;kglm')
\Vaisl-to-hip ratio
FaLfree ma.c;;s (%)
Fat ma..-;s (%)
SBP (mm Hg)
DBP (mm Hg)
He-art rate (bpm)
l TB, \pg/ml)
TNFu(pg/ml)
IL-6 (pg/ml)
CRP (~g/ml)
f, ish Oil
Mean
SEM
15
57.4
1.9
79.2
2.8
26.7
0 .9
0.8
0 .02
64.6
22
35.3
21
135.2
3.8
83.2
2.4
67.1
54.9
2.2
1.0
3.6
7.1
0 .3
0 .1
1.0
4.8
8 ~:11, body ma;.s index; SBP. S}''Stolic blood pressure; DBP, diastolic blood pressure; l
C-re!t::tivc pm1cin.
TB.~ , lcu.kotricrtt:
Ph\'tOsferol
1\tciln
SEM
14
55.7
3.0
76.5
2.6
25.6
0 .9
0.9
0.0 2
69.5
2.4
30.4
2.4
133.8
3.4
__
8 1.7
65.4
2.4
39.9
6.0
1.7
0.1
1.0
0.1
2.6
0.4
''
Combination
l\lciln
SBI
15
59.9
L2
77.9
5.8
25.9
1.4
0.9
0.03
66.1
1.8
33.9
1.8
137.9
5.7
84.9
2.9
__
67.3
46.4
7.2
1.9
0.4
t.3
0.2
>. .
0.5
''
-.
8;.: Th'Fa , tu.mor nc~rru.is flctor-alph:s; ll-6, imcrlcukin.6; CRP,
Table 2. Parfici(HUll macronulrieut and
Energy (kJ)
Fibr,: (g)
Chuleiiterul {mg)
Carboh~drate (%)
Pro tein (%)

Total fal (%)
SFA (%)'
MUFA (%)'
PUFA (%f
omega-6 PUFA (g)
omega-3 PUFA (.g)
18 : ln-3 (g)
20 : Sn-3 (g)
22 : Sn-3 (g)
22 : 6n-3{g)
d iehl r~
falty acid consumption

F'i$h Oil
Pla c-ebo
;'\lean
SEM
Mean
7493.1
22.0
219.5
43.6
21.6
1l.9
44.7
38.0
17.22
6.8
0.9
0.6
0.08
0.04
0.1
7684.7
23.0
217.2
45.1
19.9
32.8
38.3
40.7
20.8
7.0
0.7
0.5
0.0 5
0.0 1
0.1
702.0
2.5
,,
.) _ ._
2.7
0.9
2.1
2.1
1.1
2.1
0.9
0.1
0.08
0.0 3
0.0 1
O.o?
SEM
1003.6
4.2
54.4
2.3
1.6
,.
.. >
2.8
2.1
3.0
1.7
0.1
0.09
0.02
0Jl5
0Jl7
(mea n ~
SEM) using24-h food recallal baseline
Phytos:tcrol
SBI
Mean
Combina tion
Mean
SEM
7517.4
18.9
274.4
41.9
21.9
33.9
45.2
39.3
15.4
5.9
574.2
2.0
41.5
2.4
1.8
2.5
2.7
2.3
8594.7
24.0
23 1.4
47.4
17.7
33.6
42.4
1.1
16.5
7.3
1.3
0.6
0.2
0.06
1.1
0.6
0.1
0.04
0.3
0.9
0.3
0.09
0.05
0.02
0.2
I&: 3n-3, c -linolenic add: 20: 5Jt3, e ico;spenuenoic add:; "2"2 : :Sn-3, docosspem~ ooi e ~ i d: 22 : (>n-3 , docossehttaenoic ~ i d:.
Refers to pcn::er.1 a~ of !otal fat.
4 LO
0.4
794.1
2.7
37.8
2.6
1.4
2.5
2.3
1.4
1.7
1.0
0.3
0.05
0.09
0.02
0.1
Table 3. Fasting plasma lipid couccuinHions (mrnol!L) (mean l!: SEM) at baseline and post iutervcndou
Pla c~bo
Mean
Total-cholesterol
BL
BL
6.5
6.5
4.2
PI
4. 1
BL
1.5
1.5
PI
lDl-eho!esleml
Trigl)ceride
PI
HDL-cho!eslerol
Bl
PI
1.5
1.5
51: M
0.1
0.1
0.2
0.2
0.3
M~an
6.2
6.4
4.2
4.0
1.4
0.3
0.1
0.1
1.4
..
t.3
Fish Oil
SEM
0.2
0.1
0.2
0.2
0.1
Ql
0.1
Q(
Phytosterol
:\lean
6.5
5.9...
4.4
4.1
1.5
1.4
1.2
1.3
SEM
0.2
0.1
02
Q2
0.1
0.1
0.08
0.07
Combination
M~an
6.3
5.,.
4.0
3.7"
1.8
..
1.4
t.4
SEM
0.1
0.1
0.2
0.2
0.3
0.2
0.1
0.1
BL. basclin.-c (v.occk 0); PI, poo.!intcrvcmior, (w-o.:k 3 ).

There ~ocr~ n.o signifies m di ff~rcnccs bclv.~-c n g:rm.rps -a1 b.aic li rt~ (onc-v.'!ly Af\.'OV A). A signiticam ch:ang:-c from !xis.clin-c within a grot.tp is indica1cd
P<O.OI. P<O.OOI.
br P<0.05.
138
Michell e A. Micallefand l\<(anohar L. Garg
Discussion
A lthough several studies have examined the individual effects of dietary phytosterols and
omega-3 PUF A supplementation on plasma lipids, only a coup le have investigated the
combined eilects of these functional f(>od ingredients. Moreover, r isk factors other than plasma
lipid levels are affected by the combined treatment, and have been reported onl y in one recent
study [ 17] in which omega-3 fatty acids were supplemented pr imar ily in the tom1 of 22:6n-3.
The present study was designed to assess the combined effect of phytosterol and EPA-rich lish
oi l supplementation on cardiovascular risk factors in adults w ith combined hyperlipidemia.
The consumption of phytosterols in conjunction w ith EPA-rich fish oil provided the
greatest overall improvement in plasma lipid profile, compared to either of these functional
foods consumed alone. Phytosterols inhibit cholesterol absorption, thereby reducing plasma
total-cholesterol and LDL-cholesterol concentration. A number of clinical trials have
establi shed that the consumpt ion of 1.5 to 2 g daily ofphytosterols can resu lt in a 10 to 15 %
reduction in LDL-cholesterol in 3 weeks. The benefits of phytosterol consumption have been
demonstrated in normolipidemic, hyper lipidemic and stat in-dependent individuals {22]. In th is
study, we show a sign ificant reduct ion in total-cholesterol (8.0 %), LDL-cholesterol (7.5 %) and
triglyceride (5 .9 %) concentration atler 3 weeks of supp lementation w ith 2 g phytosterols dail y
tor 3 weeks. These find ings are comparable to other studies with similar phytosterol dose and
duration of supplementation. In a study by C lition et a/ [23], hyperlipidemic patients were
supp lemented w ith 1.6 g dail y of phytosterol-enr iched foods (bread, cereal, mil k and yoghurt)
for 3 weeks, show ing an average reduction in serum LDL-cholesterol of 9.0 %, w ith mil k
providing the greatest reduction (1 6%). In previous studies, the hypotriglycer idemic aftect of
phytosterols, has shown to be weak. In a meta-analysis by Naumann et a/ [24], individuals
having greater base Iine triglyceride concentrations were found to have a greater
hypotriglyceridemic response to phytosterol supplementation. In response to 2 g phytosterols
daily, reductions in plasma triglycerides of 1.0, 3.8 and 4.7 % were observed, respective to
baseline tr iglyceride concentrations of 1.0, 2 .0 and 3.0 nm101/L [24].
The hypotriglyceridemic properti es of omega-3 PUF A have long been elucidated,
however there are very few studies to date wh ich have investigated the indi vidual effects of
EPA and DHA. In a study by Mori et a/ [ 14], DHA provided the greatest reduct ion in
triglyceride concentrat ion (20 %) in hyperlipidemic adu lts. In another study a significant
" i th in-group reduct ion in tr iglyceride concentrat ion was observed (22 %) from baseli ne with
EPA supplementat ion [25]. In our study, the EPA-rich fi sh oi l group showed an 8.5 %
reduct ion in plasma triglyceride concentration and a 6.5 % increase in HDL-cholesterol.
The contradictory find ings between our study and previous fi ndings may be explained in
part by the concentration of consumed fish oil and durat ion of supplementation. In another
placebo-controlled study carried out in our laboratory, we were able to show that in
hyperlipidemic pat ients, triglyceride concentration cou ld be reduced by 22.3% (P = 0.004) in
3 weeks, with 3 g DHA-rich fish oil dail y {18]. Grimsgaard et a/ {26] reported that EPA and
DHA have s im ilar hypotriglyceridemic effects compared with a placebo . However, there are
few studi es which suggest that EPA alone can s ign ificantly red uce triglyceride and V LDLcholesterol concentration {27] and increase LDL and HDL-cholest erol {28], w ith no change in
total-cholesterol.
EPA and Phytosterols in Hyperl ipidemia
139
In combinat ion, phytosterols and EPA-rich fish oi l may offer a more comprehensive
strategy for the opt imisat ion of p lasma li pid profi le. In th is study, total-cholesterol, LDLcho lesterol and trig lycer ides were sign ificantly reduced by 9.5 1.6 %, 7.5 1.4% and 22.2
3. 1 %, respect ively and HDL-cho lesterol was inc<eased by 7. 1 2.5 %. Further analysis of
the data showed there were no sign ificant interact ion effects between phytosterol and EPArich fish oil. There is very li mited published data, which evaluates the independent and
interactive effects ofphytosterols and omega-3 PUFA. In a study by Khandelwal et a/ [ 19],
sign ificant reduct ions in triglyceride concentrat ion in response to phytosterol (enriched
yoghurt drink) and omega-3 PU FA (capsu les) alone and in comb inat ion was shown.
Compared to our study, Khande lwal showed no changes in total-cho lesterol, LDL-cholesterol
and HDL-cho lesterol. This may have been due to the di fference in part icipant populat ion,
" i th our study having a higher percentage of women (76% vs. 11 %) and a higher fasting
total-cholesterol at baseli ne. Additionall y, ou r phytostero ls were supplemented as an enriched
spread, as opposed to a yoghurt d rink, and the omega-3 fish o il supplementat ion we assessed
was predominantly a EPA-rich fish o il. The ro le ofapolipoprotein E and its various isoforms
also play a sign ificant role in lipoprotein metabol ism and may di ffer amongst participant
popu lat ions. Its influence as a source of genet ic var iability has been w idely invest igated and
infl uences ones response to di etary treatment for hyperlipidemia [29-32].
In combinat ion, phytosterols and omega-3 PU FA may offer a more comprehensive
strategy for not just opt imising circulating lip id levels, but also to provide additional health
benefi ts via anti-i nflammatory, ant i-aggregatory, ant i-hypertensive and antiarrhythmic
effects. The evidence seems to be contradictory in animal studies comparing the antiinflammatory effects of EPA and DHA [33], however in h uman cli nical trials no significant
di fference is seen between the two omega-3 PUFA [34]. In th is study, the supplementat ion
\vi th EPA-rich fish oil sign ifi cant ly reduced TNFa (P = 0.02) and had liitle affect on IL-6,
LTB 4 and CRP. The comb ination of fish oil and pbytosterols showed a significant reduct ion
in both TNFa and CRP. To date, our laboratory is the on ly research group to have
invest igated the ant i-inflammatory effects of combined phytosterol and omega-3 faHy acid
supp lementation in combined hyperli pidemia [35]. In our previous stud y combining DHArich fish oi l w ith phytosterol supplementation for 3 weeks, CRP, TN Fa, IL-6 and LTB4 were
all signi ticant ly reduced (39, I 0, I 0. 7 and 29.5%, respectively) {35].
A possible explanation for the difference in anti -inflanm1atory effects between
phytosterols combined w ith EPA as opposed to combined with DHA, could be the li mited
metabolism of omega-3 PlJF A beyond DPA, however further invest igat ion is warranted.
Gi ven a large port ion of cli nical trials are more support ive of the ant i-inflammatory propert ies
of DHA [34, 36] and the continued interest in the immuno-modulatory effects of omega-3
PUFA, further studies are needed to clarify the ant i-inflammatory propert ies of these PU FA in
different models of disease.
Condit ions associated with plasma li pid lipoprotein metaboli sm, such as hyperlipidemia,
play a major ro le in the development and progression of atherosclerot ic disease. Dietary
supplementat ion studies w ith omega-3 PUFA, demonstrate their hypotr iglyceridemic and
anti-i nflammatory propert ies, whilst supplementat ion with phytosterol esters show
hypocholestero lemic effects. The present stud y adds to th e current bod y of know ledge,
demonstrat ing a natural and efficacious combined di etary treatment for hyperlipidemia and
providing evidence of cardiovascu lar benefits of the combi ned supplementation of
phytosterols and EPA-rich fish oi l.
140
Ackn owled gem ents

This research received no speci fie grant !lom any funding agency in the publi c,
commercial or not-for-profh sectors. The authors have no confl ict of interest to disclose.
Author Contributions
MAM participated in the conception and design of the study, data coll ection, anal ysis and
drafi ing of the manuscript. MLG part icipated in the concept ion, design and coordi nat ion of
the study, provided sign ificant advice an d consu ltation and drafting of the manuscript.
References
( I] .lung UJ, Torrejon C, T ighe A P, Decke lbaum RJ. n-3 fatty ac ids and cardi ovascular
disease: mechan isms under lying beneficial effects. Am .J Clin Nutr. 2008;87: 2003S-9S.
(2] Raza JA, Babb .ID, Movahed A. Opt imal management of hyperlipidemia in primary
preventi on of cardiovascular disease. /nt .J Cardiol. 2004;97(3):355-66.
[3] Emberson .1 , \Vh incup P, Morris R, Walker M, Ebrahim S. Evaluati ng the impact of
populat ion and high-risk strategies for the primary prevention of cardiovascular di sease.
Eur Heart .f. 2004;25:484-9 1.
(4] Barzi F, Pate! A, Woodward M, Lawes CM, Ohkubo T, Gu D. A comparison of lipid
variables as predictors of card iovascular disease in the Asia pacific region. Ann
Epidemiol. 200 5; 15:405-13.
[5] Bhattacharya S. T herapy and clinical trials: plant sterols and stanols in management of
hypercholeterol emia: where are we now? Biomonthly Update. 2006 ;1 7:98- 100.
(6] Ostlund R, Lin X. Regulat ion of cholesterol absorption by phytostero ls. Curr
Atheroscler Rep. 2006;8:487-9 1.
(7] Jones PJ, Raeini-Sarjaz M, Nian ios FY, Vanstone CA, Feng N , Parsons W E.
Modulat ion of plasma lipid levels and chol esterol kinet ics by phytosterol versus
phytostano l esters . .J Lipid Res. 2000;4 1:697-705.
[8] N ies LK, Cymbala AA, Kasten S L, Lamprecht DG, O lson KL. Comp lementary and
al ternative therapies for the management of dysl ipidemia. Ann Pharmacother.
2006;40: 1984-92.
[9] Ost lund RE, Jr., Racette SB, Okeke A, Stenson W F. Phytosterols that are naturall y
present in commercial corn oil significant ly reduce cholesterol absorpt ion in humans.
Am .J C/in Nutr. 2002;75(6):1 000-4.
[ 10] Katan MB, Grundy S, Jones PJ. Efficacy and safety of plant stanols and stero ls in the
management of blood cholesterol levels. May o C/in Proc. 2003; 78:965-78.
{I I] Devaraj S, Autre! BC, Jialal I. Reduced-calorie orange juice beverage with plant sterols
lowers C-react ive protein concentrations and improves the lip id pro61e in human
volunteers. Am .J C/in Nutr. 2006;84( 4): 756-6 1.
{12] Kang .IX, Weylandt KH. Modulat ion of inflammatory cytokines by omega-3 fatty acids.
Subcell Biochem. 2008;49: 133-43.
EPA and Phytosterols in Hyperlipidemia
14 1
{13] Madsen T, Chri stensen JH, Blom !vi, Schmidt EB. The effect of dietary n-3 fau y acids
on serum concentratio ns of C-reactive protein : a dose-response study. Br .I Nutr.
2003;89(4): 5 17-22.
( 14] Mor i TA, Burke V, Puddey IB, Walls GF, O'Neal DN, Best .I D, et al. Puri tied
eicosapen taenoic and docosahexaenoic acids have di fferenti al effects on serum li pids
and lipoprote ins, LDL part icle size, g lucose, and insu lin in mildl y hyperlipidemic men.
Am .I C/in Nutr. 2000; 71(5) :108 5-94.
( 15] Kris-Etherton Plvl, Harris WS, Appel LJ. Fish consumpt ion, fish oil, omega-3 fatty
acids, and cardiovascular disease. Circulation. 2002; 106(2 1):2747-57.
[ 16] De Caterina R, Basta G. n-3 fau y acids and the innammatory response - biological
background. Eur Heart .! Suppl. 2001;3(suppl D):D42-D9.
{17] Micall ef !viA, Garg ML Beyond blood lipids: phytosterols, stati ns and omega-3
polyunsaturated fatty acid therapy fo r hyper lipidem ia . .I Nutr Biochem. 2009;20:927-39.
{18] Mical lef MA, Garg !vi.L. The Lipid-Lowering Effects of Phytostero ls and (n-3)
Pol yunsaturated Fauy Acids Are Synergistic and Comp lementary in Hyperli pidemic
lvlen and Women . .I Nutr. 2008; 138(6): I 086-90.
( 19] Khandelwal S, Demonty I, Jeemon P. Independent and interact ive effects o f plant sterols
and fish oi l n-3 long chain polyunsaturated fatty ac ids on the p lasma lipid profil e of
mi Idl y hyperlipidemic Indian adults. Brit .I Nutr. 2009; I 02:722-32.
[20] Friedewald WT, Levy RI, Fredrickson DS. Est imation of the concentration of lowdensity lipoprotein cho lesterol in plasma, w ithout use of the preparat ive ultracentri fuge.
C/in Chem. 1972;1 8(6): 499-502.
[2 1] Lepage G, Roy CC. Direct transesteri ficat ion of al l classes of lip ids in a one-step
reaction . .I Lipid Res. 1986;27 (1 ): 11 4-20.
{22] Weststrate .lA, Meij er GW. Plant sterol-enriched margarines and reduct ion of plasma
total- and LDL-chol esterol concentrat ions in nom10cholesterolaemic and mildl y
hypercholesterolaemic subjects. Eur .I Clin Nutr. 1998;52(5 ):334-43.
[23] C li fton PM, Noakes M, Erichsen N, Ross D, Ann ison G, Fassoulakis A, et al.
Chol esterol-lowering effects of plant sterols esters di ffer in mil k, yoghurt, bread and
cereal. Eur .I C/in Nutr. 2004;58:503-9.
{24] Naumann E, Plat .1, Kester A. The baseline serum li poprotein profile is related to plant
Slanol induced changes in serum lipoprotein cholesterol and triacylglyer ide
concentration . .I Am Col/ Nutr. 2008;27: 11 7-26.
[25] Buck ley R, Shewring B, Turner R, Yaqoob P, lvlin ihane AM . C ircu lating triacylglycerol
and apoE levels in response to EPA and docosahexaenoic supplementat ion in adult
human subj ects. Br .I Nutr. 2004;92:4 77-83.
{26] Grimsgaard S, Bonaa K, Han sen .1, Nordoy A. Highly purified eicosapentaenoic acid and
docosahexaenoi c acid in humans have similar triacylglycerol-lowering effects but
di vergent effects on serum fatly acids. Am .I C/in Nutr. 199 7;66(3):649-59.
(27] Rambjor G, Walen A, W indsor S, Ham s W. Eicosapentaenoic acid is primari ly
responsible for hypotr iglyceridemic effects of llsh oi l in human a. Lipid.~. 1996;3 1:S45-9.
[28] Woj enski C, S i Jver M, Walker .J. Eicosapentaen oic acid ethyl ester as an ant ith rombotic
agent: co mparison to an extract od fish oi l. Biochem Acta. 199 1; I 08 1:33-8.
(29] Dereon DM, Femstrom HA, Miller B, Krauss Rlvl. Apoli poprotein E iso form phenotype
and LDL subclass response to a red uced-fat diet. Arterioscler Thromb Vase Bioi.
1995; 15: 105- ll.
142
(30] Lopez-Miranda J, Ordovas JM, Mata P, Lichtenstein AH, Clevidence B, Judd JT, et al.
Effect o f apolipoprotein E phenotype on diet-induced lowering of plasma low density
lipoprotein cholesterol. .I Lipid Res. 1994;35: 1965-75.
(31] Mart in LJ, Connell y PW, Nancoo D, Wood N, Zhang ZJ, Maguire G, et al. Cholesieryl
ester transfer protein and high density lipoprotein responses to cholesterol feeding in
men: relationship to apolipoprotein E genotype . .! Lipid Res. 1993;34(437-446).
(32] Schaefer EJ, Licbtenstein AH, Lamon-Fava S, Contois HH , Li Z, Rasmussen HE, et al.
E!llcacy of a Nationa l Cholesterol Education Program Step 2 di et in nom1oli pidemic and
bypercholesterolemic middle-aged and elderly men and women. Arterioscler Thromb
Vase Bioi. 1995;1 5: I 079-85.
{33] Tomobe Y, Morizawa K, Tsuch ida M, Hibino H, Nakano Y, Tanaka Y. Dietary
docosahexaenoic acid suppresses inflammation and immunoresponses m contact
hypersensitivity reaction in mice. Lipids. 2000;35:6 1-9.
[34] Kew S, Mesa MD, Tricon S, Buckley R, Minihane AM, Yaqoob P. Effects of oils rich in
eicosapen taenoic and docosahexaenoic acids on immune cell composition and funct ion
in healthy humans. Am .! C/in Nutr. 2004;79(4):674-8 1.
(35] Micall ef MA, Garg Ml. Ant i-i nflammatory and cardioprotective effects of n-3
polyunsaturated fatty acids and plant sterols in hyperlipidemic individuals.
Atherosclerosis. 2008;204: 4 76-8 2.
{36] Kell ey D, Taylor P, Nelson G. Dietary docosahexaenoic acid and immunocompetence in
young healthy men. Lipid.~. 1999;33:559-66.

Edi tors: T.G. Bradleyand F.P. Vargas, pp. 143- 154
ISBN: 978- 1-62257-480-3

Chapter 7
PHYSIOLOGICAL ROLE OF E ICOSAPENTAENOIC

ACID (EPA) I N REDUCING THE CORONARY
HEART D ISEASES
Sumudu N. Warnakulasuriy a and H.P. Vasantha Rupasinghe
Department of Enviromnental Sciences, Faculty of Agriculture,

Dalhousie Un ivers ity, Truro, Nova Scotia 82N 5E3 Canada
Abstract
Omega-3 long chain polyunsamrated Fany acids (m-3 PUPA) have been a part of human diet
since the beginning of mankind. With the recent revealing of tremendous health benefits
provided by Cil-3 PUFA especially eicosapemaenoic acid (EPA), a growing progress is
occurring in understanding their biological functions and mode of action as well as
introduction of EPA-enriched value-added foods and nutraceuticals to the consumer. Seafood,
especially fish oil and marine algae as a namral source of EPA, provides promising
intervention in preventing number of diseases. The role of EPA in human growth and
development and disease prevention have been characterized by using cell cultures,
experimental a11imals and human clinical studies. The wide range of biological significance of
EPA extends to cardiovascular disease, ami-inflammatory properties, cltil dhood learning and
behaviour, adult psychiatric and neurodegenerative disorders and cancer. In vitro studies
indicate that the role of EPA in biological functions can be explained through many
mechan isms such as its interference in the physical nature of cell membranes, proteinmediated responses, eicosanoid generation, cell signaling and gene expression associated with
many cellular functions. The concurrent administration of EPA with drugs has also attracted
attention in research due to the improved physiological effects. The essentiality ofEPA for the
human li fe have been clearly shown by animal and epidemiological smdies and this chapter is
an attempt of review ing the current understanding of the role of EPA in prevention of the
coronary heart diseases.
E-mail address: v!Upasinghe@ nsac.ca; Tel : 902 893 6623; Address: P. 0 . Box 550, T!Uro. (CmTesponding au1hor)
144
Sumudu N. Wamaku lasuriya and H. P. Vasantha Rupasinghe
I. Introduc tion
Coronary Heart Diseases (CHD) are a leading cause of death in the Western world
(Massaro et al., 2008), c la iming one death every 39 seconds in the Un ited States (Kones,
2011 ). Different risk factors are in vo lved w ith the onset and progression o f CHD. Age,
gender, and genet ics are unmodi fied r is k factors and the refore, emphasis is focused on
modified risk factors such as hypertension, dys li p idemia, diabetes, obesity e tc., in prevent ion
and curing of CHD. Due to the comp lex etiol ogy, it is chall enging to find remedi es for CH D
and th ere a re many molecu lar leve l mechanisms yet to be und erstood.
Atherosclerosis is a c hronic in flammat ion in cardiovascu lar system invol ves the
fom1at ion of lesions in the arteries that are c haracterized by in flammat ion, lip id accumulat ion,
cell death, and fibrosis (Demyanets et a l., 20 11 , Hansson et a l., 2006). lt can be due to
combined act ion of severa l risk factors such as dyslipidemia, hypertension, and
hyperg lycemia (Massaro et a l., 2008). Athe rosclerot ic p laques lim its the b lood flow in
arteries, but the most severe consequence arises with the rapture of the p laque or di sruption of
a "vu lnerab le" p laque as the prothrombot ic material in the p laque exposes to the b lood and
causes sudden b lockage of the artery (li b by et al., 2009). lt leads to coronary artery disease
(CAD) and m yocardial infarct ion (M I), and myocarditis (leder et a l., 20 10) and finall y
caused heart fail ure, wh ich is a maj or cause for mortal ity in the world (Demyanets et a l.,
20 11 ). Cardia) arrhythm ia is also a common causative cli nical situat ion for cardiac arrest and
sudden death (Co lussi et al., 2007).
1.1. EPA as ro-3 PUFA for Reducing C HD

Controlling of risk factors using heart healthy foods is h igh ly focused on current research
based on heart diseases. c.o-3 PUFA such as e icosapentaenoic acid (EPA) are considered as
potential dietary constituents, wh ich can m in imize the risk !actors associated w ith CH D. Blood
pressure, p late let reactiv ity and thrombosis, p lasma trig lyceride concentrations, vascular
function, cardiac arrhythmias, and inflammation are several r isk factors, can be managed by the
di etary intake ofEPA (Calder, 2004; Roth and Harris, 20 10; Sirtori et a l., 1998).
Ev idences for the cardioprotect ive e trects of ro-3 PU FA h ave been recogn ized many
years ago (Russo, 2009). The first observation was the low mortal ity from CHD in
populations among Green land lnuits, whose maj or di et is sea fish, rich in ro-3 PUFA ( Leaf et
al., 2008). lt is also reported that Japanese, hav ing h igher in take of fish than North Americans
exh ib it lower rate of acute myocardial infarction, atherosclerosis and other ischemic
pathologies ( Russo, 2009) . lt has been showed in a study that group, wh ich consumed at least
30 g/day of fish (or two to three servings of fish per week) resulted in approx imate ly 50%
lower mortal ity rate in coronary artery diseases than the group who did not consumed fish
(K romhout et al., 1985). The first pub li shed c lin ica l trial by Burr et al. ( 1989) was to find th e
influence on fish consumpt ion on secondary p revention of myocardial infarction. Afl er two
years, There was a 29% reduct ion in mortal ity among the men w h o had been advised to eat
oi ly fish, at least 200 to 400 g p ortions tw ice weekl y compared to the men, h ad not been
adv ised. lvlany beneficial b iolog ical effects related to CH D are reported to be possessed w ith
ro-3 PUFA due to the ir antithrombotic, ant i-i nflammatory and vasodilat ion propert ies
(Calzolar i et al., 2009).
Physio logical Role of Eicosapentaenoic Acid .. .
145
1.2. Maj or Dietary Sources of EPA

EPA as well as all othe r co-3 PUFA is considered as essent ial Fatty acids wh ich cannot be
synthesised in mammali ans and therefore, it must be taken through di et. Fish o il is the major
sources ofE PA and pelagic fish living in COid waters contain h igher amounts o f EPA ( Racine
& Deckelbaum, 2007). Some species o f fish and shell fish contain E PA in h igher
concentrations (l'vlahaffey et al., 2008) and Sard ine and Hali but oi l prov ide relatively h igh
amounts o f EPA (Racine & Deckelbaum, 2007). A lthough fish is a good source of EPA, fish
do not produce themselves, EPA is produced in single cell ed marine phytop lankton. A Ifalinolen ic acid (ALA) is a plant based co-3 PUFA, whereas its conversion to EPA in human
body is li mited and a slow process (de Leiris, 2009). As the fi sh can contain environmental
contaminants such as methylmercury, polyc h lorinated biphenyls, and d ioxins, hav ing long
half-lives, long term consumpt ion o f large amounts o f fish can accumulate them in body and
cause ad verse health effects (Saremi et al., 2009). The World Health Organ ization (WHO),
and other several organi zations have suggested di etary recommendations for indi viduals to
consume 0.3 to 0.5 g o f EPA and DHA per day (approx imately two ta tty fi sh meals per
week); for pat ients w ith coronary heart d isease, I g of E PA and DHA per day;
hypertrig lyceri demic patients, 3 to 5 g ofE PA and DHA per da y (Saremi et al., 2009).
2. Physiological Role of EPA in CHD Prevention

Several mechan isms have been proposed to describe the role o f EPA in reducing CH D.
2.1. Reducing Triglyceride Levels

The mechanisms by which EPA reduce serum trig lyceri de (TG) a re not comp letely
understood (Harris et al., 2008, Krali s, 2012). In the elevated TG level, the risk for CHD is
not because o f TG themselves; it is due to other atherogen ic lipoproteins such as very low
density lipoproteins (V LDL), intem1edi a te density lipoprotein (ID L), low density li poprotein
(LD L) wh ic h are present in high concen trat ions in the condi tion o f h igh TG level (Krali s,
2012). High TG level also raises the plasma residence time of potent iall y a therogen ic
chylomicron and VL DL remnants (Harris et al., 2008). Harris (1 989) reported that fish o il co3 PUFA can reduce the fasting and postprandi al plasma TG levels, but the PUFA a bsorpt ion
is not affected sign ificantly. Studi es showed that co-3 PUFA can inh ibit the production an d
secret ion o f hepatic V LDL-TG and increase the removal ofTG from chylomicron and V LDL
particles through the up regulat ion o f enzymes such as lipoprotein lipase (M iller et al., 1993,
Bays et al., 2008). A lso, E PA is shunted into phopho lipid pathways wh ereas other fatty acids
ti ke o leate preferent iall y join w iib TG (M ill er et al., 1993 ).
EPA modulates Fatty acid ~-<lx idati on wh ich decreases the substrate level for TG
synthesis in a mechan ism involved perox isome pro li ferator - activated receptor subtypes
(PPAR) (Jump, 2002; Ren et al., 1997). Stero l regulatory element-binding proteins (SREBPs)
stimu lates the synthesis of acetyi-CoA carboxy lase- ! (ACC I) and fatty acid synthase wh ich
involves in hepat ic lipogenesis (Horton et al., 1998). EPA suppresses SRE BP- IC gene
expression and thereby results in decreased TG synthesis (Yosh ikawa et al., 2002).
146
Sumudu N. Wamaku lasuriya and H. P. Vasantha Rupasinghe
Harris (1 997 ) conducted a study to see the effect of ro-3 fish oi l (EPA and DHA) and
plant o il (A LA) on human serum li pids and li poproteins with placebo control and found that
for an intervention consumpt ion of fish o il is needed and serum trig lyceride levels were
decreased by 25% to 30%. Also, it is concluded that U>-3 PUFA have a clin icall y important
effect on serum triacylg lycerol levels especiall y in hypertriacy lg lycerolemic pa tients. A lso, it
is found that (V LDL) concentrat ions decrease, littl e rise in LDL cho lestero l and HDLcho lestero llevel was unaffected. In a study conducted in pat ients with type 2 di abetes, fish oil
supp lementat ion signi ficantly lowered trig lycerides and raised LDL-cho lesterol, no
si gn ificant effect on total cho lestero l and g lycemic contro l (Montori et al., 2000). Whereas,
Gollo (1 998) reported that ro le of plasma triglycerides in atherogenesis is shown to be
controversia l as di ~ferenl studies gave paradox ical results. There are some studies generat ing
paradox, for an example, the G ISS I (Gruppo ltali ano per lo Studio d e ll a Sopravvivenza
nell' lnfarto miocardi co) study results showed on ly a slight reduction in trig lyceride levels and
there were no clin icall y sign ificant changes in cho lesterol ( Russo, 2009).
2.2. Anti-Thrombotic Effect

Alteration in platelet aggregation and process related to coagulat ion and fibr inolysis can
be resu lted by w-3 PUFA and also prolonging ihe template bleeding time an d may exert some
beneficial effect on erythrocyte flexibility (Knapp, 1997). Shahar et al., (1 993) showed that
the blood levels of several coagu lation factors may be modi lied by fish o il ro-3 PU FA. There
is a posit ive effect of w-3 PUFA on prevent ing platelet aggregation. Platelet aggregation
causes the development of arterial th rombosis after fom1ing the plaques (H o lub, I 988). EPA
inh ibits th e synthesis of arachidon ic acid (AA) and replaces ii in membrane phospholi pids.
There by ti ssue levels of AA is lowered. Eicosanoids 2-series (prostaglandi n 2-PGl2 an d
tromboxane A2 - TXA2) shows a w ide range of effects on metaboli c pathways wh ic h are
related to atherosclerosis and AA is the precursor o f2-series eicosanoids. Specially, TXA2 is
responsible for platelet aggregation and vasoconstriction. 5-Li poxygenase metabo liies such as
leukotri enes have an effect on inflammation and atherogenesis. Although there are some AA
derived metaboli tes such as prostacyclin wh ich act as vasodil ato rs and oppose platelet
aggregat ion, the imbalance of theses AA metaboli tes create a proatherogenic state (Harris et
al., 2008). E icosanoid 3-series wh ich is d eri ved by EPA is less vasoconstrictive and create
less platelet aggregation compared w ith the eicosanoid 2-series produced from AA (Calder,
2004). EPA shows a compet itive inh ibit ion towards the conversion of AA into endoperox ides
a! the level ofcyclooxygenase (Holub, 1988) .
2.3. Ant i-Hypertensive Effect

lncrease in systemic arte rial compli ance due to incorporation o f EPA into membrane
phospholipids (Nestel et al., 2002) and improvement in endothelial function by EPA (Ch in et
al., 1995) are two mechan isms introduced for blood pressure lowering effect of EPA. Morris
et al., (1 993) observed a dose dependent effect on blood pressure in consumpt ion of w-3
PUFA in fish oil , - 1.3/ -0.7 mmHg at doses per 3 g per day or less, -2.9/ - 1.6 mmHg at 3.3 to
7g per day and -8. 1/ -5.8 mmHg at 15 g per day. Although there was no efFect observed on
Physio logical Role of Eicosapentaenoic Acid .. .
147
blood pressure in healthy subjects, a sign ificant effect was observed in the group of
hypertensive patients and a non-sign ificant decrease in patients w ith atherosclerot ic
card iovascu lar di sease. Gele ij nse et a l., (2002) reported about a meta analysis done with 36
randomized trials wh ich showed a lowered systo li c b lood pressure by 2. 1 mm Hg (P < 0.01)
and di asto li c b lood pressure by 1.6 mm Hg ( P < 0.01) w ith the median dose of3.7 glday E PA
plus DHA. Meta-analysis conducted by Appe l et al., ( 1993) conclude d that die ts b igb in w-3
PUFA (> 3 g per d ay) reduced b lood pressure in patients w ith untreated hypertension.
Another meta-analysis is reported by Mozaffarian et a l., (2005) found that consumpt ion of
fish o il, medi an d ose of 3.5 glday EPA p lus DHA lowered the heart rate by 1.6 bpm in 30
randomized tr ia ls. Bonna et a l., (1 990) reported that 6 g/day of ro -3 PU FA dietary
supp lementat ion in hypersentive patients lowered systo li c b lood pressure by 4.6 mm Hg and
di asto li c b lood pressure by 3.0 mm Hg, wh il e the control w ith corn o il did not show any
effect on b lood pressure.
2.4. Anti-Arrhythmatic Effect

Although further research is required to understand and confirm the molecular
exp lanation for the anti-arrhythmic effects o f ro-3 PUFA, it has been suggeste d from animal
mode ls and cardiomyocytes cultures, ability of EPA to influence the act ivity of myocyte
sarcolemma ion channels (sodi um and L-type calc ium) is the reason for its an6-arrhythmic
effects (Russo, 2009). Massaro et al., (2008) and Kris-Etherton et a l., (2002) expla ined using
several reported studies that ro-3 PUF A inh ib it sodium channels w h ich makes longer relative
refractory period and increase the vo ltage required for membrane depolarizat ion (Massaro et
al ., 2008). A lso, ro-3 PUFA mainta in the integrity o f l-type c a lcium channels as well and
thereby cytosolic calc ium overload dur ing the ischemic stress is prev ented (H allaq et a l.,
1992, Kris-Ethe rton et al., 2002). A lso, Russo (2009) ment ioned about o ther mechan isms
proposed in di fferent reports; increase in the threshold of ventricu lar fi brillation, increase in
heart rate var iabili ty, and reduce in isch emic damage. The actual effect can be a synergistic
interaction of those mechan isms (Russo, 2009). it is found that ro-3 PlJFA are incorporated
into myocardia l cell membranes (Harris et a l., 2004) and have the ab ility of affect ing
eicosanoid production and ion -channel function (Leaf et a l., 2003).
Experiments have shown the decreased inj ur ies to heart tissues and abnormal hea rt
rhythms with ro-3 PlJ FA in dogs and cats undergone stimu lated heart attacks. A lso, it is
reported that ro-3 PlJ FA prevented tachyarrhythmaias in neonata l ventricular muscle cell s in
rats (Serami, 2009). In a study conducted by Christensen et a l., ( 1996) to see the effect of
di etary ro-3 PlJ FA in surv ivors of lvll, it was found that subjects showed an increase in heart
rate. The increased parasympathetic cardi ac tone is reflected by an increased heart var iability
wh ich increases the ventricular fibri ll at ion thresho ld. Infusion of EPA in spontaneously
beating isolated rabbit heart gave inotrop ic an d chronotrop ic e fTects (Dhein et a l., 2005). X iao
et a l., ( 1995) reports in an experiment carried out w ith rat cardi omyocytes, ro-3 PUFA
suppressed both L-type calc ium channels and sodium channels. Grimsguard et a l., (1 998)
conducted an intervent ion study w ith the di etary supp lementat ion w ith EPA ethy l esters (4 g
per day) for the healthy non smok ing men aged 36 to 56 years and concluded that there is an
influence ofEPA on heart rate.
148
Sumudu N . Wama kulasuriya and H. P. Vasantha Rupasinghe
Cl in icall y observed most common card iac a rrhythmia is atri al fibri llation wh ich causes
stroke and heart fail ure (H arris et al., 2008). Low incidence o f postoperat ive atrial fibrill ation
was observed by Cal'o et al., (2005), in the patients w ith coro nary artery bypass graft surgery
who were administrated ro-3 PUFA . C lin ica l experiment per formed w ith 402 patients w ith
irnp lantable cardioverter defibr ill ators (lC D), prov iding 2.6 g/day EPA + DHA w ith placebo
controll ed showed a signi ficanl t ime delay in their first lC D discharge ( Leaf et al. , 2005).
Consumpt ion o fro-3 PU FA is beneficial fbr post M l patients (Harris et al., 2008).
2.5. Anti-Ath eroge nic Effect

Eritsland et al. ( 1996) conducted a study with patients w ith coronary artery b ypass
graft ing giving 4 g per day o f fi sh o il concentrate and w ith antith rombotic treatments. On ly a
few patients with increasing relat ive change in serum phosphol ipid ro-3 P UFA. A lso
concluded that ro-3 PUFA supp lementat ion reduces the vein graft occlusions.
Di fTerent mechan isms have been propo sed for the in fl uence o f ro-3 PUFA treat ing on
atherogenesis. The in nammat io n associated w ith atherosclerosis is mit igated by ro-3 PU FA
by inh ibit ing the 5-lipoxygenase pathwa y in neutrophil s and mo nocytes an d decreasing the
pro duct ion o f inter leuk ine- 1 a nd interleuk ine-6. Kr is-Etherton (2002) proposed th at ro-3
PU FA interrupt in the arach idon ic acid react io ns by replacing A.A in cell mem branes and
inh ibit ing c ycloox ygen ases wh ich are res ponsible for pro-inflammat ion. Abe et al., ( 199 8)
suggested that ro-3 PUFA could change the metabo li sm of cell adhesion mo lecules . They
prevent the adhesion o f monocytes to e ndothelial cell s, a process med iated b y leukocyte
and vascular cell adhesion mol ecules (Calzolari et al. , 2009 ). By decreasing th e adhesion
mo lecu le, the ce ll ular mo vements into th e vessel wall int ima ma y be slowed down,
red ucing th e atherosclerot ic plaque format io n. A lso, these PUF A are th ought to protect
endothelium by inh ibiting ox idat ive stress wh ich causes the atherosclerotic vascular lesions
(Co ll ussi, 2007) .
3. Conclusion
EPA can be introduced as a d ieta ry intervent ion for the reduction o f risk factors
associated w ith CH D through th eir abil ity to act as ant ithrombati c, ant ihypertensive,
anti ath erogen ic, and anti arrhythmic agents. Both prima ry and secondary prevention o f CHD
can be addressed through the intake o f E PA in required doses . However, further resea rch is
needed to beller understand the molecu lar level mechanisms in providi ng cardio protection, as
some contradi ctory observations and d ifferent mechan isms are p roposed for the physiological
action o f EPA.
Table I. Mechanism of a er ion ofEPA

Mc-chaniim
Redu-.;;ing triglyoerides
levels
Anti-thrombo tic effect
Anti -h yp~rten si, e effect
Anti-arrhrthmatic effL'>
Anti-atherogenic eff~;_
Remarks
~1arine w-3
PUFA consumption lowered .serum trigly'--.erid: le-vd by 25% to
30%.
lowere-d Lriclyceride levels in type 2 diabetes with ftSh oil supple-mentation.
Fish oil (!)3 PliFA modify the levds of .several ooaculation fa"-'"tors in blood.
w-3 PUFA supplementation d:creas.W hemostatie markers of atherosclerosis.
w-3 fish oil has dose dependent effect o n blood pressure. Hrpolensi'oe effect o n
hyperten-sive patients.
Reduction in blood pressure with high dosed ro-3 PUFA d iel~ in hypertensive
patients.
6g/d<t}' <:>-3 PUFA lowered S)stolic and diastolic blood pressure in hypertensive
patients.
lncreas..'"lll he-art rate-was o bserved in survivorsof MI with dietary
!llpplemenlation ofw-3 PUFA.
EPA influences the heart rat~ in health)' -subj ects.
Fish rich diet on post tvll patients shmved rttluoed cardia-;; death.
Ventricular tadt)'Cardia in patients with '-'-l!.rdio\terter d:fibrillato rs with infusion
ufm-3 PUFA.
w-3 PUFA supple-mentation reduces the vein b'rafl ood usions patientS with
oomnary artery bypass grafting.
q tokine-stimulated adhesion molecule expression was inhibited in endothelial
oells, but in lesse,r extenl than DHA
Rc(crcncl!i
Harris, (1 997)
Montori Cl aL, {2000)

Shalur e t al., (1993)
Jolun<on <l al.,( l999)
tv1orris et at ( 1993)
Appd <tal., ( 1993)
B>nna e tal., ( 1990)
Ch risten s~n et al..
(l996)
Grimsguaard et al., {l 998)

Burr e t al., (1989)
Schrepf e t al., (2004)
Eritsland et al.. (1996)

d!-Caterina d al . ( 1998)
PA
(o>.J PUFA)
I
I VLOL.I.DL
1AA m
!TO
cdl !nm:lbra!H'J
f RDL
I jTXA2 I
Hypouiglyccmic effec:~
L _ _ __
II
___J
PrCkSWIOids ). SC:rid'
(P(if3 and TXAs)
b;~b.ibit
; .lipoxyaeru~
pathway
II
Anri-thrombotk tfftct
Prt\"'('n1
""
LNkotrintts
Sstdu
Elec:uophrsiolosical
tfftcts
tdhtiiOft
lnltibitl:on
llntdituldn.tl &
l>fpbtdrt
.a.gptg;uion &
\'uodilation
\
irlttdtukint -6
And-infbmm.u ory effect
Anlih)'Pertett.sivt
<ffttt
1
Anri.cllerogmic tfl"ea
11
,;\nli.Mtbythm..aric cfrec:t
1/
.....------:-------,
Cardio prottc:don
..
Physiological Role of Eicosapentaenoic Acid .. .
!5 1
References
[ I ] Abe, Y., E I-Masri, B., Kimball, K. T., et al. 1998. Soluble cell adhesion molecules in
hypertriglyceridemia and potent ial significance on monocyte adhesion. Arterioscler
Thromb Vase Bioi. 18, 723- 73 1.
[2] Appel, L. J., Mill er, E. R., Seidler, A, J., et al. 1993. Does supplementation of diet "~ th
' tish oil' reduce blood pressure? A meta-anal ysis of controlled clinical trials. Arch
Intern Med. !53, 1429- 1438.
[3] Bays, H. E., T ighe, A. P., Sadovsky, R., and Davidson, lvl. H. 2008.
Hypertriglyceridemia is a risk factor for ath erosc lerotic coronary heart disease. Expert
Rev Cardiovasc Thero. 6, 39 1-409.
[4] Bonaa K. H ., Bjerve, K. S., Straume, B., et al. 1990. Effect of eicosapentaenoic and
docosahexaenoic acids on blood pressure in hypertension. A population-based
intervent ion trial from the Tromso study. N Engl .I Med. 322, 795-80!.
[5] Burr, M., Gilbert, J. F., Holliday, R. M., Elwood, P. C., Febily, A. lvl., Rogers, S., et al.
1989. Effects of changes in fat, fish, and fibre intakes on death and myocardial infarction
diet and infarction trial: Diet and infarction Trial (DART). Lancet. 334,757-76 1.
[6] Cal'o, L., Bianconi, L., Colivicch i, F., et al. 2005. n-3 Fatty acids for the prevent ion of
atrial fibrillat ion after coronary artery bypass surgery: a randomized controll ed trial. .I
Am Call Cardia/. 45, 1723- 1728.
[7] Calder, P. C. 2004. n-3 Fatty acids and cardiovascular disease: evidence explained and
mechan isms exp lored. C/in Sci. 107, 1- 11.
[8] Calzo lari, 1., Fumagalli, S., Marchonni, N. & Di Bar i, M. 2009. Polyun saturated falty
acids an d cardiovascular disease. Otrr Pharm Des. 5, 4094-4 102.
[9] Chin, J. P, & Dart, A. M. 1995. How do tish oi ls affect vascu lar funct ion? C/in Exp
Phannacol Physiol. 22, 7 1- 8 1.
[ 10] Christensen, J. H., Gustenhoff, P., Korup, E., et al. 1996. Effect of fish oi l on heart rate
variab ili ty in survivors of myocardial infarct ion: a double blind randomised controlled
trial. BA-1.1. 3 12,677- 678.
[ ll ] Colussi , G., Catena, C., Baroselli , S., Nadali ni, E., Lapenna, R., Chiuch, A & Sech i, L
A. 2007. Omega-3 Fauy Acids: from biochemistry to their clinical use in the prevent ion
of cardiovascular disease. Recent Patents on Cardio vascular Drug Disco very. 2, 13-2 1.
[ 12] de Caterina, R., Bernini, W., Carluccio, M. A., Liao, J. K. & Li bby, P. 1998. Structural
requirements for inhibit ion of cytokine-induced endotheli al acti vat ion by unsaturated
fatty acids. J Lipid Res. 39, 1062- 1070.
[ 13] de Leiris, J., de Lorgeri l, lvl., & Boucher, F. 2009. Fish oi l and heart health . .I
Cardiovasc Pharmacal. 54, 378-384.
[ 14] Demyanet s, S., Huber, K. and Woj tal , J. 2011. Inflammat ion and the cardiovascular
system. Eur Surg. 43, 78- 89.
[ 15] Dhein, S., M ichaeli s, B., & Mobr, F. W. 2005. Antiarrhythmic and electrophysiological
effects of long-chain omega-3 polyunsaturated Falty acids. Nauny n Schmiedebergs Arch
Pharmacal. 37 1, 202- 2 11.
[ 16] Eritsland, J., Amesen, H., Gronseth, K., et al. 1996. Effect of dietary supplementat ion
with n-3 fatty acids on coronary artery bypasses graft patency. Am .I Cardia/. 77, 3 1- 36.
152
8umudu N. Wamaku lasuriya and H. P. Vasantha Rupasinghe
[ 17] Geleijnse, J., Giltay, E., Grobbee, D., Donders, A., Kok, F. 2002. Blood pressure
response to tish o il supp lementat ion: meta regression analysis of randomized tr ials . .I
Hypertens. 20, 1493- 1499 .
[ 18] Gotto Jr., A. M. 1998. Triglyceride as a r isk factor for coronary artery disease, Am . .f.
Cmdiol. 82, 22Q- 25Q.
[ !9] Grimsgaard, S., Bonaa, K. H., Hansen, J. B., et al. 1998. Effects of high ly purified
eicosapentaenoic acid and docosahexaenoic acid on hemodynamics in humans. Am .I
C/in Nutr. 68, 52- 59.
[20] Hansson, G, K. & Libby, P. 2006. The inunune response in atherosclerosis: a doub leed ged sword. Nat Rev /mm uno/. 6, 508- 5 19.
[2 1] Harris W. 1989. Fish oils and plasma lipid and lipoprotein metabo lism in humans: a
cri t ical review . .! Lipid Res. 30, 785- 807.
[22 ] Harris, W. 8. 1997. n-3 Faity acids and serum lipoproteins: human studi es. Am .I C/in
Nutr. 65(8uppl): I 6458 - 1654S.
[23 ] Harr is, W., Sands, S., Windsor S., et a l. 2004. Omega-3 !atty acid levels in transplanted
human hearts: effect of supp lementation and comparison with erythrocytes. Circulation.
I 10: 1645- 1649.
[24] Harris. W. S., M ill er, lvl., T ighe, A. P., Davidson, l'v!, H., Schaefer, E, J. 2008. Omega-3
fatty acids and coronary heart disease risk: cli nical and mechan istic perspect ives.
Atherosclerosis. 197, 12- 24.
[25] Holub, B. J. 1988. Current Review: Dietary fish oils containing eicosapentaenoic acid
and the prevent ion of atherosclerosis and thrombosis. CM4.f. 139, 377-38 1.
[26] Horton, J. D., Bashmakov, Y ., 8 himomura, !. & Shimano, H. I 998. Regulat ion of sterol
regu latory element binding proteins in livers of fasted and refed mice. Proc. Natl. A cad.
Sci. USA 95, 5987-5992.
[27] Johansen, 0., Seljeflot, !., Hostmark, A. T, et al. 1999. The effect of supp lementation
with omega-3 Fatly ac ids on so luble markers of endotheli al function in pat ients w ith
coronary heart disease. Arterioscler Thromb Vas Bioi. I 9, 168 1- I 686.
[28] Jump, D. B. 2002. The b iochemistry of n-3 polyunsaturated fatly acids . .I Bioi Chem.
277, 8755- 8758.
[29] Karali, D. G. In Fish oil: production, consumption and health benefits. Dijk, M. V.;
Vitek, J.; Nova Science Pulishers, Inc. Hauppauge, NY, 2012; 353-366.
[30] Knapp, H. R. 1997. Dietary Fatty acids in human thrombosis and hemostasis. Am .I C/in
Nutr. 65 (Suppl), I 6878- 16988.
[3 1] Kones, R. 20 I I. Primary prevent ion of coronary heart disease: integrat ion of new data,
evolving views, revised goals, and ro le of rosuvastati n in management. A
comprehensive survey. Drug Des Devel Ther. 5, 325-380.
[32] Kris-Etherton, P. M, Harris, W. S., & Appel, L J. 2002. American Heart Association.
Nutrit ion Committee. Fish consumpt ion, fish o il, omega-3 fatty acids, and
cardiovascular disease. Circulation. 106,2747- 275 7.
[33] Kromhout, D., Bosschieter, E. B., & Coulander, C . I 985. The inverse relat ion between
fi sh consumpt ion and 20-year mortali ty from coronary heart disease. N Engi .I Med. 3 12,
1205-1209.
[34] Leaf, A., A lbert, C., Josephson, M., et al. 2005. Preventi on of fatal arrhythmias in highr isk subjects hy fish o il n-3 fatty acids. Circulation. I 12,2762- 2768.
Physiological Role of Eicosapentaenoic Acid .. .
153
[35] Leat; A., Kang, J., X iao, Y ., & Bill man, G. 2003. C linical prevention of sudden cardiac
death by n-3 polyunsaturated fatty acids and mechanism of prevent ion of arrhythmias by
n-3 fish oils. Circulation . 107,2646-2652.
[36] Leaf, A., Kanga, J. X . & X iaob, Y . 2008. Fish oil fatty acids as cardi ovascular drugs.
Curr Vase Pharmacal. 6, 1- 12.
[37] Leder, C., Z iegler, l\1!. and Gawaz, M. 20 10. Modulat ing immune responses and
inflammat ion. Semin Thromb Hemost. 36, 21 9- 222.
[38] Libby, P., Ridker, P. M. & Hansson, G. K. 2009. Inflammat ion in ath erosclerosis: from
pathophysiology to pract ice . .I Am Col/ Cardia/. 54, 2129- 2138.
[39] Mahaffey, K. R., C lickner, R. P. Jeffries, A. J. 2008. Methylmercury and omega-3 faHy
ac ids: eo-occurrence of dietary sources with emphasis on fish and shellfish.
Environmental Res earch. I 07, 20- 29.
[40] Massaro, M., Scodili i, E., Carluccio, M. A., Caterina, R. 2008. Basic mechanisms
beh ind the effects of n-3 fatty acids on cardi ovascular di sease. Prostag Leukotr Ess. 79,
109- 11.
[4 1] Mill er, M ., Motevalli , M., Westphal, D ., Kwiterovich, Jr. P. 0 . 1993. Incorporat ion of
oleic acid and eicosapentaenoic acid into glycerolipids of cultured normal human
.fi broblasts. Lipid.~. 28, 1- 5.
[42] Montori, V . M., Farmer, A., Woll an, P. C., et al. 2000. Fish oil supp lementation in type
2 di abetes: a quantitative systematic review . Diabetes Care. 23, 1407- 14 15.
[43] Morris, M. C ., Sacks, F., & Rosner, B. 1993. Does fish oil lower blood pressure? A
meta-analysis of controll ed trials. Circulation. 88, 523- 533.
[44] Mozatr arian, D., Geelen, A ., Brouwer, 1., et al. 2005. Effect of fish oil on heart rate in
humans: a meta-analysis of randomized controll ed trials. Circu lation . 112, 1945- 1952.
[45] Nestel, P, Shige, H, Pomeroy, S, et al. 2002. The n-3 fatty acids eicosapentaenoic acid
and docosahexaenoic acid increase systemic arterial compli ance in humans. Am .I C/in
Nutr. 76, 326-330.
[46] Racine, R. A. & Deckelbaum, R. J. 2007. Sources of the very-long-chain unsaturated
omega-3 faity acids:eicosapentaenoic acid and docosahexaenoic acid. Curr Opin C/in
Nutr. I 0, 123- 128.
[47] Ren, B., The len, A., Peters, J., Gonza lez, F., & Jump, D. 199 7. Polyun saturated fatty
ac id suppression of hepati c fatty acid synth ase and S l 4 gene express ion does not require
perox isome proli ferator-act ivated receptor alpha . .I Bioi Chem. 272,26827-3 2.
[48] Roth, E. M . & Harri s, W. S. 20 10. Fish oil fbr primary and secondary prevent ion of
coronary heart di sease. Curr Atheroscler Rep. 12, 66- 72.
[49] Russo, G. L. 2009. Commentary-Dietary n-6 and n-3 polyunsaturated fatty acids: From
biochemistry to cli nical impli cat ions in cardiovascular prevent ion. biochemical
p harmacology. 77, 93 7-946.
[50] Saremi, A . & Arora, R. 2009. The utility of omega-3 fatty acids in cardiovascular
disease. Am .I Ther. !6, 421 -436.
[5 1] Schrepf, R., Li mmert, T., Weber, P.C., The isen, K . & Sell mayer, A . 2004. Immediate
effects of n-3 fatty acid infusion on the induction of sustained ventricular tachycardi a.
Lancet. 363, 1441- 1442.
[52] Shahar, E., Folsom, A. R., Wu, K . K, et al. 1993. Associati ons of fish intake and di etary
n-3 polyunsaturated fatty acids with a hypocoagulable profi le. The Atherosclerosis R isk
in Communit ies (A R!C) Study. Arteriase/er Thromb. 13, 1205- 1212.
154
Sumudu N . Wamakulasuriya and H. P. Vasantha Rupasi nghe
[53] Si rtori, C . R., Crepaldi, G., Manzato, E., lvlancini, M., Rivell ese, A., Paol ett i, R ., eta!.
1998. One-year treatment with ethyl esters of n-3 Fatty acids in pat ients w ith
hypertriglyceridemia and glucose intolerance Reduced triglycer idemia, iota! cholesterol
and increased HDL-C w ithout glyc.e m ic alterations. Atherosclerosis. 137, 4 19-427.
[54 ] Xiao, Y. F, Kang, J. X, Morgan, J. P, & Leaf, A. 1995. Blocking eiTects of
pol yunsaturated Fatty acids on Na- channels o fneonatal rat ventricular myocytes. Proc
Natl Acad Sci USA. 92, 11000-11 004 .
[55] Yoshikawa, T., Shimano, H., Yahagi, N., et al. 2002. Polyunsaturated fatty acids suppress
sterol regu latory element-binding protein l e promoter act ivity by inh ibition of liver X
receptor (LXR) binding to LXR response elements . .I Bioi Chem. 277, 1705-1711 .

Edi tors: T.G. Bradley and F.P. Vargas, pp. I 55- 163
ISBN: 978- 1-62257-480-3

20!2 Nova Science Pub lishers, Inc.
Chapter 8
H YPOCHOLESTEROLEMIC AND H YPOLIPIDEMIC

EFFECTS OF EMULSIFIED SURIMI FEEDING IN MICE
Teruo Matsushita, Ry usuke Tanaka,
Hideto Fukushima ami Yutaka Fukuda
Department of Food Science and Technology, National Fisheries Univers ity, Japan
Abstract
Fish oil, comaining eicosapemaenoic acid (EPA) and docosahexaenoic acid (DHA), shows
prevemive effects against various diseases. 'Surimi'" is a thick paste of fish meat, which is the
cooking material used m make "kamaboko," a processed Japanese seafood produce However,
the main nmriem of surimi is fish protein, and the comem of fish oil is very low. We have
developed an emulsified surimi in wh ich added fish oil is formed into fine panicles and
un iformly distribmed th roughout the fish protein. In the presem study, we studied the effects
of emulsified surimi on hypercholesterolemia and the absorption of EPA and DHA through
mice imestines in order m confirm a fumre role for emulsified surimi in functional foods.
Hypercholesterolemia was induced in mice by the imake of a diet comaining cholesterol (2%)
and cholic acid (0.5%), and the effects of emulsified surimi feeding on hypercholesterolemia
in this disease model were examined. The emulsified surimi was lyophilized, and the
powdered emulsified surimi was added m the diet for mice in the experiment. The mice's
serum cholesterol level was clearly increased by consuming the cholesterol-added diet for 9
days. Furthermore, the increase of the serum cholesterol level, induced by the consumption of
the cholesterol-added diet, was significamly inhibited by the addition of a powdered
emulsified surimi to the diet. In addition, the EPA and DHA levels in the livers of the
powdered emulsified surimi-fed mice were significamly increased, compared with the control
mice livers. The results of the presem study suggest that imaking emulsified surimi-used food
products might be used to exploit disease preveming properties.
Introduction
Fi sh oil contain ing n-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (E PA,
20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), shows preventi ve effects against var ious
di seases, including hyperlipidemia and thrombosi s (1 -5], and have been recommended for
156
Teruo Matsush ita, Ryusuke Tanaka, Hideto Fukushima et al.
intake in daily diets. However, fish is unsuitable for infants and elderly peop le who have
di fficulty eating it due to the presence of bones. W ith such a considerat ion, boneless types of
foods, which retain the health benefi ts of fish, are needed. "Surimi" is a th ick paste of fish
meat {6-7] which is the cooking or processi ng material used to manufact ure " Kamaboko," a
tradit ional Japanese fish cake [8- I 0]. However, th e main nutrient of surimi is fish protein, and
the content of 6sh oil is very low. Therefore, we have developed an emulsi fi ed surimi in
which added fish oil is fom1ed into fine partic les and un ifom1ly distributed throughout the
fish prot ein. In the present study, we examined the effects of the emu lsified surimi on mouse
hypercholesterolem ia, and the absorption ofE PA and DHA th rough the intestines of mice, in
order to confim1 a future role for emu lsi fied surimi in functional foods.
Materials and Methods

Materials a nd Animals
Dry, powdered emu lsified surimi was o btained from Fuj imitsu Co. Ltd . (Nagato,
Yamaguch i Pref., .Japan), prepared by the meth od of Okazak i et al. [ I I, 12]. In brief, after
adding pur ifi ed fish oil containing EPA and DHA to Surimi fish protein, the emu lsified
surimi was further prepared by emu lsi fying it under vigorous mixing condit ions of a high
rotation speed. The powderizat ion of the emulsilied surimi was perfom1ed using a freeze-dry
apparatus. The lipid content of the powdered emulsified surimi was approximately 40%.
1vlale BALB/C mice (seven weeks old) were purchased from Kyudo Co., Ltd. (Tosu,
Saga Pret:, Japan). The mice were housed in individual cages kept under Ctlndit ions of 2326"C w ith a 12-hour li ght/dark cycle until used in the foll ow ing examination. Food and water
were freely avai lable.
T he Effect of Emu lsified Surimi Feedings on 1\ormal

and H ypercholesterol emic Mice
(i) Animals and Experimental Desig n
After an acclimation period of about one week, the mice were randomly di vided into four
groups (group I [n = 5], group 11 [n = 5], group Ill {n = 5] and group IV [n = 5]). Group I was
given /VfF (95%) (K BT Oriental Co. Ltd., Tosu, Saga Pref., Japan) as a basal di et + casein
(5%). Group 11 was given 1v!F (95%) + emu ls ifi ed surimi (5%). Group nI was given a high
cho lestero l diet (95%) + casein (5%). Group IV was given a high c-holesterol di et (95%) +
emulsifi ed surimi (5%). The composit ion of the high cholestero l diet was M F supplemented
" i th 2% cholesterol and 0.5% choli c acid. T he diet and water were freely avail ab le. In th is
regard, the upper li mit of the diet consumpt ion was 4.0 g/day/ mouse. The administrat ion tem1
for th e above-ment ioned diets which included emulsifi ed surimi or its control was 9 days. On
the last day, a blood sample was taken from a heart puncture and the li ver was excised under
anesthesia w ith di ethyl ether. To obtain the serum, the coll ected blood sample stood fbr 30
minutes in a test tube at room temperature and centrifuged f(lr at 1,900 g for 20 minutes. The
excised liver was washed w ith ice-cold saline and stored at -30' C unt il used .
Hypocholestero lemic and Hypo Iipidemic Effects of Emulsified Surimi Feeding...
157
All of th e animal experiments were performed with pem1ission of the "Committee for
Use and Care of Laboratory An imals" of National Fisheries Un iversi ty, Japan and in
comp liance with the "Guideline for An imal Experiments in Research Institutes under the
jurisdi ction of the Ministry of Agriculture, Forestry and Fisheries, Japan."
(ii) Determination of Lipid Levels in t he Liver

The hepatic lipids from I g of liver were extracted according to the method ofFolch et al.
( 13]. T he obtained extract was diluted to 10 ml with a ch lorofomv'methanol (2: I) solution.
The solut ion was retained as the sample. The total amount (w/ w) of hepat ic li pids was
measured by drying the solvent of the sample solution and weighing the residue.
The lipid class (triglyceri de, cholesterol ester, cholesterol, free fatty acid and
phospholipid) anal ysis was carried out according to the method of Peyrou et al. ( 14] with
some modificat ions. Three mg of the obtained hepati c lipids were dissolved in 600 ~t L of a
chlorofomv methanol (2: I) solut ion. Two ~tL of the solution was spotted onto pre-heated S-Ill
chromarods with a 5 ~t L micro-dispenser (Drummond Scient ifi c Co., Broomall, PA, USA).
After spott ing, the rods were placed in the developing tank unt il the developing sol vent
reached 10 cm. The composit ion of the developing solvent was hexane-ether-formic acid (60:
5: 0.1 5, v/ v). The rods were then dri ed w ith Roddryer (TK-8, latron Laboratories, Inc.,
Tokyo, Japan) for one minute. The spots in the rods were analyzed by a hydrogen flameionizat ion detection (HF ID) scan using an !A TROSCAN l'v!K-5 anal yzer (!atron
Laboratories) . The hydrogen and air flow rates of the HFID were 160 and 2,000 ml/ minute,
respectively. The scann ing speed was set at 30 cnv'minute.
To analyze fatty acid composit ion, the extracted lipids were transmethylated by
saponification followed by using a modification described by lchihara et al. [ 15]. A lipid
samp le (ea I 0 mg) and I mg of heptadecanoic acid, as an internal standard in a screw-capped
glass tube, were hydrolyzed w ith 0.75 ml of0.5 % KOH in methanol at i00C for 9 minutes.
One mL of 14% BF 3 was added to the react ion mixture in methanol at 100C for 7 minutes.
One mL of water and 2.5 m L of saturated NaC I solution were added to the solution. The
mixture was vortexed and centri fuged at 2,000 g for I 0 minutes. The upper layer contained
fatty acid meth yl esters (FAMEs). These weretransferred to Sep-Pak Silica-630 mg (Waters,
Mi lford, USA), pre-washed w ith hexane and then eluted w ith 10 mL hexane/diethyl ether
(96:4). The eluted solution was evaporated to dryness using a centrifuged concentrator
(fA IT EX, Koshigaya, Japan) and the FAMEs were dissol ved in 200 ~t L of acewne for a gas
liquid chromatography (G LC) analysis. The G LC system was a gas c.hromatograph (G-6000;
HITACHI , Japan) equipped with a flame-ionization detector and a capill ary column (TCFFAP, 30 m x 0.25 nm1 i.d.; GL Science, Japan). The column temperature was programmed
for a li near increase of 2c min 1 from !80 to 23oc. The injection and detect or port
temperatures were 250 and 250C, respect ively. T he FAMEs on the chromatogram were
ident ified and calcu lated by convent ional methods using the retention time of standards.
(iii) Determination of Lipid Levels in Serum

The total cho lesterol level was assayed by the cholesterol oxidase-DAOS method using a
Cholestero l 4est kit (Wako Pure Chemical Industries Lid., Osaka, Japan ) according to the
158
manufacturer's protocol. The triglycer ide level was assayed by the GDP-DAOS method using
the Triglyceride -test kit (Wako) accordi ng to the manufacturer's protocol.
Data and Statis tical Ana lysis

Data is expressed as the mean standard deviation. A statist ical analysis of the mult iple
comparison was done with Dunnett 's test, using the software "Excel Statist ics" (Esumi Co.
Ltd., Tokyo, Japan).
Results and Discussion

The emulsified surimi was lyophili zed and the powdered emulsified surimi was added to
the diet. The same amount of casein powder was added to the control diet instead of th e
emu lsified surimi. Table I shows the composit ion of the experimental diets for the four
groups of mice (1, 11, Ill and IV). The li pid class analysis shows that cholesterol and
triglyceride contents were much higher in the high cholesterol diets ( Ill and IV) and
emulsified surimi-added diets (11 and IV), respectively (Tab le 1). The fatty acid analysis
shows that the contents ofEPA and DHA were higher in th e emulsified surimi-added diets ( 11
and IV) than in the contro l di ets (I and Il l), as shown in Table I. Large di fferences associated
\vi th the other component s in the experimental diets were not observed (Table I ).
Table I. Composition oft he experimental diets

IJ
Basal
diet
Genera l component (%)
Moisture
Protein
Lipid
Carbohydrate
Ash
Lipid class (mgfg)
Triglyceride
Cho lesterol
Phospholipid
o-3 polyunsaturated fatty acid
(mg/g)
EPA (20:5 n-3)
DHA (22 :6 n-3)
EPA+DHA
El' A : cic.osapcntacnoic acid,
DHA: doeosahexaenoic acid,
rtl : not detected.
7.3
27.4
Basal diet
+ emulsified
surimi
7.3
Ill
IV
High
High cholesterol diet
cholesterol
+emulsified surimi
diet
7.I
26.9
6.8
53.6
7. 1
25.2
8.5
53.6
5.6
5.6
54.4
5.8
25.7
6.7
54.4
5.8
34.7
nd
15.3
50.2
nd
17. I
27.2
24.5
16.5
4 1.7
24.7
18.0
0. 12
3. 9 1
4.03
0.63
6.97
7.60
0.11
2.75
2.86
0.59
6.30
6.89
5.0
Hypocholestero lemic and Hypo Iipidemic Effects of Emuls ified Surimi Feeding.. .
159
The effects of the emulsi fied surimi feed ings on body weight gain, I iver weight and food
intake in the experimental mice are shown in Tab le 2. The feedings of the high cholesterol
diet for 9 days slightly decreased the body we ight gain as compared to the feedings of the
basal diet, probab ly due to the reduction of the total f(>od intake in mice. On the other hand,
the feedings of the high cholesterol di et sign ificantly increased the li ver weight of mice as
compared to the feedings o f the basal di et. Feedings of emulsi fied surimi showed almost no
effects on body weight gain, liver weight or food intake in mice. During the experimental
period, no abnormal symptoms were observed in mice of either experimental group.
Table 2. Effects of Emulsified Surimi feedings on body weight gain,
liver weight and food i ntake i n mice
I
11
Ill
IV
Basal diet
Basal diet
+emulsified
surimi
High cholesterol
diet
High cholesterol
diet
+emulsified surimi
23. 1+1.3 (ns)

25. 1:.:1 .0 (ns)
1.91.7 (ns)
24.0:d .0 (ns)
24.40.6 (')
0.4+ 1.1 (ns)
25.2+0.5 (NS)
25.80.5 Cl
0.60.6 (NS)
1.5 1+0.09 (ns)
2.03+0. 11 (')
1.880. 15 (NS)
3 1.03.5 (ns)
26.5+1.8 ( )
28. 1+2.5 (NS)
Body weight (g)

Day I
23.81.4
Day 9
25.71.1
Ga in
1.91.1
Liver weight (g)
Day 9
1.6 1+0.07
Total intake of bait (g)
Day I-Day9
32.81.2
..
mc.an standard deviation (n=5);

'' 1'<0.0 I vs basal diet, ns: not signilk ant vs basal diet;
"P<O.O I vs high cholesterol diet, NS: not significant vs high cholesterol diet.
Table 3. Effects of Emulsified Surimi feeding on se.rum choleste.rol

and t riglyceride levels i n mice
Serum
cholestero l
(mg/ 1OOm l)
Serum
triglyceride
(mg/ 1OOm l)
IV
High cholesterol
diet +
emulsified surimi
11
Ill
Basal diet
Basal diet+
emulsified surimi
High cholesterol
diet
122.1+11.6
106.43.7 ( )
183.738.8 ()
122. 1+11.6()
178.5+14.2
129.9+20. 1 ( )
..
123.634.4 ()
103.922.0 (NS)
mean standard deviation (n=5)

P<o.o 1, ' 1'<0.05 vs basal diet.
'P<0.05 vs high cholesterol diet. :-.IS: not signillc.ant vs high cholesterol diet.
Hypercholesterolemia in mice was induced by intaking a di et that contained cholesterol

(2%) and chol ic acid (0.5%), and the effects of emulsified surimi feedi ngs on the
hypercbolesterolemia in th is disease model were examined. As shown in Table 3, the serum
160
cholesterol level of mice was clearly increased by intaking the high cholesterol diet for 9
days. The serum cholesterol level was significant ly lowered by feedings of emulsified surimi
in both the high-cholesterol-diet and the basaJ-djet mice. In addit ion, the serum triglycerid e
level was also lowered by feedi ngs of emulsifi ed surimi . These results suggested prevent ive
or therapeutic functions, associated w ith emulsified surimi for hypercho lesterolemia and
hyperlipidemia, simil ar to a number of reports show ing that fish o i I containing EPA and DHA
exerts various [unctions of physiological regu lation {1-5, 16-22].
Ta ble 4. Effects of Emulsi fied Surimi feeding on hepatic lipid content
and hepatic lipid class levels i n mice
Basal diet
11
Basal d iet
+em ulsified
su rimi
Ill
High
cholesterol
diet
..
Hepatic lipid content
(%)
4.68+0.35
4.82+0.26 (ns)
5.8 10.62 ( )
Hepatic lipid class

(mg/g)
Triglyceride
Cholesterol ester
Cholesterol
Free fatty ac id
Phospho lipid
12.2+2. 9
1.0+0.4
1.20.3
nd
43.75.4
12.1+1.3 (ns)
0.60.3 (ns)
1.5+0.6 (ns)
nd
43.1 ' 2.9 (ns)
8.33.9 (ns)
18.22.4 ( )
1.60.2 (ns)
0.10.2
29.8+9.2 ( )
..
IV
High cholesterol d iet
+emulsified su r imi
5.73+0.44 (NS)
11.42. 9 (NS)
12.5+2.8 c=J
1.50.1 (NS)
nd
3 1.85.8 (NS)
rr:J : not detected

mean standard deviation (n=5)
"1'<0.0 I, ' P<0.05 vs basal diet, ns: not significant vs basal diet.
"P<0.05 vs bigh cholesterol diet, NS: not significant vs high cholesterol diet.
Since the serum levels o f cholestero l and triglycer ides were affected by the feedings of
emulsifi ed surimi , li pid analyses were performed in the next stage on the li vers of the mice.
Tab le 4 shows the data of the total lipid content and lipid class analysis in the Jivers of mice.
Al though the hepat ic total li p id contents and phospho lipid levels of the mice fed the high
cholesterol diet, (group 11[) respectively, compared to those of the mice fed the basal d iet
(group 1), were sign ificant ly elevated and decreased, respectively, s ign ificant di fferences of
total lipid contents and phospho li pid leve ls between groups Ill and IV were not observed
(Table 4). A simil ar tendency of change in hepatic triglyceride levels was observed (Table 4) .
Al though hepat ic cholesterol ester levels of those mice fed the high cho lestero l ctiet were also
signi ticantly elevated compared to those of the mjce fed the basal djet, feedings of emulsi tied
surimi sign ificant ly (P < 0.05 versus group Il l) suppressed a rise in the hepatic cholestero l
ester level o f th e group IV mice (Table 4).
To contim1 the ro le of EPA and DH A in the emulsified su rimi's funct ions for
hypercholestero lemia and hyperlipidemia expressed in the present study, the absorption and
accumulat ion of EPA and DHA through the intestine into the mouse body were est imated by
measuring either EPA and DHA levels in the li ver or intake amoun ts of these fatty acids lr om
the di et. Table 5 shows the anal ysis data associated w ith the hep atic fatly acid contents in
mjce of the test groups. The hepatic contents o f n-3 fatty acids, such as EPA and DH A, were
Hypocholesterolemic and Hypo Iipidemic Effects of Emuls ified Sur imi Feeding.. .
16 1
higher in the mice fed the emulsifi ed surimi-(groups II and IV) than the control mice (groups
I and [J 1). Total intake amounts of EPA plus DHA fo r 9 days were calculated to be 18.6 0. 7
mgfmouse (group 1), I 09.1 12.2 mgfmouse (group 11), 13.4 0. 9 mglmouse (group lll ), and
I 06.2 9. 1 mglmouse (group IV) according to the data shown in Tables I and 2. The increase
in EPA plus DHA intake induced by emulsi fied surimi feedings were 90.5 mglmouse for the
basal diet groups ( I and H) and 92.8 mglmouse for the high cholesterol diet groups ( Ill and
IV). On the other hand, the contents of EPA plus DHA in the livers of mice after feeding on
the experimental di ets for 9 days were 6.48 0.35 mglmouse (group 1), 11.1 7 2.4 4
mglmouse (group 11 ), 5. 78 0.34 mglmouse (group 11 [), and 12.88 + 1.07 mglmouse (group
IV) according to the the data shown in T ables 2 and 5. The increase in hepat ic EPA plus DHA
levels induced by emulsi fied surimi feedings were 4.69 mglmouse for the basal diet groups (l
and !I) and 7. 10 mgfmouse for the high cholesterol diet groups (Ill and IV). From th is data,
the absorpt ion and hepat ic accumu lation rates of EPA plus DHA induced by the feeding of
emu lsified surimi were est imated to be 5.18 % for the basa l diet groups (I and II) and 7.66%
for the high cholesterol diet groups ( I! I and IV). These val ues might be reasonable because
the absorbed EPA plus DHA could be distributed to other organs and t issues, as well as the
li ver, and excreted from the body.
Table 5. Effects of Emulsi fied Surim i feedi ng on hepatic fatty acid levels in mice
Basal diet
IV
High cholesterol
High cholesterol
diet
di et
+emulsified
Ill
11
Basa l diet
+emulsified
surimi
Hepatic fatty acid (mg/g)

16:0
7.960.61 8.03+1.07 (ns)
16:l(n-9)
0.540.27 0.490.40 (ns)
18:0
3.740.50 3.87+0.44 (ns)
18:l(n-9)
5.50+1.21
5.70+0.95 (ns)
18:2(n-6)
4.464.03 7.46+1.00 (ns)
18:3(n-3)
0.090.05 0.140.02 (ns)
20:1(n-9)
0.140.08 0.140.03 (ns)
20:2(n-6)
0.080.04 0.08+0.02 (ns)
20:3(n-6)
0.460.06 0.42+0.07 (ns)
20:4(n-6)
3.480. 16
2. 120.6 1 ( )
20:5(n-3) (EPA)
0.1 2+0.06
0.63:~:0. 1 2 (")
22:4(n-6)
0.070.02 0.07+0.02 (ns)
22:5(n-3)
0.2 10.02
0.390.06 ( )
22:6(n-3 ) (DHA)
6.74+1.37 (')
3.9 10.07
Total
30.974.50 36.495.19 (ns)
n-3 , total
4.470. 14 8.07+1.55 (')
8.55+4.05 10.1 5+1.63 (ns)
n-6, total
n-9, total
6.1 81.25 6.3 2 1.3 1 (ns)
saturated, total
11.760.80 11.951.25 (ns)
..
..
surimi
..
5.65+0.32 (')
1.350.26 ( )
2.1 40.1 4 ( )
I 1.44 I. 18 ( )
6. 170.88 (ns)
0. 11 0.0 1 (ns)
0.220.07 (ns)
0.20+0.03 (')
0.590.03 ( )
I.950.23 (')
0. 11 +0.03 (ns)
0.050.0 1 (ns)
0. 140.04 ( )
2.7---:ro.22 (" )
32.931.97 (ns)
3.11 +0.26 (')
8.970.80 (ns)
13.001.4 1 (')
7.85+0.42 (')
....
..
6.260.43 (")
1.030. 18 (NS)
2.16o.22
9.981.28 (NS)
6.700.33 (NS)
0. 180.03 ("")
0.230.03 (NS)
0. 12+0.04 ()
o.37+o.o7
1.240.58
0.59+0. 11 ("")
0.050.02 (NS)
0.360.06 ("")
6.3oo.6o
36.251.4 1
7.460.74 ("")
8.490.46 (NS)
11.231.33 (NS)
9.070.48
n
n
n
n
n
mean standard deviation (n=5) .. P<O.O I, ' P<0.05 vs basal diet, ns: not significant vs basal diet. ''P<O.OI,
' 1'<0.05 vs high cholesterol diet, NS: not signilkant vs high c.holcstcrol diet.
162
As emulsi fied surimi contains large amounts of fish prote ins, the rol e of fish proteins, as
" 'e ll as !ish oil, might be considered in part in the effects of emulsified surimi feedings on the
serum total cholesterol and triglycer ide levels in mice.
This study is a un ique report on emulsi fied sur imi, an intem1ediate mater ial to manufacture
Kamaboko, a traditional Japanese seafood, which may possess hypocholesterolemic and
hypolipidemic effects. As a si milar li ne of story, it has also been reported that " Kazunoko,"
" h ich is a salted, yellow herring roe product and a traditional Japanese seafood, shows
reduction effects in serum total cholesterol and tr iglyceri de levels in mice [23].
Conclusion
The results of the present study suggest !hat the intake of emu lsi fied surimi-used food
products might be used to exploit disease preven ting properties, and would be especiall y
suitable for the diets of infants and elderly people, as well as the general popu lat ion.
Acknowledgme nts
We thank Ms. Noriko Fuj io, Mr. Takashi Nishioka and Mr. Futoshi Noguch i for their
techn ical assistance. Th is study was in part supported by the research grant (innovation for
regional development) (2008-2009) trom the Ministry of Economy, Trade and Industry of Japan.
References
[ I ] Woodman R. J., Mori T. A., Burke V., Puddy l. B., Watts G. E., Beilin L. J. (2002).
E ffects of purifi ed eicosapentaenoic an d docosahexaenoic acids on glycemic control,
blood pressure, and serum li pids in type 2 diabetic patients w ith treated hypertension.
A m .I C/in Nutr, 76, 1007- 10 15.
[2] Buckley R., Shewring B., Turner R., Yaqoob P., lvl inihane A . M. (2004). C irculating
triacylg lycerol and apoE levels in response to EPA and docosahexaenoi c acid
supplementat ion in adult human subj ects. Br .J Nutr, 92, 477-483.
[3] Harris W. S. ( 199 7). n-3 fatty acids and serum li poproteins: human studi es. Am J C /in
Nutr, 65, l 645S- l 654S.
[4] Cobiac L., Cli fl on P. Jv!., Abbey M., Belli ng G . B., N estel P. .1. (1 99 1). Lipid,
li poprotein and hemostat ic effect s o f fish vs fi sh-oil n-3 fatty acids in mildl y
hyperlipidemic males. Am .I Clin Nutr, 53, 12 10- 12 16.
[5] Roche H. M., Gi bney M. J. (2000) . Effects of long-chain n-3 pol yunsaturated fatty acids
on fast ing and post prandial triacylglycerol metabolism. Am .! C/in Nutr, 71, 232S-237S.
[6] Poowakanjana S., Mayer S. G ., Park J. W. (20 12). Opt imum chopp ing condit ions for
Alaska poll ock, Pacific whiting, and threadfin bream surimi paste and gel based o n
rheological and raman spectroscopic anal ysi s . .I Food Sci, 2012 Mar 6. doi: 1 0. 1111~ .
750-384 1.20 I I.
[7 ] Balange A. K., Benjakul S., lvlaqsood S. (2009). Gel strengthen ing effect of wood
extract on surimi produced from mackerel stored in ice . .I Food Sci, 74(8), C6 19-627.
Hypocholestero lemic and Hypo Iipidemic Effects of Emulsified Surimi Feeding...
163
[8] Jafurpour A., Gorczyca E. M. (2008). Alternat ive techniques for producing a quality
surimi and kamaboko from conmHln carp (Cyprinus carp io) . .I Food Sci, 73, E4 15-424.
[9] Chen H-H. (2000). Effect of non-muscle protein on the them1ogelat ion of horse
mackerel surimi and the resultant cooking tolerance of kamaboko. Fisheries Science, 66,
783-788.
[! 0] Kawashima T., Ohba A., Arai K. (1 973). Studi es on muscu lar proteins of 6sh-X II I.
Relat ionship between the amount of actomyosin in frozen surimi and the quality of
kamaboko from the same material in A laska-pollack. Nippon Suisan Gakkaishi, 39,
120! - 1209, ( in Japanese).
[ I I ] Okazaki E., Noda S., Fukushima H., Fukuda Y. (2006). Improvement of th e physical
propert ies of heat-induced surimi gel by !ish oil emulsi fication. ll/ippon Suisan
Gakkaishi, 72, I 093- 1098, (in Japanese) .
[1 2] Japanese Patent: Registration Number JP, 3 11 8556,B.
[ 13] Folch, J., Lees, lvl., Stanley, G. H. S. ( 1959). A simple method for the isolat ion and
puri fication of total li pids from an imal t issues . .I Bioi Chem, 226 , 497-509.
[ ! 4] Peyrou, G., Rakotondrazafy, V., lvlouloungui, Z., Gaset, A. (1 996). Separation and
quanti tat ion of mono-, di-, and triglycerides and free oleic acid using th in- layer
chromatography with flame-ionizat ion detect ion. Lipids, 3 1, 27-32.
[ 15]1ch ihara K., Fukubayash i Y. (20 10). Preparat ion of fatly acid methyl esters for gasliquid chromatography . .I Lipid Res, 5 1, 635-640.
[ 16] de Goede J., Geleijnse J. M., Boer J. M., Kromhout D., Verschuren W. M. (2010).
Marine (n-3) fatly acids, fish consumpt ion, and the 10-year risk of fatal an d nonfatal
coronary heart di sease in a large population of Dutch adults w ith low fish intake. J Nutr,
140 , 1023- !028.
[ 17] Billman G. E., N ishiji ma Y., Belevych A. E., Terentyev D., Xu Y., Haizlip K. M.,
lv!onasky M. lvl., Hiranandani N., Harris W. S ., Gyorke S., Cames C. A., Janssen P. M.
(2010). Effects of di etary omega- 3 fatly acids on ventr icular funct ion in dogs w ith
healed myocardial infarct ions: in vivo and in vitro studi es. Am .I Physiol Hea rt Circ
Physiol, 298, H 12 19- H 1228.
[ 18] von Schacky C., Harris W. S. (2007). Cardiovascu lar benefits of omega-3 fatty acids.
Cardiovas cular Research, 73, 3 10- 3 15.
[ 19] S imopou los A. P. (2002), Omega-3 fatly acids in inflanm1ation and autoimmune
diseases . .I Am Col Nutr, 2 1, 495- 505.
[20] Harris W. S. (2007), Omega-3 fatly acids and cardiovascu lar di sease: A case for omega3 index as a new risk factor. Pharmacal Res, 55, 21 7- 223.
[2 1] Simopou Ios A. P. (1 999). Essent ial !ally acids in health and chronic di sease. Am .I C/in
Nutr, 70 (suppl), 560S- 569S.
[22 ] Barre D. E. (2007) The role of consumpt ion alpha-linolenic, eicosapen taenoic and
docosahexaeno ic acids in human metabo lic syndrome and type-2 diabetes - A mini
review, .I Oleo Sci, 56, 3 I 9-3 25.
[23] Higuchi T., Sbirai N., Suzuk i H. (2006). Effects of dietary herring roe lipids on plasma
li pid, glucose, insu lin, and adiponectin concentrations in mice. .I Agric Food Chem, 54,
3750-3755.

Edi tors: T.G. Bradley and F.P. Vargas, pp. I 65- 175
ISBN: 978- 1-62257-480-3

20!2 Nova Science Pub lishers, Inc.
Chapter 9
IDENTIFICATION AND CHARACTERIZATION

OF N-6 AND N-3 FATTY ACID DESATURASE
AND ELONGASE FROM ACANTHOPAGRUS SCHLEGELJJ
1
Sun Hee Kim I , .long Sug Park I , Yo Soon .fang,
So Yun Kint', .Jung-Bong Kim 1, Ky ung Hee Roh 1,
Hy un Uk Kim 1, Ky eong -Ry eol Lee' and .long Bum Kim 1'
1
National Academy of Agricultura l Science,

Rural Development Admin istration, Seodun-Dong, Suwon, Korea
2
East Sea Environment research Department, KORDI,
Hujung-ri, Jukbyeon-myun, U\i in-gun, Gyeongbuk, Korea
Abstract
Polyunsamrated fatry acids (PUf.A.s), especially eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) have significantly beneficial effects on human health. T he
primary dietary sources of these fatty acids are marine fish. The current high use of fish and
fish oil leads ro research rowards the development of a viable alternative sustainable source of
PUFAs. We focused on genes encoding the primary PUFAs biosynrhetic activities and
characterized them. Farry acid delta 6- desarurase (D6DES) and elongase (ELOV L5) are key
enzymes in the synthesis of PUFAs. Two plasmids pYES2 and pYES3 were used ro
cotransform into NVSc I for the coexpression of D6DES and ELOV L5 in yeast, respectively.
The corransformed yeast cell, designated as pY2-D6D and pY3-FAE produced y-linolenic
acid (G LA) and di-homo-y-li nolenic acid (DG LA) from exogenous linoleic acid (LA) and
stearidonic acid (STA) and eicosatetraenoic acid (ETA) from a -linolenic acid (A LA). In these
results, the recombinant A. schlegelii D6DES and ELOVL5 show activiry on borh n-6 and n-3
fatty acid substrates. Thus, these genes may play a critical role in the bio-production of borh n6 and n-3 PUPAs and could be good sources for PUFAs synrhesis in engineered oilseed crops.
Keywords: Polyunsaturated fatty acids, Delta 6-desaturase, Fatty ac id elongase A. schlegelii.
E-mail address: j ong957 1@ korea.kr; T el: +82 31 299 1702, Fax: +82 31 299 1672. (C01Tesponding author)
166
Sun Hee Kim, Jong Sug Park, Yo Soon Jang et al.
Introduction
Fish li pids are the best source of long chain polyunsaturated fatty acids (PUFAs),
including arachidonic acid (A RA), eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA). These PUFAs thus have beneficial heal th effects in humans that are particu larly
related to neurodevelopment { 1], the treatment of inflammatory and auto immune diseases {2]
and reduced risk of sudden cardiac death [3]. In view of the major roles of PUFAs in human
physiology, th e enzymes involved in thei r biosynthesis have been recentl y aliracted by
grow ing interest. PUFA biosynthesis is catalyzed by sequential desaturat ion and elongat ion
reactions, wh ich intro duce double bonds ai speci tic positions and add two-carbon subun its
onto their fauy acyl imem1ediates, respecti vely (Figure 1).
Pla nts
18:0 stearic acid
!.c.
desaturase
18:1 oleic acid
A 12 desaturase
18:2 linoleic acid (LA)
w3 desaturase
----------,1
(w6= n6)
~.c. 6 desaturase
18:3 y -linolenic acid (GLA)

' FA elongase
18:3 a -linolenic acid (ALA)
(w3=n3)
' A 6 desaturase
20:3 di-homo y-linoleic acid

! As desaturase
18:4 stearidonic acid (STA)
20:4 arachidonic acid (AA)
20:4 eicosatetraenoic acid
1FA elongase
22:4 docosatetraenoic acid
~ FA elongase
1A desaturase
20:5 eicosapentaenoic acid (EPA)
1 elongase
22:5 docosapentaenoic acid
1 elongase
5
FA
FA
24:5 tetracosapentaenoic acid
f.c.desaturase
24:6 tetracosahexaenoic acid
1Peroxisomal ~-oxidation
Mammals
22:6 docosahexaenoic acid (DHA)
Figure I. Proposed pathways of polywtsamrated fatty acid (PUFA) biosynthesis from a-linolenic acid
(A LA, C l8:3) and linol eic acid (LA, C l8:2). Solid thick lines represent the delta 6-desarurase and
elongases involved in this work. :VIodified from [ 14].
Identi ficat ion and Characterization ofn-6 and n-3 Fatty Acid Desaturase.. .
167
The enzyme, delta 6- desaturase (D6DES) modi fies fauy acid structure by introducing a
double bond at the delta 6 positi ons. The D6DES react ion is thought to be a rate li miting step
in de novo long chain PUFAs syn thesis [4, 5].
In addition, elongases of very long-chain (ELOVL) fatty acids are essent ial enzymes for
increasing the chain length of fatty acids d uring the biosynthesis o f PUFAs. In mammals,
seven members of the ELOVL famil y tem1ed ELOVLI-7 have been ident ified {6-8] .
ELOV L2 and ELOVL5 add two carbons to their respective C 1s, Cw or C 22 PUFA substrates.
The ELOVL5 preferent ially elongates C 1!VC2o PUFA substrates whereas the ELOVL2 has
speci fi city for ~JC22 PUF A substrates {9-11 ] .
Acanthopagrus schlegelii is a marine fish s pecies (()r (()Od that contains h igher level o f
DHA than EPA {12] and has great aquaculture potential in As ia. A lthough some major
advances in the cloning and man ip ulat ion o f n-6 and n-3 fatty acid desaturase and elongase
genes from various o rganisms have heen made over the last few years, the isolation o f more
effect ive genes that will generate th e desired fatty acid in oi lseed crops is st ill needed {13]. In
th is study, we reconst ituted the pathways for ETA and DG LA biosynthesis considered
alternat ive sources o f PUFA in Saccharomyces cerevisiae by eo-expression o f the genes
encodi ng delta 6-desaturase, AsD6DES [ 14] and fatty ac id elong ase (F AE; AsELOVL5) [ 15]
from A. schlegelii. We discuss the poten tia l for eo-expressing !:.6 desaturase and AsELOV L5
to produce very long chain po lyunsaturated fauy acids (V LCPUF As) in oi lseed crops.
Materials and Methods

Fish Materials
Muscle or live r tissues from black seabream (Acanthopagms schlegelii), wh ich contain
high levels o f PU FAs, were obtained from the Korea Ocean Research and Development
Institute. These t issues were frozen in liqu id n itrogen and stored at -8oc until RNA extraction.
E Coli and Yeast Strains

E. coli DH5a (New England Biolabs) was used to construct the recombinant plasmids.
Saccharomyces cerevisiae INVSc l (lnvitro gen) was used as a host strain to express
heterologous genes.
Vector Construction
The codi ng regions o f delta 6-desaturase and fatty acid elongase genes were ampli fi ed by
RT -PCR using a template o f total RNA extracted from A. schlegelii. The primers used for the
amp!i ficat ion were introduced at Kim et al. [ 14, 15].
The full-l ength AsD6DES cDNA was re-ampli fied with the primer set A (Table I) to
enable Hindii l and Xho l digest ion. For full-length FAE (AsELOVL5) cDNA, primer set B
(Tab le I) was re-amp li fied and digested with BamH l and eaRL These cDNA fragments
were then gel-puri fied w ith the Q iagen purification Kit (Chatsworth, CA), and ligated into the
correspond ing sites o f pYES2 and pYES3 to generate pY2-D6D and pY3-FAE, respect ively.
!68

Ta ble I. P rimers used fo r construct io n of t he pY2-D6 D and pY3-f A E
Kinds
[>rimer
Set
Primer Name
AsD6D Hindl ll-F

pY2-D6D
AsD6D X11ol-R
AsELOJIL5 Bam i-U-F
pY3-FAE
AsELOJIL5 Eco RI-R
Ta
CCG AAG CTT ATG GGA GOT
GGAGGCCA
CO CrC GAG TCA TTT ATG GAG
ATA AGC ATC CAG C
CO GGA TCC ATG GAG ACC TIC
AATCACAA
CO GAA T TC TCA ATC CAC TCT
CAG TIT CIT GTG T
68'C
65' C
Restriction enzyme sites are underlined.

ll1c data include sequences and annealing temperatures (Ta) lo r primer pairs.
Yeast Transformation and Functional Characterization of AsD6DES and

As E LOV L5
The pY2-D6D-pY3-FAE and pYES2/pYES3 vectors were introduced into
Saccharomyces cerevisiae INVSc l using the lith ium acetate method and the yeast cells were
then plated on minimal medi um plates lacking uracil and tryptophan. The selected
transforn1ants were further gro,vn in minimal medi um lacking uracil and tryptophan
supp lemented with 2% glucose and incubated at 28C overnight. The cultured cells were
diluted to an OD60o of 0.4 in mini mal medium containing 2% galactose, and 0. 1% tergitoiNon idet P-40 (Sigma, Si Louis, MO) w ith or without 0.5 nu\il of the substrate fatty acids LA
or A LA, respect ively. These cells were then incubated at 2oc for three days and then at l 5C
for a further three days as described by Qiu et al. [ 16] .
The cells were harvested by centri fugat ion at 4,000 rpm for 5 min and the eel I pell ets
were washed tw ice w ith water. The pellets were freeze-dried and stored at -80C until fatty
acid analysis was perforn1ed.
Fatty Acid Analysis

Yeast ce ll s were lyophilized in a 50 ml tube. fi ve milliliters ofMeOH:CHCJ, (2: 1, v/ v)
and l mg PDA (pentadecanoic acid in MeOH) as an internal standard were added to th e
lyophilized samples. Fally acid methyl esters (FA1v!Es) were prepared using li pid extract ion
method and analyzed by gas chromatography according to our previous studi es ( 14]. FA1\fEs
were identi fied by reference to a well-characterized commercial standard peak (Sigma, St.
Louis, MO) based on the GC retent ion t ime and quant ified us ing computer software. A ll
analyses was per forn1ed in tripli cate and rep licated three t imes. Methanolic base (SigmaAidricb Canada Ltd., Oni. Canada) and a FAME standard (S igma St. Louis, MO) were used
as re lerence standards.
Identificat ion and Characterization ofn-6 and n-3 Fatty Acid Desaturase.. .
169
Results
Sequence Analyses of As D6DES and AsELOVL5
The ORF ofAsD6DES was shown to be 1338 bp, wh ich specified a protein of 445 amino
acids (GenBank under the accession number Bankii 1227570). The protein sequence included
all the characterist ic features of microsomal fatty acid desaturases, includ ing two
transmembrane regions, three hist idine boxes and N-tem1inal cytochrome b5 domain [ 14].
Table 2. Identity matrix showing the results of a pnir-wise comparision between the
identities of the amino acid sequenc.e of the fish and human fatty acid desaturases
S:1D6D
98
SsD6D
78
OmD6D
75
76
94
DrFAD2
68
67
65
66
As06D
Sa06D
77
Ss06D
Om060
DrFAD2
Ss05D
HsFA D2
Data arc percentage o f amino acid residucs that are identical.
SsDSD
77
77
91
92
64
HsFAD2
65
65
65
65
65
63
HsDSD
60
60
61
61
58
60
62
Table 3. Identity matrix showing the results of a pair-wise c.o mparision between the
identities of the amino acid sequence of the fish and human fatty acid elongases
SaE.L
()
TmE.L
OVLS
93
92
SsE1o
>'15b
SsE1o
>'15a
OrnE
LO
t\SELOV L5
98
83
83
83
SaELO
82
82
81
84
TmELOVL5
83
83
SsEiovl5b
92
91
SsEiovl5a
98
OmELO
DrELOV L5
HsELOV L5
SsELOVL2
Data are percentages o f amino acid rcsiducs that are identical.
DrEL
OVLS
74
74
76
75
76
76
HsEL
OVLS
71
71
71
70
72
72
71
Ss EL
OVL2
57
57
57
56
56
56
HsEL
OVL2
57
57
58
54
54
54
58
58
56
56
71
A pair-w ise comparison among fish desaturase sequences showed the ammo acid
sequen ce predicted by the A. s chlegelii desaturase ORF showed greatest identity to that of
gilt-head bream delta 6-desaturase (Spams aurata, SaD6D) (98%), 78% identity to that of the
At lantic salmon (Salmo solar, SsD6D), 75% ident ity to that of the rainbow trout
(Oncorhyn chus mykiss, OmD6D), 68% to that of the zebra fish (Donio rerio, DrFAD2), 65%
to that of the human (Homo sapiens, HsFAD2), and 77% ident ity to the Salmo salar delta 5desaturase (SsD5D), respectively (Table 2). These results strongly suggest that the AsD6DES
gene encodes a delta 6-desaturase invo lved in the synthesis of y-linolenic acid (GLA) and
stearidonic acid (STA) in A. schlegelii.
The ORF ofAsELOVL5 was shown to be 885 bp, wh ich specifi ed a protein of294 amino
acids (GenBank under the accession number gbl218654). The protein sequence included
170
characterist ic features of microsomal fatty acid elongases, including a single hist idine box
redox centre motif(H XXH H) and mu ltiple transmembrane regions [ 15]. AsE LOV L5 shows a
98% sequence identity, the highest found, w ith a putat ive fatty acyl elongase from Sparus
aurata (SaELO), a 93% ident ity to the ELOVL5 (gbiACZ559301) of Thummus maccoyii, and
a 71 -83% homology w ith TmELOV L5-Iike proteins from mammals (Homo sapiens and
Danio rerio) and fishes (Oncorhynchus mykiss and Salmo salar).
However, th is protein shows a relatively low (57%) identity with mammal ian and fish
ELOVL2-I ike proteins (Homo sapiens and Salmo sa/m-) (Table 3). These results suggest that
AsELOV L5 is an elon gase that targets C 18 and C 20 desaturated fauy acids.
Functional Ana lysis for the Coexpression of AsD6DES and AsE LOV L5
To con fl m1 these funct ions by eo-expression of the genes encod ing delta 6-desaturase,
AsD6DES [ 14] and fatty ac id elongase (FAE; AsE LOV L5) [ 15] from A. schlegelii, the ORF
of AsD6DES cDNA was subcloned into the pYES2 vector at the Hin d JJI and Xho l sites and
the AsELOVL5 cDNA was introd uced into the pY ES3 vector w ith BamHJ an d EcoR I under
the control of the inducible GALl promoter, respectively.
Standard
LA
_ lA...
16:0
1 ~:0
I
ALA
DGLA
Gr
ARA
EA
Q)
pY21pY3
Ill
LA
(LA)
Q.
Ill
...
Q)
lk
IU.
JU\.
A
'
pY2D6D/pY3-FAE
(LA)
LA
.lt.
~
10
GLA
.~
20
DGLA
A
30
*11
40
50
mn
Retention ti me ( m in)
Figure 2. Fatty acid profiles of GC analysis from Eransgenic Saccharomyces cerevisiae I~VScl. This
shows profiles of Ehe faEty acids in Erans formed yeasE expressing pY2-D6D-pY3-FA E and empty vecwr
pYES2JpYES3 as a comrol in Ehe presence of LA subsEraEe. The new peaks appeared in Eransformams
are marked wiEh asEerisks in Ehe profile of farry acids compared wiEh sEandard faEEY acids.
ldenti ficat ion and Characterization of n-6 and n-3 Fatty Acid Desaturase.. .
171
Using the lith ium acetate method, the result ing constructs, pY2-D6D and pY3-FAE, were
then cotrans fom1ed into S. cerevisiae rNVScl. The coexpression of AsD6DES and
A~E LOVL5 in a single strain was then induced by the addit ion of galactose as the carbon
source 10 the growth medium, wh ich was also supplemented with LA or A LA. Subsequent
fully acid anal ysis indicated that the exogenous LA or A LA had been incorporated into the
ti pids of the transfom1ed yeast harboring pY2-D6D-pY3-FAE. Gas chromatography analysis
further revealed novel peaks in the yeast transfom1ed w ith pY2-D6D-pY3-FAE wh ich
corresponded to convert LA via y-linolen ic acid (GLA, C 1s:; n-6) to di-homo-y-l ino len ic acid
(DG LA, C2<u n-6) (Fi gure 2) and to convert ALA via stearidonic acid (STA, C 18, 4 n-3) to
eicosatetraenoic acid (ETA, Czo: n-3 ) (F igure 3).
These peaks were absent from yeast harb ori ng the empty vector (pYES2-pYES3) as
control. These resulls further indicate that the della 6-fatty acid desaturase and fatty acyl
elongase can successfu ll y convert incorporated LA via GLA to DGLA and ALA vi a STA to
ETA, respect ively.
As sho"n in Figure 2, the fatty acid prolile of the pY2-D6D-pY3-FAE-expressing yeast
cells grown in medi um supp lemented with lino leic acid (LA, C 1s,2 ("
9 12
)),
had a dist inguishable
extra fatty acid peak compared with the pYES2/pYES3-expressing control cells.
Standard
18:0
E.PA
- J.l
JlJJ.
STA
~ JA_J. . _ _ .L__ ~-- "L _nA-'

U
AUJ
Ill
ALA
Ill
pYZ/pY3
(ALA)
r:
0
Q.
Ill
...Ill
Q
....
11.
dh
_J(
1t
pY2-060/pY3-FAE
AU
(AU)
STA
E.TA
it.,
io
~lj
2.0
30
40
so
...
Retention time (min)
Figure 3. Fatty acid profiles from GC analysis of transgenic Saccharomyces cerevisiae INVScl. This
shows profiles of the fatty acids in transformed yeast expressing pY2-D6D-pY3-FAE and empty vecror
pYES2/pYES3 as a comrol in the presence of ALA substrate. The new peaks are marked by asterisks in
the fany acid profiles and compared with standards. Other minor peaks are the result of the elongation
of endogenous fatty acids.
172
Sun Hee Ki m, Jong Sug Park, Yo Soon Jang et al.

This pea k showed a retent ion time identical to that o f they-linolenic acid (GLA, C 1s:>
6 9 12
(" ' ' ))
and di-homo-y-lino len ic acid (DGLA, C 20,, (t. 8' 11 '
14
))
standard in the upper panel
(Fi gure 2).

Th is new ly synthesized GLA and DGLA in the trans f()ml ed yeast cell s also re presented
0.5% and 0.7% o f the total fatly acid content, respect ively (Ta ble 4), indicat ing that th e
introduced de lta 6-desaturase eflicient ly converts LA to GLA and G LA to DG LA ( 1.4% and
58.3%, respectively; Table 4).
In addi tion, the pY2-D6D-pY3-FA E showed stronger catalytic act ivity towards tl-
~ no l en i c acid (A LA, C 1s:> (<> 9' 12' 15 )) and can convert A LA via stearidonic acid (STA, C 1s:4
( "' 6'9 ' p- I -'))
to e .tcosatetraeno .tc act. d (ET A,
c2o:4 (t. 8' 1114' 17)) (F.tgure 3) . ST A
and ET A was
produced to levels of about 3.2% a nd 4.3% of the total fatty acids in the transfo m1ants,
respecti vel y and the substrate conversion from A LA to STA reached as 6.1% and ST A to
ETA reac.h ed as 57% (Table 4).
These results confi m1 that AsD6DES is a delta 6-faity acid desaturase that can introduce
a double bond at posit ion 6 o f C 18,2 ( <>9 12) and C 18 ,;
the delta 6-desaturated fatty ac ids G LA C 18,3 ( "
6 9 11
)
9 12 15
(" )
substrates, and thereby produces
and STA C 18,4 ( o.6 9 1215 ), respect ively.
Table 4. Fatty acid compositions (% w/w) of the total lipid contents

from yeast transform ants harboring the cont rol plasmid pYES2/pYES3
or t he recombinant plasmid pY2-D6D-p Y3-FAE
Fatty acid
16:0
16: I
18:0
18:1 (n-9)
18:1 (n-7)
18 :2 (LA, n-6)
18 :3 (G LA, n-6)
18 :3 (ALA, n-3 )
18:4 (STA, n-3 )
20 :3 (OG LA, n-6)
20:4 (ETA, n-3)
.6.6 desaturation (%)*
Elon gation ofCI8 (%)*
p Y ES2/p Y ES3
+LA
+ALA
17.0+0. 1
12.20.2
21.8+0.3
14. 10.1
6.6: 0.1
9. 10.2
14.2+0. 1
I 0.90.2
0.90.2
37.0+0.2
NO
NO
NO
NO
NO
0.60. 1
NO
NO
55.5+0.2
NO
NO
NO
pY2-D6D-pY3-FAE
+LA
15.80.2
19.80. 1
8.60. 1
15.40. 1
5.0+0.2
34.20.1
+ALA
I 0.6+0.2
15.8+0. 1
5.5+0.2
12.4+0. 1
0.50. 1
NO
NO
0.70.2
NO
3.70. 1
NO
NO
48.70.2
3.20. 1
NO
4.30. 1
1.4
58.3
6. 1
57.0
Eac.h value is the mean SD from three independent experiments.

:-.10 , nOI dCICCied.
7LA and ~ALA, exogcn ously supplied J'au y acids.
l hc percentage o f FA elongation was c.alculatcd using u1c following fonnula: (product area/ (product area
substratc area) x I00].
173
The AsE LOV L5 showed that the act ivity of fally acyl elongase that can add two carbons
6 9 12
6 9 11 15
to their respect ive C1s substrates, G LA C18:> ( <> ' ' ) and STA C 18,. ( <> ' ' ' ), thereby
14
produces the elongated fally acids DGLA, C2<u (t.8 n ) and ET A, C 20, 4 (t.811 1417),
respectively. Our data further indi cate that AsD6DES and AsE LOV L5 can reconst itute both
the n-6 and n-3 pathways in a hetero logous system .
Disc ussion
With the increase in nutritional health awareness among humans, tish oil is in high
demand as a maj or source of PUF As. However, th is has now become an issue in tem1s of the
depletion and heavy metal contaminat ion of natural fish resources. Hence, it is important to
elucidate and exp loit the de novo PUFA biosynthesis pathways and therefore man ipulate the
key enzymes involved in opt imizing the act ivity of these pathways in fish. Ident ification of
the genes encoding the key enzymes involved in PUFA synthesis w ill enable reverse
metabolic engineering in p lants to prod uce V LCPUFAs. The biosynthesis of VLCPU FAs
involves alternating desaturat ion and elongat ion react ions. Th is biosynthesis scheme is not
speci fi e or restri cted to llsh but is a feature of the "standard" (i.e. non po lyketide synthesis)
route to PUFA.
Polyunsaturated laity acids (PUFAs) are important in humans as they are precursors of the
prostaglandins and are therefore essent ial for patients who su!rer from diabetes, cancer, aging,
and infection {18]. In our previous study, the synthesis ofSTA and DGLA from ALA and GLA,
respectively, has already been successfully demonstrated w ith speci fic single-step delta 6desaturase (AsD6DES) and fatty acyl elongase (AsELOVL5) from A. schlegelii in transgenic
yeast [1 4, 15]. Our isolation and functional characterizat ion of an essent ial delta 6-iatty acid
desaturase and fatty acyl elongase from A. schlegelii was sign ificant as it has the potential to be
used in the future metabolic engineering of polyunsaturated fatty acids in plant.
In our present study, we coexpressed AsD6DES and AsELOV L5 in a single strain that
was grown in the presence of LA or A LA. The recombinant S. cerevisiae carrying the pY2D6D-pY-FAE produced 0.5% G LA and 0.7% DG LA and 3.2% STA and 4.3% ETA of total
fatty acid s by eo-expression of delta 6-desaturase (pY2-D6D) and fatty acyl elongase (pY3FAE) genes from A. schlegelii (Table 4). LA was first desaturated to G LA by AsD6DES; the
desaturated product was subsequently e longated to DGLA (Figure 2). ALA was desaturated
lO STA by AsD6DES; the desaturated product was subsequent ly elongated to ETA (Fi gure 3).
However, recombinant S. cerevisiae single gene-expressing the delta 6 desaturase { 14] and
fatty acy l elongase [ 15] gene from A. schlegelii produced 11.8% G LA, 21.6% STA and 8.8%
!XiLA of total fatty acids, respect ively. The proportion of G LA, STA and DG LA
accumulated in the recombinant S. cerevisiae carrying the pYES2-AsD6DES or pYES2A~E LOV L5 was higher than that the recombinant S. cerevisiae carrying the pY2-D6D-pY3FAE. Th is indicates that the genes encoding desaturase and elongase may be su itable for
producing desirable PUFAs by genetic manipulat ion. On the other hand, th e strain expressing
the pY2-D6D-pY3-FAE showed an increase in C 1s,1 (n-7) (Table 4) as well as proport ions of
C2o 1(n-9) and C 20 1 (n-7) (Figure 2 and 3) [15]. They all were most likely fom1ed by
A~ELOV L5-catal yzed elongat ion of the yeast monounsaturated fatty acids ( 15] .
174
In summary, we present here the reconstitution of ET A and DGLA biosynthesis in S.

cerevisiae by eo-expression o f the genes encoding delta 6 -desaturase and fatty acid elongase
from A. schlegelii. In addition, the successfu l reconstitut ion of PUFAs synthesis in yeast
represents an encouraging step in the engineering strategies to produce V LCPUFA such as
EPA and DHA with a comb ination of other PlJF A-related genes in plants. Since DHA can be
biosynthesized direct ly by M desaturase from DPA, the production of DHA might be
possible by eo-expression of AsE LOVL5, ~4 and ~5 desaturase in certain crops.
Acknowledgme nts
Th is work was supported by grants from the National Academy of Agricultural Sci ence
(PJ006745), the Rural Development Admin istration, Republi c of Korea. S.H. Kim was
supported by a 2012 Post-doctoral Fellowsh ip Program of Nat ional Academy of Agricu ltural
Science, the Rural Development Admin istrat ion, Republi c of Korea.
References
( I]
{2]
[3]
{4]
{5]
Smi thers, L. G., Gibson, R. A. , Mc Phee, A ., and Makrides, M. (2008). Effect o f longchain polyunsaturated faily acid supplementation of pretem1 infants on disease rick and
neurodevelopment : a systemat ic review of randomized contro lled trials. American
Journal of Clinical Nutrition, 87, 9 12- 920.
Proudman, S. M., C leland, L. G., an d James, M. J. (2008). D ietary omega-3 fills for
treatment of inflammatory joint disease: efficacy and ut ility. Rheumatic Disease Clinics
of North America, 34, 469-479.
Metcalf, R. G., Sanders, P., James, lvl. J., et al. (2008). Effect of dietary n-3
po lyunsaturated fatty ac ids on the inducibility of ventricular tachycardia in patients w ith
ischemic cardiomyopath y. American Journal of Cardiology, I 0 l (Sup pi 6 ), 758- 76 1.
Brenner, R. R. (1 974). The oxidat ive desaturation o f unsaturated fatty acids in animals.
Molecular and Cellular Biochemistry, 3(Suppl I), 4 1-52.
Horrob in, D. F. (1 993). Fatty acid metabol ism in health and disease: the role of " -6-
desatu rase. American Journal ofClinical Nutrition, 5 7, 732S-737S .

[6] Leonard, A . E., Pereira, S. L., Sprecher, H., et al. (2004). Elongat ion of long-chai n fatty
acids. Progress in Lipid Research, 43, 36-54.
{7] Jakobsson, A., Westerberg, R., and Jacobsson, A . (2006). Fatty acid elongases in
mammals: their regulation and roles in metabolism. Progress in Lipid Research, 45,
237 -249 .
[8] Tamura, K., Mak ino, A ., Hullin-Matsuda, F., et al. (2009). Novel lipogenic enzyme
ELOVL7 is invo lved in prostate cancer growth th rough saturated long-chain fatty acid
metabolism. Cancer Research, 69, 8 133-8 140.
(9] Leonard, A. E., Bobik, E. G., Dorado, J., et al. (2000). Clon ing of a human cDNA
encodi ng a novel enzyme invo lved in the elongat ion of long-chain polyunsaturated fatty
acids. Biochemical Journal, 350, 765- 770.
{10] Leona rd, A. E., Kelder, B., Bobik, E. G., et al. (2002). ldentiticat io and expression of
mammalian long-chain PUFA elongat ion enzymes. Lipids, 37(Suppl 8), 733- 740.
175
{I! ] lnagaki, K., Ak i, T., Fukuda, Y., et al. (2002). Ident ification and express ion o f a rat
fatty acid elongase involved in the biosynth esis of C l 8 fatty ac ids. Bioscience
Biotechnology and Biochemistry, 66(Supp l 3 ), 613- 62 1.
( 12] Jeong, B. Y., Choi, B. D., Moon, S. K., et al. (1 998). Fatly acid composit ion of 72
species of Korean fish. Journal of Fisheries Science and Technology, I, 129- 146.
{13] Robert, S. S. (2006). Production of eicosapentaenoic and docosahexaenoic acidcontain ing oils in transgen ic land p lants for human and aquacu lture nutrit ion. Marine
Biotechnology, 8, I 03- 109.
{14] Kim, S. H., Kim, J. B., Kim, S Y., et al. (2011). Functional character izat ion of a delta 6desaturase gene from the black seabream (A canthopagrus schlegeli). Biotechnololgy
Letters, 33, 11 85- 11 93.
{15] Kim, S. H., Kim, J. B., Jang, Y S., et al. (2012). Isolat ion and funct ional
character izat ion of polyunsaturated fatty acid elongase (AsELOV L5) gene from hlack
seabream Acanthopagrus schlegeli. &otechnololgy Letters, 34, 26 1-268.
( 16] Qiu, X., Hong, H., and MacKenzie, S. L (2001). Identification of a M fatly acid
desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexaenoic
acid by heterologous expression in Saccharomyces cerevisiae and Brassica j uncea.
Journal of Biological Chemistry, 276, 3 156 1-3 1566.
{17] Agaba, M. K., Tocher, D. R., Zheng, X., et al. (2005) . C lon ing and funct ional
characterisat ion of polyunsaturated fatty acid elongases of marine and freshwater
teleosts fish. Comparative Biochemistry and Physiology B, 142, 342- 352.
( 18] Lu, H., Li, J-N., Chai, Y-R., et al. (2009). Identificat ion and characterization of a novel
66-fatty acid desaturase gene from Rhizopus nigricans. Molecular Biology Reports, 36,
229 1-2297.

Editors: T.G. Bradley and F.P. Vargas, pp. 177-203
ISBN: 978- 1-62257-480-3

2012 Nova Science Publishers, Inc.
Chapter /0
FRACTIONATION AN D CONCENTRATION
OF OMEGA- 3 BY MOLECULAR DrSTILLATION
Pablo Rossi', Nelson Ruben Gros.w, Marfa del Carmen Pramparo
and Valeria Nepote
Universidad Nacional de Rio Cuarto
Univers idad Nacional de C6rdoba (LMBJV- CONICET)
Abstract
Certain oils are a rich source of compounds called omega-3 , which are polyunsaturated fatty acids that play an essential role in the human diet because of their ability to
prevent disease. These acids participate in the production ofhom10nes involved in several physiological systerns regu lating pain and moisture, while ma intain ing adequate
blood pressure and optima l levels of cholesterol and promoting nerve transmission.
Especia ll y two fatty acids, both omega-3 type, the 5,8, 11,1 4, 17-eicosapentaenoic acid
(EPA, 20:5w3) and the 4, 7, I 0,1 3, 16, 19-docosahexaenoic acid (DHA, 22 :6w3) are important functional constituents of the hwnan body.
The comp lexity in the p reparation p rocess of concentrated omega-3 compounds
is mainly based on the sim ilar physicochemical properties of the fatty acids. In this
sense, it is d iffi cult to separate them from the other fatty acids in the oil.
Simple fractionation processes do not discriminate between different po lyunsaturated fatty acids present in an oily mixture, so it is necessary to evaluate the different
available alternatives. Nowadays, at conm1ercial production scale, molecu lar distillation is the most widely used technique.
Molecular distillation is a special type of very high vacuum disti llation, which
takes place in an apparatus constructed so that the distance that the mo lecu les must
travel between evaporation and condensation is smaller than their mean free path. In
this way, it is possible to avo id an excessive exposure of the product to deteriorating
conditions. This feature makes molecu lar distillation a good a lternative for application
in the fractioning process of omega-3 fatty acids.
The main objective of this chapter is to present a rev iew where it is discussed, in
a theoretical and experimental way, the process of fractionation and concentration of
ethy l esters of omega-3, using the falling film molecu lar distillation .
'E-mail address: prossi@iog.unrc. edu.ar
178
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
it is important to highlight that for obtain ing a concentrate of omega-3, it is fi rst
necessary to perfom1 a rransesterification stage for ach ieving the separation of the carbon chain of the glycerol mo lecu le. In this reaction, the most favorable condition is
achieved by working at high temperatures close to the boil ing point of the ethanol.
:vtoreover, the conversion of the reaction increases with increasing the catalyst concentration within the studied ranges.
As esters of omega-3 have an intem1ed iate vo latil ity value between those of esters
oflight fatty acids and the esters ones, it is required to perfurm two stages of molecu lar
distillation. Work ing at low temperatures, both stages resu lt in a low separation effi ciency. On the other hand, working at very high temperatures the separation efficiency
is not favorable, but an optimal point in the process can be found.
Several works have shown mathematical models which op timize molecular distillation process, aiming to find conditions which a llow a good recovery of ethy l esters
ofomega-3 fatty acids with a h igh degree of purity. Models also allow analyz ing the
infl uence of different operating variables.
Keywords : Omega-3, EPA, Molecular Distillation, Optimization Processes.
I.
Introduction
Omega-3 com pounds are polyunsaturated fatty acids wh ich play an essemial role in the
human diet because of th eir ability to prevent disease. These acids participate in the production of hormones involved in several physiological systems regulating pain and moisture,
while maintaining adequate blood pressure and optimal levels of cholesterol and promoting
nerve transm ission. Recent research on the effects of these omega-3 acids in humans show
concrete proof of their ami-inflammatory power and their important effect in the treatment
of cardiovascular diseases, cancer, Alzheimer's and artt1ri tis [ 1).
Especially two fatty acids, both omega-3 type, the 5,8, 11 , 14, 17-eicosapentaenoic acid
(EPA, 20:5w3) and the 4,7, I0, 13, 16, 19-docosahexaenoic acid (DHA, 22:6w3) are important
fi.1nctional constituents of the human body. The marine oil is one of the richest sources of
omega-3. Table I shows the typical fatty acids found in some marine oils.{2) Crude oil is
tt~uall y refined, especiall y if it is for edible purposes, in order to improve its chemical and
organoleptic characteristics, eliminating some compounds that give to the oil undesirable
smell, color and/or taste {3).
Omega-3 acids are characterized by a high level of insaturation. Fatty acids in the form
of trans isomer, inhibit prostaglandin formation and increase the level of LDL cholesterol
in the blood, so these isomers have undesi rable effects in heal th. Instead, fatty acids in the
form of cis isomer do not have these undesirable properties. Omega-3 molecules found in
nature have double bonds cis, so their consumption is not harm ful (4, 5).
Jn general, Omega-3 acids are sold as soft ge l capsules, aqueous emulsions or mixed
with vegetable oiIs [6). However, the consumption of these capsules is not well digested by
many human organisms, mainly because of the organoleptic propert ies of the oil.
The development of concemrated omega-3 compounds would make it possible to have
a product that comains the nutraceut ical of imerest, having less total fatty acids by daily intake, so it would be more accepted by human organism. l'he complexity of preparing these
concentrated compounds is mainly due to: ( I) fatty acids are found as triglycerides and they
Fractionation and Concentration of Omega-3 by Molecular Distillation
179
Table 1. Typical fatty acids fou nd in certai n com mercial marine oils I %p/pl
""'
<"
<J
C l4:0
Cl5:0
Cl6:0
C 16: I
<J
UJ
8
I
17
13
18
C l 8:0
C 18: I
10
3
16
I
9
1
19
12
18
10
3
II
10
17
14
12
17
10
13
5
22
3
2
13
I
10
7
2
I
16
2
21
c 17:0
Cl8:2
Cl8:3
C l 8:4
C20: I
C22: 1
C20:5
C22:5
C22:6
Others
2
2
14
2
9
13
2
15
14
22
2
8
13
I
I
3
4
3
16
2
9
7
14
14
I
1
3
2
3
17
15
12
3
13
15
19
15
6
7
17
12
18
10
14
7
I
1I
6
1
14
11
9
14
6
2
22
6
must be separated from the glycerol molecule, (2) fatty acids have similar ph ysicochemical
characteristics, fact that difficult their separation, and (3) they are highly labile compow1ds.
Commercial production of high enriched omega-3 compounds is nowadays a big challenge for research. Single fractionation processes do not discriminate between different
polyunsaturated fatty acids. Therefore they are not useful for th is enrichment. Several
methods for preparing enr iched fractions of omega-3 have been developed. However, some
methods are only appropriate for small scale preparations due to their high cost. The proper
technique for small-scale preparations include a controlled winterization (7), a molecular
di stillation (8), a fractionation of the uric phase {9) and a resin silver chromatography ( I0].
Another feas ible technique but very expensive, is the supercritical extraction with carbon
dioxide ( 11 ). Nowadays, at commercial production scale, molecular distillation is the most
promissory technique.
1.1.
Molecular Distillation
Molecular distillation or short path distillation is a special type of very high vacmun disti llation, wh ich takes place in equipment constructed so that the di stance that the molecules
must travel between evaporation and condensation is smaller than their mean free path.
This technology is suitable for separation, purifi cation and concentration of th ermolabile
substances with low vapor pressure {8, 12). As molecular distillation separates compounds
180
by their difference in volatility, esters of fatty acids have better separation efficiency th an
unesterified fatty acids since the reaction causes a decrease in the boi Iing point of the fatty
acids.
Currently, there are not avai lable simulators for molecular distillation operations since
it is not a conventional operation ( 13, 14]. Moreover, it is considered an emerging technology at industrial scale and it is based on di fferent separation theories to those used in
conventional distillation, since for working conditions phenomena is governed by different
molecular theories. On the oth er band, there is not much information about the physical,
chemical and rheological properties of the organic compounds that are found in the oil ( 15].
Molecular distillation is based on the evaporation of the components of a mixture, usually in the form of a falling fi lm in contact with a heated surface and with a subsequent
condensation on a cold surface very close to the previous one. The distance between those
surfaces is small er than the mean free path of the molecules. The main feature of th is
operation is the working pressure, in the order of w- 1 - 100 Pa. Under th ese working
conditions, the relative volatility of the components increases and the operating temperature is reduced, al lowing th e separation of compounds at a lower temperature. At these low
operating pressures, molecules that leave the evaporation surface do not practically suffer
an impact ( 16].
Molecules tha! leave the evaporation surface must travel a short way before their condensation, and they access to the condenser surface without delay. Instead, in conventional
distillation it is established an evaporation-condensation equilibrium. Therefore, in molecular distillation, very high evaporation rates are established, thereby reducing !he residence
time of the molecules in the process. Under these two conditions, short residence time and
low temperantre, thermal decomposition of the components is largely prevented and the
separation occurs at acceptable technologically rates (8].
The degree of separation reached in a molecular distillation process is not only a function of the relative volatilities of the components ( 17, 18]. When the liqui d evaporates,
th e vapor-liquid interface is cooled and, for a given mixture, the composition of the more
volatile elements decreases. This phenomena leads to the appearance of driving forces
wh ich produce diffusive mass and heat transfer (19).
lt has been investigated the influence of the feeding ternperantre on tt1e evaporation
efficiency (20]. A mathematical model of molecular distillation, without considering the
resistance to the transference in the vapor phase and discretizing the liquid phases of evaporation and condensation, considering a moving boundary, presented an analysis of the main
variables that influence in the performance of a faUing fi lm evaporator (2 1, 22).
Distilled liquid flows with a fi lm thickness determined by the liquid load and its viscosity. That fi lm is agitated by the action of a roller wiper. Due to surface evaporation,
temperature and concentration gradients are established. The main function of the roller is
to offset those effects, mixing the fi lm and favoring heat transfer to other layers r icher in
the more volatile components. The actual situation falls between two flow regimes: laminar (with semi-parabolic rate distribution and tempera,ture and concentration gradients) and
turbulent (ideal mixing, with no gradients of temperature and composition) f23, 22].
Further advances in theoretical modeling have been reported. Cvengros has used a
theoretical model to analyze th e molecular distillation operation for binary mixtures and
has studied the influence of some variables involved in th e process [20]. Bose and Pal mer
181
showed that the existence of concentration and temperature gradients in the liquid phase
decrease the separation efficiency (24). Bhandarkar and Ferron worked on heat and mass
transfer in a liquid fi lm on a tapered centrifugal evaporator (25). Batistella and Maciel have
provided comparative results of efficiency and performance between fall ing fi lm molecular
di stillation and in centrifugaI equipment [ 14). Phenomena of heat and mass transfer that
take place in a molecular distillation operation are more complex than those in conventional
distillation. The effect of the pressure of an inert gas in a molecular evaporator was also
studied (26).
The modeling for an industrial scale has not been developed yet. In order to develop
economically viable tech nologies on an industrial scale it is necessary to perform processes
of high production capacity {4).
1.2.
Prepara tion of Samples for Molecular Distilla tion
First of all, it must be carried out a stage of preparation of the raw material before
processing it in the molecular distillation. ln this step oil rich in fatty acids linked to glycerol
molecules will be transformed into ethyl esters of fatty acids.
Alkyl esters of fatty acids have many uses in the textile, cosmetic, food and synthetic
lubricants industries. Particularly, methyl esters of fatty acids are ones of the most commons, and they are employed in the production of amides and long chai n alcohols, and
more complex esters. The product ion and consumption of th e methyl esters of fatty acids
are increased due to their use in biodiesel process. On the other hand, ethyl esters are widely
used in tt1e food industry (27).
Alkyl esters of fatty acids are obtained by an esterification reaction from free fatty
acids, and also from vegetable or marine oils. ln that reaction, glycero l is replaced by
a short chain mono-al cohol through a transesterifi cation reaction. Tl1e transesterification
reaction is carried out in different ways. The most used method is th at which occurs in
a catalyt ic medimn, alkaline or acid, in a liquid phase. The reaction rate of the alkal ine
catalyzed reaction is higher and it requires a lower temperature (28, 29]. The presence
of two immiscible final phases (aqueous and lipid) makes that a good performance of this
reaction may be highly dependent on operating variables used, and th e composition of the
lipid raw material (30). The final products of the transesterifi cation are ethyl esters of
fatty acids and glycerol (31 , 32). After the transesterifi cation process, it is possible to use
the molecular distillation, prior glycerol is separated before being fed to the concentration
process {33).
An alternative to the alcoholysis is to perform a hydrolys is to produce fatty acids. However, eth yl esters have a higher vapor pressure than the acids one (34]. Esters make possible
to work at a lower vacuum and lower is the cost of vacuum. The traditional transesterification reaction is performed with methanol, but in products for human consumption, ethano l
is used sinee it is not danger to health [5).
The transesterifi cation reaction is shown in Figure I, where Rh R2 y R;l are different
carbon chains.
According to all what was discussed above, it is most convenient to su~j ect the raw material to a prior transesterification stage through an alcoholysis reaction. The recommended
catalyst is alcoholic potass ium hydroxide (KOH). After that reaction, the product has an
182
R1COOCH 2
~COOCH
Catalyst
+ 3CH3CHpH
HOCH2
HOCH
R3 COOCH2
HOCH2
Triglyceride
Glycerol
R, COOCH2CH3
R 2COOCH 2CH 3
~COOCH 2CH3
Ethyl esters
Figure I. Transesterification reaction.

alkal ine pH, and it must be neutralized and removed from the product In th is way it must
be used an acid to neutral ize the catalyst, with out deteriorating the qual ity of the product. A
highly recommended acid is the citric acid which is very effective in retarding the oxidative
deterioration of lipids in foods and it is commonly used in th e oils after the deodorization
stage (35, 36).
For a maximum conversion in tt1e esterification reaction, and thus achieve good yields
in the obtained esters, it is necessary to perform an optimization of the operation parameters wh ich have greater infl uence. To achieve this it is required a further experimental
development to monitor the progress of the reaction, for example, by measuring the concentration of free glycerol, HPLC or Thin Layer Chromatography (37) and, combined with
the change of time, th e concentration of mono, di and triglycerides and/or the concentration
of the esters of fatty acids (28, 6].
Due to the complex kinetics of alcoholys is, because of the vari ability of fatty acids provides a large munber of different reactive compounds, optimization is a topic of current
interest. To fi nd the optimal conditions for the esterification different mathematical and
statistical methods can be used, jointly with experimental data. These tools, if they are
jointly used, would allow decreas ing th e cost of experimental trials to achieve the optimization of a highly complex system. In recent years the development of alternative methods
for solving nonlinear systems has made significant contributions in th e field of simulation
and process control. These methods include genetic algorithms, fuzzy logic and Art ificial
Neural Networks {38, 39, 40].
l.3.
Object ives
The main objective of th is chapter is to present a review in which the process of fractionation and concentration of ethyl esters of omega-3 has been analyzed theoretically and
experimentally, us ing falling fi lm molecular distillation.
Specifically, it is presented an analys is of the main physicochemical properties of
omega-3 fatty acids. Moreover, it is shown a mathematical model which descri bes the
process of fractionation of fatty acids by molecular distillation, and the optimization of
the fractionation process, evaluating the influence of the operating variables that affect the
purity and the final yield of the process.
2.
183
Experimental lVIethodology
In this section it is initially discussed the treatment of the raw material and the required
equipment for fractionating the fatty acid ethyl esters. it is also anal yzed the need for an
esterification stage and the most favorable experimental conditions to carry it out. Final ly, it
is anal yzed the molecular distillation process of ethyl esters of fatty acids, and th e technical
characteristics of the molecular distillation are described.
2. I.
Description of the Raw Material
The process requires the use of refined and deodorized oil. This oil is a triglyceride
mixture wherein the carbon chains are predominantly myristic acid, palmitic acid, oleic
acid, EPA and DHA. The ethyl esters of the fed oil are obtained from th e transesterification
\\i th ethanol.
2.2.
Experimental Procedure
The process comprises a reaction stage, a step of separation by density difference and
two separation stages by molecular distillation. In Figure 2 it can be seen the process flowsheet. In the ini tial stage the reagents, oil and ethanol, are mixed. Moreover, a sui table
amount of catalyst for th e esterification is added to the mixture. Subsequent ly the mixture is conditioned by stirring and heating. When the operating condi tions are achieved,
the transesterification reaction is performed. After th is reaction, two phases are obtained:
a lipid phase, consisting essentiall y of ethyl esters of fatty acids, and an aqueous phase,
consisting of unreacted glycerol and etl1anol. This mixture is separated by decantation or
centrifugation. The lipid phase is subjected to molecular distillation in two stages, each one
at di fferent operating conditions.
Oil
MO-l
MO-ll
Ethyl etttrs
Ethanol
j'_
/"
lY J Mixer
....
Heavys
Reactor
Glycerol
Lights
Omega--3
Figure 2. Flowsheet of the obtention process of concentrated compounds of omega-3 . DM1: Stage I of the Molecular Distillation, DM-11 : Stage 11 of th e Molecular Distillation process.
184
2.2.1.
Esterification Stage
The most impo11am operating variables in the alcoholys is reaction are the reaction temperature, the dosage of catalyst and the amount of alcohol.
The temperature range varies from 50 - 78 'C, using a heated and stirred reactor. The
catalyst employed in the reaction is alcoholic KOH in concentrations of 0.4 - 1.6 %pp- 1
The reaction time can vary from one to two hours, working with a vollUne ratio of
ethanol/mass of oil between 1 ml g- 1 a 3 ml g- 1 (41, 28, 42, 43).
The reaction can be monitored, for example, by the quantitative determination of the
mass fraction of total glycerol in the lipid phase, wh ich is tl1e product of th e esterification.
These fractions are measured us ing standard analyt ical techniques {44).
As it is shown in Figure I, each mole of reacted triglyceride liberates one mole of
glycerol, so it can be deduced that:
( I)
where,
n~AG y n&r are the initial moles of triglycerides (TAG) and glycerol (GL) respectively;
JlifGL is the molecular weight of glycerol;
i~ the mass fraction of glycerol initially bound to the triglyceride molecule;
w&L
mm is tt1e mass of the sam pie.

The conversion of a chemical reaction x for a non-steady state process is defined as the
di fference between the reacted moles of limiting reagent in an initial and final instant and
the initial moles. it can be symbolical ly expressed as:
()
X=
71TAG
f
- 71 TAG
o
71
(2)
TAG
Mass loss in the lipid phase because of the loss of glycerol is approximately equal to
the mass of alcohol which binds to the esters. Analyzing this behavior, with th e equality ( l)
equation (2) can be written as:
X ::::
wbL
wo - wf
GL
woGL
GL
(3)
where
is the mass fraction of glycerol bound to the triglyceride molecule at the end of
the reaction.
On the oth er hand, as the number of moles of unreaoted triglycerides (TAG) are equal
to the moles of glycerol still remaining bound, the mass fraction of TAG can be cal culated
as follows:
f
f NfrAG
wTAG
-- wGL
1\,fGL
(4)
Experimental ly it is observed that the higher th e operating temperature and the higher
the concentration of catal yst with in the appropriate ranges of work (37), the higher is the
conversion of the reaction and the lower the amount of TAG remaining unreacted. The
process is usual ly operated at a temperature close to 78 'C which is the normal boiling
point of the ethanol, and at a concentration ofKO H of 1% p p- 1 (45].

2.2.2.
185
Molecu lar Distillation Stage
Molecular distiller consists of an evaporator enveloped by a closed cylindrical heating

jacket and an internal condenser. Fluid descends tl1rough the cylinder a~ a fi lm, it evaporates
partially and evaporated molecules condense in the internal condenser (20) .
The wiping or scraping of the liquid fi lm on th e evaporation area of a molecular evaporator is the key of an efficient operation, and the correct distillation of a thermosensitive
substance without suffering them1al risks. In a scraped-surface evaporator the liquid in
the fi lm of the evaporated surface is continuously mixed and distributed on the cylindrical
surface of th e evaporator. Scratching creates better conditions for heat and mass transfer
(46).
In the fi lm, th ennal gradients are generated as a result of the intense evaporation, and
stirring generall y mitigates th is effect. In the evaporation surface, resinous substances deposits are produced. Roller ensures proper distribution of those deposits, making it possible
to process highly viscous compounds [47). Figure 3 shows a diagram of the equipment.
Feed
Roller-wiper
Condenser
Heating
media -
Distillate
Figure 3. Molecular Distiller diagram.

Figure 4 shows the order of magnitude of the vapor pressure, wl1ere the t1igher the vapor
pressure of a certain compound, the eas ier its evaporation.
lt is demonstrated that it is possible to separate the ethyl esters of fatty acids by a
distillation process where the separation is driven by a molecular ratio of relative volatilities
expressed in equation 5 (48, 22].
(5)
where,
is i molecular relative volatility in respect of j;
and PV j are the vapor pressure of i and j, respectively;
Jl!fi and li!Ij are the molecular weight of i and j , respectively.
Cl:ij
pv;
186
Pablo Ross i, Nelson Ruben Grosso, M aria de l Can nen Pramparo et al.
1 00rr=====~==.---r-----------~
- Palmitic acid
- Oleic acid
80 EPA
.... DHA
Figure 4. Comparison of the vapor pressures of the ethyl esters.

Ethyl esters of omega-3 are compounds with intermediate volatility in respect of the
remai ning compoun ds of the mixt ure. Therefore, two distillation stages are performed. The
more volatile compounds are separated in the first stage and the omega-3 in the second one.
Operating temperamres range from 60 - 150 ' C. At lower temperatures, lower is
the separation efficiency. On th e other hand, higher temperatures cause changes in the
structures of the com pounds molecules. In order to work in those conditions, it is necessary
to maintain a working pressure of between 0.07 - 40 Pa. Lower pressures do not all ow the
desired formation of condensate and h igher pressures do not all ow a good evaporation (3 1).
3.
Phenomenological Modeling
The transport equations that describe the mo lecular evaporation process in the fi lm are
obtained us ing the corresponding global mass balances for each component. Constin nive
equations complete the phenomenolog ical model ing, in th is Langmuir- Knudsen equation is
ttsed to predict the rate of evaporation of each component in the surface fi lm. This equation
has been deduced from the kinetic theory of gases (8).
Micov proposed a ntrbu lent fi lm model which represents a fall ing fi lm ideally mixed
where the concentration of the fi lm is constant in the radial direction and the tem perature
is constant and equal to the temperature of the heating surface (22]. This mode l has the
following assumpt ions:
I. Steady state condition throughout the process;
2 Low condensation temperature to prevent re-evaporation of the condensing surface;

3. Rectangu lar coordinate system for describing the variables of th e fi lm, because the
fi lm th ickness is very smal l compared to the radius of the cylindrical evaporating
surface;
4. Radial gradients of tem perature and composition are negligible along the machine;
187
5. The influence of the feed temperature is negligible.

Molar flux density for the components, N j , is expressed as a function of the rate of
evaporation, kj , as follows:
' .
(6)
J~'
VJ -- x
J . f<J
where,
Nj is tt1e molar flux of j component;
Xj is the mole fraction of j component the evaporation surface;
kj is the evaporation rate of j component.
The evaporation rate of each component per unit of area of the evaporation surface, kj,
is evaluated by the Langmuir- Knudsen expression as follows:
k J -
P vj(TS )
R M jT '
J2"
(7)
wl1ere,
ys is the absolute temperature of the evaporation surface;
P vj is tt1e vapor pressure of j component;
J\!Ij is the molecular weight of j component;
R is the universal constant of gases;
" is the ratio between the circumference and the diameter.
As a result of the application of differential mass balances for each component, the
respective equations of molar flow variation with the vertical position y, are obtained as it
i.~ shown below:
(8)
where,
Ij is the molar fl ow of j component;
Re is the radius of the cylindrical surface where the fi lm descends;
11 is the vert ical position of the fall ing fi lm.
The sum of the molar flow contributions of all th e components involved allows calculating the value of the total fall ing flow as follows:
n
(9)
where,
n is the total number of compounds in the mixture;
I is the total molar flow.
Boundary condition relating to the system of differential equations is formulated as
presented below:
y = 0, Ii = Ijl,
( I 0)
where
fJ is the feed molar fl ow of j
component.
This model is mathematically complex due to it demands the simultaneous solution of

many equations as compounds involved in the mixture.
188
3.1.
Ra tio of Flows for a Multicomponent Mixture
From a mass balance for i and j components, the equations system is:
di
Ij
_2. = - 2ro ReA:j
2::: Ik
dy
v=o.
(I I)
y = 0,
(12)
k= l
where n is the number of compounds in the mixture.

Dividing tl1e equations term by term:
dij = A:j I j .
di;
A:; I ;
f}
j =
( 13)
j '
The expression ( 14) is taken

kj
Ctjj =
(14)
1... .
"t
where c'ji is defined as the Relative Molecular Volati lity of j component respect to i [49).
Using equation (7) the foll owing ratio is calculated:
Equation ( 15) shows that Ctj; varies with t'l1e change of Ts . Using equation ( 14) the
expression is reduced to:
dij
Ij
di; = cxji J;
rtl
Ij = 13 .
l; = fY .
( 16)
Solving the di fferent ial equation it is obtained the expression ( 17).
I; )!/<>;.
~J = ( I9
(17)
and the expression (9) can be written as:

( 18)

3.2.
189
Analysis of the Turbulent Mode l
In order to analyze the performance of the ntrbulent molecular distillation model for a
mult icomponen t mixture, it is necessary to study the phenomenon of molecular evaporation
of a pure compotmd.
The mass balance for a pure compound is:
di
- =-21TRck .
dy
.
v= o,
I = I 0,
( 19)
where f l is the molar flow of the pure component in the feed flow.
A dimens ionless variable B is defined as th e relative position in the fal ling fi lm and is
expressed as B = f, and 1 as the relative flow of the compound in the falling fi lm and is
expressed as 1 = frr. Replacing these variables in ( 19) allows arriving to (20) {50) .
di
21rRcLk
- =dB
I0
B = 0,
j = 1
(20)
where,
J is the dimensionless molar flux of the compound on the molar fl ow which is fed;
L is the length of the falling fi lm;
B is the dimensionless position of the fall ing fi lm.
The heating area can be calculated as As = 21r Rc L. Therefore:
-
dB
= -- 0-
'
B = 0,
I = 1,
(2 1)
k is defined as the Molecular Coefficient of Evaporation of the pure componen t and it is
expressed as i<(TS ) = A'~\tl . This coefficient relates the evaporated flow of the component compared to the fed flow, for a given geometry and a specific working temperature.
Therefore, the express ion (21) is as follows:
(dliB. -- - \
f_,, B = 0,
A~ it can be seen,
I. = l.
(22)
depends on the working temperature. As the turbulent model

assumes that the process behaves as an isothermal system, k is constant independently
of the relative position of the fal ling fi lm. Therefore, it is not necessary to do an energy
balance.
Solving (22), the relative flow of the pure compound along the fall ing fi lm can be calculated as follows:
J = 1 - i<B.
(23)
Equations (22) and (23) show that for a given operating temperamre and in presence of
a pure component, the flow has a linear behavior with respect to the position of the fal ling
fi lm.
190
For a multiple components mixture, equation (8) for a i component must be used.
(24)
where Xi
i.~
the mole fraction of i component in the fal ling fi lm and it is defined as:
(25)
Combining equations (24) and (25), it can be arrived to the following expression:
dJ.
I;
dy
2::: Ij
- ' = - 27i RcA:i ...,,::-=-.,
(26)
j=l
Taking equation ( 17), the follow expression can be obtained:

(27)
It is expressed as B =
t Replacing these variables leads to (28).

(28)
Heating area appears as A-' = 2r; RcL. As A and A:i are constant, they can be considered
into the summation in the denominator, so the equation is:
(29)
As it was noted above, I<j = 7 y O:ji = Jt;-, the equation can be expressed through
the Molecular Coefficient of Vaporization of j component and the Molecular Relative
Volatility between i and j, so the equation can be expressed as follows:
'
A' k
==
Kj
'' J'
NA:j A:;
JO
T.":
j
"-J
(30)
(30) is replaced in (29).

'
li
B = 0,
i; =
1.
(31 )
Fractionation and Concentration ofOmega-3 by Molecular Distillation
191
Equation (31) has an analyt ical resolution [5 1) as it can be seen below:

n
j=l
(l _ J? "i)
'K j
= B.
(32)
Applying expression ( J 7), the behavior for any j component can be known.
(33)
The i component chosen will act as a key compound, where it behavior will be analyzed
in the mixture from th is analys is and it can be understood the behavior of other components.
Jn the model, the influence of the operating temperature rs as variable operational is
highlighted. lt can be demonstrated that for a given value of B, an in crease in the operating
temperature results in a decrease in the molar flow of the falling fi lm for the compound
analyzed. This is the same to talk about an increase in the evaporation flow.
Another variable of interest is the composition ofi in the fal ling fi lm and it is defined as
(25). Its value depends directly on the falling fi lm flows. This is a variable of interest wh en
analyzing the purity level that the process reaches.
This model, because of its dimension less, is independent of the scalar flows involved
and of the size of the equipment. So, it only depends on the intensive variables. This fact
makes it possible to use the model to analyze the process independent ly of,the studied scale,
and so, to ut il ize this anal ysis to carry out a change of scale.
The model results in an implicit function between the flow rate of th e i component and
the relative position in the fal ling fi lm B. This fact represents a great math ematical simplification, since the original model requires the solut ion of simultaneous di fferential equations
as many compounds involved in the mixrure. Meanwhile, the model obtained is reduced to
the resolut ion of a single equation. Even so, equation (32) is an implicit nonlinear eq uation
wh ich requires a numerical solution. To avoid this situation there are two numerical solution alternatives. The first is to use the differential equation (31). A suitable method is the
fourth order R ung~Kutta meth od. The second alternative is the numerical solution of the
implicit funct ion of equation (32), using hybrid methods for solving non-linear equations.
In these methods favorable characteristics of various methods are combined (52).
3.3.
Parameters of Interest
The characteristics of th e falling fi lm are related to the operation of th e equipment. The

fi lm can be described by the flow profile, the th ickness of the fi lm, the average residence
time of the fluid imo the equipment, the heat needed to achieve the required evaporation flow
and half the mean vapor pressure of the mixture wh ich determ ines the order of magn imde
of the working pressure.
The ave rage speed of a falling fi lm can be obtained from th e momentum equation
{23, 50]. The result is as follows:
(34)
where:
v is the average speed of the fluid;
192
p is the mass density of the fluid;
g is the acceleration of gravity;

h. is the fi lm thickness;
11 is the fluid viscosity.
Moreover,
Wy =
(35)
l;(y)M;
i= l
wl1ere,
W is the mass flow of the residue stream;
I; is the mo lar flow of each i component in the falling fi lm.
(36)
where Re is the radius of the fi lm.

The mean residence time can be obtained through the phenomenological modeling of a
full ing fi lm. In th is way, the continuity equation must be applied (23, 50). This equation is:
op
- + \i'pv = 0.
ot
(37)
where 11 i~ the fluid speed.

A~
the fi lm is in liquid phase, it can be assumed that the density p is constant, so:
\i'v = 0 ===> v = con stant .
(38)
Speed is defined as the change of position of the fi lm front over time.

V =
oy
fJt :
t = 0,
1J = 0.
(39)
Jntegrating equation (39), assuming that the fi lm moves at an average speed ii, leads to:
- -y .
V=
(40)
The average retention time (tr) is defined as:

Volume
L
tr - Vo lume flow - -ii
(4 1)
Substituting (34) in (36), it is possible to obtain the falling film thickness ins ide the
equipment as it is s!1own below:
3vW11
2rrgRcp '
(42)
Fract ionation and Concentration of Omega-3 by Molecular Distillation
193
where v is the kinematic viscos ity defined as I/ = 1L/ p.

The necessary heat to achieve the evaporation is calculated by perform ing an energy
balance (53, 54).
(43)
Q = MI
where,
Q is the heat received by the fi lm;
b.H is the enthalpy ch ange of,the system.
Although the s implified mode l does not provide the feed temperature, the macroscopic
energy balance can contemplate the heat necessary to heat the feed to the working temperature. On the fall ing fi lm system, we find that;
Q = DHn + WFiw - FFip
(44)
where,
D, W and F are the molar flows of distillate, res idue and feed respect ively,
Fin, Fiw and lip is the specific enthalpy of each stream.

Taking a reference state regarding to a liquid phase and to the feed temperature, results in :
(45)
where,
c;
is the heat capacity of the mixture from k stream;

b.Hf:l is the latent heat of the mixture.
An est imation of the ideal working pressure can be made by Langmuir- Knudsen equa-
tion, which yields:
Pr =
1\!!M
L'"'fj
Pvi
X;
. I
=
i'vf
(46)
- '
wl1ere,
Pvi is the vapor pressure of the -i component;

1\!Ii and !11M are the molecular weight of the i component and of tt1e mixture, respectively;
and Pr is the vapor pressure generated by the mixture, which also represents the working pressure that is reached, as a function of the position.
An average val ue of Pr can be est imated through the following expression (55) :
L
f
Py
P(y) dy
0
L
J dy
0
(47)
194
Pablo Ross i, Nelson Ruben Grosso, Maria de l Can nen Pramparo et al.
3.4.
Obtaining the 1\ecessary Properties for the Model
Thermodynamic and rheological properties of the esters of pure fatty acids are avai lable
in literature or th ey can be estimated through specific correlations (56, 57]. The mode l
requires critical propert ies, vapor pressures, dens ities of the pure components in a liquid
phase and the densi ty of the liquid mixture. Vapor pressure as a function of the temperature
can be cal culated employing a method developed by Ceriani [34].
Oil propert ies can be detenn ined knowing its average molecular ch aracteristics, wh ich
can be predi cted by means of saponification index and iodine index [5]. Th is procedure is
described below :
Using the saponification index to obtai n the average molecular weight.
Saponification index is defined as th e amount ( in m9) of KOH needed to neutralize
all the fatty acids derived from complete saponification of an oil in one gram sample.
For example,
The saponification index of a sam ple is 181.44 m9T<OH g-1, and the equ ival ent
weight of KOH is 56.1 9 eq - 1 . Relating these values:
181.44m9t<OH9-I
.
- :l
-1
_1 ,
=
1.078
10
m
ol
r
AG
9
,
3 eq !<OH m ol H v6.1 91' eq - 1
where 1.078 w - :l mol-rAG9 - 1 is th e number of moles reacted in one gram of

sample. As the sample is a mixture of TAG, each molecule consumes tt1ree moles
of KOH. This ratio indicates the number of moles of TAG reacted, so the mo lecular
weight of TAG is obtained by:
1
1
92
7
69
l
- TAG - 1.078 10- 3 molr .-tG9- 1 - . m o.
Using iodine index to obtai n the average number of double bonds per mo lecu le of
triglyceride.
The iodine index is defined as the amount of iodine ( in grams) wh ich reacts with 100
grams of oil.
For exam pie,
The iodine index of a sample is 176.61 9T2 lOOg - 1, the we ight equivalent of h is
127 g eq - 1 and every double bond (DB) consumes two equivalents of / 2 .
1. 76fH 9t2 g- l
I = 6 .953 . w - :l DB 9 - 1 .
2eqJ 2 DB- 1 127 91 2 eqj.,
where 6.95a w - :l DB g - 1 is the number of double bonds reacted in one gram

of sample. As the sample is a mixture of TAG, the amount of double bonds per
mole of TAG can be calculated. On the other hand, the molecu lar we ight of TAG is
927.6 k g km ol- 1 So, there are :
927.6 gm.ol - 1
6.953 10- :l DB g- 1 = 6 .5double bonds per mole of TAG .

3.5.
195
Application of the Turb u lent Model to t he Case of St ud y
Fed oil is obtained through transesterification with eth anol. By means of gas chromatography about 15 different compounds are often detected. These peaks may be characterized
by the chain length of similar fatty acids. Only a few of those compounds are fotmd in high
proport ions (above the 5%w w- 1) , and th ey can therefore be grouped into fatty acids of
similar chai n length representatives of the mixture. This allows to use the turbulent mode l
only to those compounds, reducing the number of mass balances to be applied in the model
[58).
A~ it was discussed above, the most important operational variable is the working temperature. In order to analyze this behavior it is necessary to analyze the influence of ornega3, because they are key compounds in the separation [ 18].
Simulation shows that omega-3 flow increases in the distillate and decreases in the
residue when increasing the operating temperature. This is because, higher the temperature,
higher the compounds volatil ity, and there is a higher trend to move to the vapor phase (53).
lt is also seen that the composition of omega-3 increases in the distillate when increasing
the temperature up to a certai n temperature in wh ich the ethyl esters of fatty acids heavier
than the omega-3 begin to evaporate.
4.
Process O ptim ization
To optimize th e process the main goal is enrich ing the distillates in the compounds of
interest. In the distillate from Stage I (D 1) it is desired to recovery those compounds lighter
th an ornega-3. On the oth er hand, in the distillate of Stage 11 (D 2 ) it is attempted to obtain
the highest recovery rate of ethyl esters of omega-3 (w3). The optimized process is shown
in Figure 5.
MD-I
F
MD-11
01
/'
,. /
xxxx
IX XXX
I
02
"\,
./
"
./
W1
...
Figure 5. Process to optimize.
W2
...
196
The optimization function is:
(48)
where,
is the weights proposed for each parameter;
wf is the mass fraction of the i compound in A: stream;
Rec are the respective recoveries calculated as:
11.;
D2 D2
Ww:l
Rec..u:l = -y
F '
111
(49)
w:l
R ecLiv
1/J.D'
Liv
.F
Dl
F .
(50)
u;Li1.1
The theoretical model is a multidimensional non-linear model. The optimization is
obtained through the implementation of numerical methods for the non-linear mult idimensional problem which was generated. For this reason, it is required to use a nwnerical
optimization method such as Gauss- Newton, Conj ugate Gradient, Levenberg- Marquardt,
Nelder- Mead, among others. Nelder- Mead optimization method guarantees convergence
and has the advantage of being simpler than the other methods (59).
4.1.
Analysis of Temperature as the Most lnliue ntial Ope rat'ing Condition
Based on Rossi et al tests (58) results of the process optimization show an optimal
point in T 1 = 120 'C and T 2 = 1<10 ' C considering evaporation temperatures as the most
influential variables. The point of minimization of th e objective function leads to a value of
mass composition of omega-3 in th e distillate of Stage 11 of 80%pp- 1 and the amount of
product obtained is near 40% in respect of feeding.
The resulting model (58) has a good response in omega-3 concentration and in the flow
ratio against temperature changes at each stage. The content of omega-3 in the omput of
Stage ll is increased when tt1e temperature of Stage I is increased. This is a consequence of
the separation of light compounds which are tmdesired in Stage I. Moreover, the amount of
distillate in Stage I is increased and the distillate in Stage 11 decreases when increasing T 1
This fact implies a decrease in the recovery ofomega-3 in the distilled material of Stage 11
(D 2 / F). Considering this behavior, it is necessary to find the optimum condition of ptD'ity
of omega-3 jointly with d1e recovered amount. According to the temperature of Stage 11,
recovered omega-3 increases wh ile higher is the temperature T 2 .
The temperature of the distillation of omega-3 should be less than 140-150 'C to avoid
th ermal changes of labile compounds. These results are comparable with the work ofLiang
[31]. Liang determined an elimination curve for esterified marine oil employing multistage
molecular distillation at different temperatures between 50 and 150 C. At lower temperantres than 130 'C, final product had high concentrations of light compounds (fatty acids
with less than 18 carbons) . At temperatures between 130 and 150 'C, the final product had
t1igh concentrations of omega-3.
According to Liang, the separation of ethyl ester oils from squid visors oi l in a range
of 10 ' C, leads to a higher concentration of omega-3 in the range of 20 'C. However, it is
197
preferable to perform the distillation at intervals of 20 C. In th is way, the number of stages

is decreased and operating costs are reduced. it can be observed that the concentrations of
the ethyl ester of omega-3 increases, but its yield decreases with the number of distillation
stages. Final ly, it is only achieved a high concentration of omega-3 ethyl esters in the distillate fraction when feeding a raw material enriched in EPA and DHA. This fact determines
the need to use the first distillation stage to separate the lighter components.
4.2.
Analysis of t he l nlluence of t he Feed Temperature
The resul ts of different studies [20, 49, 14) indicate the advantages and effectiveness
of preheating the feed stream to temperatures close to the temperature of the surface of
the fi lm under steady state conditions. Otherwise, a port ion of the evaporation surface
is used for post-heating, and th e evaporation temperature in th is region is lower than the
boiling temperature of the fi lm surface. Within a certai n range, independently of the inlet
temperature val ue, the developed temperature profi le reaches an asymptotic val ue. This
phenomenon always occurs regardless of th e used model, approximated or not. Tl1ere is a
substantial temperature gradient between the wall temperature and the surface temperature
of the fi lm when temperature is asymptotically reached. The gradient represents about
10 I< mm- 1 and it is mainly determ ined by the physical parameters of the fluid (thermal
conductivity, viscosity).
The surface temperature of the fi lm is a crucial factor for the rate of evaporation, and
it represents the operation temperature in th e evaporator. There is no reason to preheat the
feed beyond that temperature. Preheating the feed stream has the addi tional techno logical
importance that all ows an intensive degassing of the inert elements before reaching the
evaporation surface, and it reduces the risk of entrainment. it should be noted that the
preheating of the feed stream must be economical, without triggering th ermal damage and
without the presence of oxygen (at vacuum) (20) .
4.3.
Alternative Molecular Distillation Techno logy with Recycle
As it was shown, both th e experience and the used model give encouraging results
when applying th is procedure to produce omega-3 . Although this chapter is not intended
to a profitability and process costs anal ysis, it is essential to consider that two molecular
distillation equipment or a process where one distiller is used to perform the two stages can
be expensive.
As th e process is focused in the distillation stages, it can be considered a disti llate reflux
system, similar to a conventional distil lation process (18), only employing a molecu lar
distillation stage.
To study the performan ce of the recycling process shown in Figure 6, a mass must be
included in the turbulent model as fol lows:
Fl = F - LD.
(5 I)
Fl = D l+ W,
(52)
Dl = D - LD ,
(53)
198
LD
M O~ I
Fl
/-,
,..
....
lXXX
01
"
./
w
Figure 6. System diagram with recycle.

RJ = LD
Dl
(54)
where,
F l is the molar process feeding flow;
LD is tt1e process recycle molar flow;
D l is the process distillate molar flow;
D is the disti llate molar flow of the distiller;
F is the molar flow fed to the distiller;
vV is the residue molar flow of the distiller;
Rj is the reflux ratio, which relates the molar recycle flow to the di stillate molar flow
of the process.
Simulating the complete process including a recycle stream has shown that operating
results are improved when flows and compositions increase. But if a high recovery and
purity of omega-3 is desired to be achieved, these improvemems are not enough to replace
a two stages process.
5.
Conclusions
In order to obtain a concentrate of omega-3, it is necessary to perform a transesterification stage for achieving the separation of .the carbon chain of the glycerol molecule. ln
such reaction it was foun d that the most favorable condi tion is achieved by working at high
temperatures. In th is case, the temperature was th e boiling point of the eth anol. On the
other hand, the conversion of tt1e reaction increased with increasing to an asymptotic val ue
the catalyst concemrat ion, which was alcoholic hydroxide potassium.
As the omega-3 have an intermediate volatility value between light and heavy ethy l
esters of fatty acids, separation process requires two stages of molecular distillation.
A math ematical model to optimize the molecular distillation process was shown, looking for conditions th at all ow good recovery of ethyl esters of omega-3, with a high degree
Fract ionation and Concentration of Omega-3 by Molecular Distillation
199
of purity. The model also allowed analyzing the influence of different operat ing variables.
The model consists of simplifications that allowed anal yzing s imultaneously severa l
compotmds . it was found that such s implifications satisfactory fitted the real phenomenon.
References
fi) Kinsella, J. ( 1986). Food components with potent ial therapeutic benefits : the n - 3
polyunsaturated fatty acids of fish oil. Food Technology, 28, 89- 97.
{2) Bimbo, A. P. (2000) . Pautas para la clasificacin del aceite de pescado comestible. Libro
de Oro de A &G - 10 Aniversario, Tomo 11, 555- 563.
[3) Yal enzuela, A., Nieto, S., & Uauy, R. (2002). Desafios tecnologicos para evaluar
acidos grasos n - 3 polii nsaturados en aceites rnarinos de uso alimenticio y farmaco logico.l.ibro de Oro de A&G. 10 A ni versario, Torno 11, 572- 58 1.
{4] Hui, Y. ( 1996). Bailey:~ industrial oil and f at products. N .Y, USA: John Wiley Sons,
Inc.
f5) Gunstone, F. D., Harwood, J. L., & Dij kstra, A. J. (2007). The lipid h@dbook. Tay lor
& Francis Group.
[6) Torres, C . F., Nettekoven, T. J., & Hi ll, C. G. (2003). Preparation of purified acylglycerols of eicosapentaenoic acid and docosahexaenoic acid and their re-esterification
'vi th conj ugated linoleic acid. Enzyme and Microbial Eeclmology, 32 ( I), 49- 58.
{7) Gunstone, F. D., McLaugh lan, J., Scrirngeour, C. M., & Watson, A. P. (1976). Improved procedures for the isolation of pure oleic, linoleic, and linolenic acids or their
methyl esters from natural sources. Journal of the Science of Food and Agriculwre,
27 (7), 675-680.
f8) Weissberger, A. ( 1951). Destillation under high vacuum in technique of organic chemistry. fnterscience Publishers, IV, 495-602.
[9) Ackman, R., Ratnayake, W., & Olson, B. ( 1988). The basi c fatty acid composition of
atlantic fish oils: potent ial simi larities useful for enrichment of polyunsaturated fatty
acids by urea comp lexation. JAOCS, 65, 136- 138.
f!O] Adlof, E. E. ( 1985). The isolation ofornega3 poly unsaturated fatty acids and methy l
esters of fish oils by silver resin chromatography. JAOCS, 62, 1592- 1595 .
fi I)
Lin, T., Chen, S., & Chang, A. (2006). Enrichment ofn-3 PUFA contents on triglycerides of fish oil by lipase-catalyzed trans-esterification under supercritiwl conclitions. Amsterdam, PAYS-SAS: Elsevier.
(I 2) Prarnparo, M., Prizzon, S., & Martinello, M. (2005). Estu dio de la purificacion de
acidos grasos, tocoferoles y esteroles a part ir del destilado de desodorizacion. Grasas
y Aceites, 56 (3), 228- 234.
200
fl3] Rossi, P. C., Gayol, M. F., Grosso, N. R., Pramparo, M. C., & Nepote, V. (2012).
Desarrollo e implementacion de un modelo simplificado de destilaci6n molecular de
multicomponentes a un software comercial. In Congreso Latinoamericano de Ingenieria y CieJwias Aplicadas ( CL/CAP).
fJ 4)
Batistella, C. B., & Maciel, M. R. W. ( 1996). Modeling, simulation and analys is of

molecular distillators: Centrifugal and falling film. Compwers & Chemical Engineering, 20 (Supplement 1), S 19- S24.
(15] Schweitzer, P.A. ( 1997). Handbook ofseparation techniques fOI d1emical engineers.
(3rd ed ed). McGraw-Hill,.
(16) Stephan, K. ( 1992). Hem transfer in condensation and boiling. Berlin, Heidelberg,
New York, USA: Springter-Verlag.
{I 7) Kawala, Z., & Stephan, K. ( 1989) . Evaporation rate and separation factor of mo lecular
di sti llation in a falling fi lm apparatus. Chemical Engineering & Technology, 12 ( I),
40~13.
fl 8) Treybal, R. ( 1989). Mass
Tran.~fer Operations.
(3era edici6n), 461-463.
(19) Vansant, E. ( 1994). Separation technology. Elsevier.

{20) Cvengros, J., Lut isan, J., & Micov, M. (2000). Feed temperature influence on the
efficiency of a molecular evaporator. Chemical Engin eering Journal, 78 ( I), 61- 67.
(2 1) Pramparo, M., Bonino, F., Gatica, E., & Martinello, M. (2003). Modelado y anlisis
de la destilacion molecular de pellcula descendente. A cws del XIII Congreso sobre
Mtodos Numericos y sus Aplicaciones (ENIEF 2003), I, 1692-1701.
[22) Micov, M., Lutisan, J., & Cvengros, J. ( 1997). Balance equations for molecular distillation. Sep(u:cuion ScieJ?ce and Technology, 32 ( 18), 305 1- 3066.
{23) Bird, R., Stewart, W., & Light foot, E. ( 1992). Fenmenos de transport e. Barcelona,
Espaiia: Reverte.
(24) Bose, A., & Pal mer, H. J. ( 1984). Influence of heat and mass transfer resistances on the
separation efficiency in molecular distillations. Industrial & Engineering Chemistry
Fundamenwls, 23 (4), 459-465. doi: I0. 102 J/i I 000 16a0 14.
{25) Bhandarkar, M., & Ferron, J. R. ( 199 1). Simulation of rarefied vapor flows. Industrial
& Engineering Chemistry Research, 30 (5), 998- 1007. doi: I0. 1021/ ie00053a023.
(26) Lutisan, J ., & Cvengros, J. ( 1995). Effect of inert gas pressure on the molecular disti1lation process. Separation Science and Technology, 30 ( 17), 3375-3389.
[27) Basiron, Y., & Ahmad, S. (2002). Palm oil, coconut oil and palm kernel oil: Issues
and future prospects. In Procee~lings of the 2002 World Oleochemiml Conference,
Barcelona, Spain.
Fract ionation and Concentration ofOmega-3 by Molecular Distillation
201
(28) Man, V., Konar, S., & Boocock, D. (2004). The pseudo-s ingle-phase, base-catalyzed
transmethylation ofsoybean oil. JAOCS, 8 1 (8), 803- 808.
f29) Haas, M., Scott, M., Manner, N. W., & Foglia, A. T. (2004). In situ alkaline transesterification: An effective method f or the production offauy acid esters from vegetable
oils, Vo l. 8 1, Heidelberg, ALLEMAGNE: Spr inger.
f30) Sh ao, P., He, J . Sun, P., & J iang, S. Process optimisation for the production ofbiodiese l
from r apeseed soapstock by a nove l method of short path disti llation. Biosystems Engineering, I 02 (3) , 28 5- 290.
p i ] Liang, J., & Hwang, L. (2000). Fractionation of squid visceral oi l ethy l esters by
short-path disti llation. JAOCS, 77 (7), 773- 777.
f3 2) Ooi, C., Choo, Y., Yap, S., Basiron, Y., & Ong, A. ( 1994). Recovery of carotenoids
from palm oil. Journal of the American Oil Chemists ' Society, 7 1 (4), 423-426.
(33) Batistella, C. B., & Maciel, W. M. R. ( 1998). Recovery of carotenoids from palm oil
by molecular di stillation. Compwers & Chemical Engineering, 22 (Supplement J),
S53-S60.
{34) Ceriani, R., & Meirell es, A. J. A. (2004). Predicting vapor-liquid equi libria of fatty
systems. Fluid Phase Equilibria, 2 15 (2) , 227- 236.
{35] Aliakbar ian, B. Dehghani, F., & Perego, P. (2009). The effect of citr ic acid on the
phenolic contents of olive oi l. Food Chemistry, J 16 (3) , 6 17-623 .
(36) Cerretani, L., Bendini, A., Poerio, A., & Tosch i, T. G. (2009). Citric acid as coadj uvam: improvement of the antioxidant act ivity of edible olive oils. AgroFOOD
industry hi-tee!!, 19 (3), 64-66.
f37) Ross i, P. C., Barchies i, M. E., Gayol, M. F., Grosso, N. R., Pramparo, M. C., &
Nepote, V. (20 J2). studio de la reaccion de ester ificacion de aceite de calamar para
la obtenci6n de omega-3 . In Congreso Latinoamericano de lngenieria y CieJ?cias Aplicadas (CL/CAP).
l38) Bulsar i, A. B. ( 1995). Neural Networks for Chemical Engineet:~. Elsevier Science Inc.
{39) Rahman, A. M., Chaibakhsh, N., Basr i, M., Sai leh, A., & Abdu l Rahman, R. (2009).
Application of art ificial neural network for yield prediction of lipase-catalyzed synthes is of dioctyl adipate . Applied Biochemistry and Biotechnology, 158 (3) , 722- 73 5.
f40) Ying, Y., Shao, P., J iang, S., & Sun, P. (2009). Artificial neural network analyis of
immobilize~! lipase catalyzed sy nthesis of biodiesel from rapesee~i soapsock, (Ch . I,
pp . 1239- 1249). JHP Internationa l Federation for Information Process ing. Boston :
Spr inger.
{4 1] Darnoko, D., & Ch eryan, M. (2000). Continuous product ion of pal m methyl esters.
Journal of the American Oil Chemists ' Society , 77 ( 12), J 269- J 272.
202
Pablo Rossi, Nelson Ruben Grosso, M aria de l Can nen Pramparo et al.
(42) Facioli, N., & Barrera, D. (2002). Opt imization of direct acid esterification process of
soybean oi l deodorizer distillate. Grasas y Aceites, 53 (2), 206- 2 12.
f43) Lotero, E., Liu, Y., Lopez, D. E., Suwannakarn, K., Bruce, D . A., & Goodwin, J. G.
(2005). Synthes is of biodiesel via acid catalys is. Industrial & Engineering Chemistry
Research, 44 ( I 4), 5353-5363 . doi: I 0. 102 J/ ie04915 7g.
f44) AOCS. ( I 994). AOCS Officials Methods & Recommended Practices of the Anw-ican
Oil Chemists Society. AOCS Press.
{45) Rossi, P.C., Nepote, V., Grosso, R., Renaudo, C., & Pramparo, M. (2010). Uso de
redes neuronal es art ificiales para la opt imizacion del proceso de esterificac ion de materiales lipidicos. In VI Congreso Argemino de lngenieria Quimica (CA IQ 20 10) .
f46) Shao, P., J iang, S. T., & Ying, Y. J. (2007). Opt imization of molecular distillation for
recovery of tocopherol from rapeseed oi l deodorizer distil late us ing response surface
and artificial neural network models. Food and Bioproducts Processing, 85 (2), 85-92.
f4 7) Cvengros, J ., Pollak, S., Micov, M., & Lut isan, J. (200 I). Fi Im wiping in the mo lecular
evaporator. Chemical engineering journal, 8 1, pp. I .
{48) Douglas, J. ( 1988). Conceptual Design of Chemical Processes. Me Graw Hi ll.
(49) Lutisan, J., Cvengros, J., & Micov, M. Heat and mass transfer in th e evaporating fi lm
of a molecular evaporator. Chemical Engineering Journal, 85 (2-3), 225-234.
(50) Deen, W. ( 1998) . A nalysis q( Transport Phenomena. New York, Oxford.
(5 I) Perko, L. (200 I) . Di}}'eremial Equations and Dy namical Systems. New York .
{52) Heath, M. T. (2002). Scientific Compwing: An Introductory Survey . NY: McGraw
Hi ll.
{53) Modell, M., & Reid, R. C. ( I 983). Thermody namics and Its Applications. PrenticeHall INC.
{54) Reklaitis, G. & Schneider, D. ( 1983).1ntroduction to Material and Energy Balances.
New York, USA: John Wiley & Sons.
(55) Ruiz, P. ( 1995). Ccllculo vectorial. Prentice Hall Hisponoamericana SA.
[56) Reid, P. J. ( 1987). The Properties of Gases and Liquid~. (4th ed.). Manfenix.
(57) Perry, R., Green, D., & Maloney, J. (2007). Perry:~ Chemical Engineers Handbook.
Eighth Edition. McGraw Hi ll.
{58) Rossi, P.C., Pramparo, M. C., Gaich, M. C., Grosso, N . R., & Nepote, V. (2011).
Optimization of mo Iecular disti11 at ion to concentrate ethyl esters of eicosapentaenoic
(20:5 ?-3) and docosahexaenoic acids (22:6 ?-3) us ing s implified phenomenologica l
modeling. Journal of the Science of Food and Agriculture, 9 1 (8), 1452-1 458.
203
l59) Constantinides, A., & Mostoufi, N. ( 1999). Numerical method~ for chemical engineers
with MatLab applications. Prent ice Hall.
I NDEX
alcohol w nsumption, 132
alcohols, _1 8 1
Ll;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;iA
ii;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;ll-_ algae, x,
14 3
'"
a-linol enic acid (ALA), , ;, 57, 165
accelerator, 6 1
allele, I 02
access, 28, ISO
allergy, 13 1
acetone, 157
alters, 34, 69, 89, 94, 97
acetylcholine, 44
American Heart Association~ 48, 152
acid, vii, viii, ix, xi, 3, 6, 8, 9, I 0, 11 , 12, 13, 15, 17,
American Psychiatric Association, I 19, 124
18, 19, 22, 24, 26, 27, 31 , 32, 33, 34, 35, 36. 38,
amincs, 33
39, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51 ' 52,
amino, 3, 65, 78, 169
53, 54, 56, 57, 58, 64, 66, 67, 68, 69, 70, 72, 73,
amino acid, 3, 65, 169
75, 76, 78, 79, 85, 86, 87, 88, 89, 90, 9 1,92, 93,
amino acids, 65
94, 95, 96, 97, 99, 100, 103, 104, 110, Ill , 11 2,
- ~' 4, :J' 7
amnC.S:Ia,
113, 11 4, 115, 116, 11 8, 124, 125, 126, 127, 130,
amplitude, 87
132, 133, 135, 13 7, 139, 14 1, 145, 146, 148, 152,
amygdala, 4, I 7
153, 157, 158, 160, 161 , 163, 165, 166, 167, 168,
amyloid beta, 36
169, 170, 17 1, 172, 173, 174, 175, 177, 178, 18 1,
anatomy, 37
182, 183, 199, 20 1, 202
angiogen esis, ix, 99, 100, 101 , 107, 109, 11 5
active compound, 86
anisotropy, 96
acute lcukemia, I 03, I 12
annealing, 168
acute promyelocytic leukemia, 6 1
A."'OVA, 133, 136
ADA, 48, 50
anti-canc.e r, I 0 I, 110
additives, 130
antidepressant, viii, 75, 76, 77, 78, 83, 85, 86, 92,
adhesion, 130, 148, 149, 151
11 9, 125
adiponectin, 163
antidepressants, viii, 75, 77, 78, 86, 95
adipose, I 04 , 11 3, 11 4
anti-inllammatory drugs, 32, 116
ad.i pose tissue, I 04, I 14
antioxidant, 34, 43, 44, 45, 46, 84, I 06, 20 I
aduiU100d, 14, 32, 49
antipsychotic, 122
adults, x, 50, 54, 57, 67, 95, I 00, I 03, 120, 129, 131 ,
antitumor, ix, 99, I 00, I 05
138, 14 1, 163
anxiety, 19, 20, 22, 31 , 46, 11 8, 123
adverse e flects, 64, 65
anxiety disorder, 123
advertisements, 131
apoptosis, vii, I, 7, 14, 18,20, 2 1, 25, 27, 29, 31 , 60,
aerobic exercise, 6 7
61, 7 1,83,84
allectivc disorder, 127
appetite, 58, I 18, I I 9
AFM, 95
aquac.u hurc, 167, 17 5
age., 2, 10, 11, 16, 22, 53, 54, 57, 58, 69, 76, 132
Argentina, 75
agencies, 50
arrh)thmia, 144
aggregation, 40, 42, 146
arteries, 144
aggression, 123
artery, 115, 144, 148, 151
aggressiveness, I 04
arthritis, 49, 178
agonist, viii, 2, 12, 31 , 33
ascites, 96
akinesia, 2
Asia, 44, 96, 140, 167
Alaska, 162
asparmte, 87
11
,o,
206
assessment,
Lndex
16, 22, 23
a~trocytes,
6, 44, 78, 84, 97

a~trogliosis, 20
atherogenesis, 146, 148
atherosclerosis, 130, 144, 146, 148, 149, 152, 153
atherosclerotic plaque, 148
atrial llbilllation, 148, 151
atrophy, 23, 42
autoimmune diseases, 166
autoimmunity, 125
aversion, 131
awareness, I 73
bonds, 194
bone, vii, viii, 4 7, 49, 51 , 52, 53, 54, 55, 56, 5 7, 58,
59, 60, 6 1' 62, 63, 64, 65, 66, 67, 68, 69, 70, 72,
73
bone biology, 70
bone cancer, 59, 60, 61 , 65
bone cells, 61
bone lbrm, 49, 57, 61 , 63, 67, 70
bone growth, 54
bone marrow, 5 7, 60, 63, 64, 69
bone mass, 51 , 52, 53, 54, 55, 5 7, 62, 64, 67, 68, 69,
70
bone mineral content, 59
bone resorplio~, viii, 47, 52, 5 7, 61, 63, 64
11
' - - - - - - - - - - - - - - - - - - bone
bones,volume,
55, 58, ,59,
g 61 , 156
bacteria, 58
borderline personality disorder, 122
basal ganglia, 3, 19, 23, 33, 36, 3 7, 45
brain,vii, l, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 15, 17, 18,
base, 168
19, 20, 2 1' 22, 24, 27, 28, 29, 30, 32, 33, 35, 36,
888, 5, I0, 28
37' 38, 39, 40, 42, 44, 45, 46, 66, 78, 80, 8 1, 83,
Beck Depression Inventory, 120
84, 85, 87, 88, 89, 90, 91 , 92, 93, 94, 96, 97, 117,
beet; 94
11 8, 12 1, 124, 125, 127
behavioral change, 31
damagc, --
n s)
bram
bencticial eflbct, xi, 8, 15, 22, 23, 30, 51 , 55, 70, 84,
brain stniCture, 124
105, 106, 119, 12 1, 122, 140, 146, 165
breakdown, 55, 5 7
benefits, vii, viii, ix, x, 7, 14, 35, 4 1, 47, 50, 60, 62,
breast cancer, 60, 7 1, 102, I 04, I 07, I 09, 110, I l l,
64, 65, 76, 106, 109, 117, 11 9, 129, 130, 138, 139,
11 2, 113, 115, 11 6
143, 152, 156, 163
by-products, 26
benign, I 04
benzodiazepine, 92
bioavailability, 7, 68, 94
11
biochemical processes, I 00
biochemistry,43, 55, 127, 151 , 152, 153
cachexia, ix, 4 1, 60, 99, I 05, 106, I 09, 110, I l l,
biodicsel, 18 1, 20 I, 202
113, 114
biological activity, 63
CAD, 144
biological processes, viii, 47, I 00
calcium, viii, 4 7, 49, 51 , 52, 53, 55, 56, 57, 58, 61 ,
63, 65, 68, 69, 70, 82, 87, 97, 147
biomarkers, 5 7, 102, 104, 112
biomcc.hanics, 64
calorie, 16, 17
cancer, ix, x, 4 1, 52, 59, 60, 99, I 00, I 0 I, I 02, I 03,
bioregulators, 110
biosynthesis, 11 , 39, 64, 166, 167, I 73, I 74, I 75
104, 105, 106, 107, 109, 110, I l l, 113, 114, 115,
bipolar disorder, 76, 77,9 1, 93, 95,96, 12 1, 125
125, 143, 173, 178
canc.er progression, I 02, I 05
bladder cancer, I 03, I 12
bleeding, 146
candidates, 7
bleeding time, 146
capillary, 157
blood, xi, 13, 15, 28, 55, 5 7, 62, 63, 76, 87, 89, 90,
capsule. 131, 133
94, 102, 103, 107, 110, 119, 124, 126, 132, 140,
carbohydrate, 16, 133
14 1, 144, 146, 147, 149, 151, 153, 156, 162, 177,
carbohydrates, 17, 65
carbon, xii, 25, 78, 8 1, 86, 17 1, 178, 179, 18 1, 183,
178
blood clot, 13
198
blood Jlow, 144
carbon atoms, 8 1
blood pressure, xi, 63, 132, 146, 147, 149, 151 , 153,
carbon dioxide, 78
162, 177, 178
carcinogenesis, I 00, 107, I 08, I I I
card noma, I 13
blood supply, I 07
blood vessels, I 07
cardiac arrest, 144
blood-brain barrier, 4 1
cardiac arrhythmia, 144, 148
body c'omposition, 132
cardiomyopathy, 174
body fat, 17
cardiovascular disease, x, 7, 51 , 65, 1 2 5~ 140, 14 1,
143, 147, 151 , 152, 153, 163, 178
body mass index, 16, 17, 131 , 132, 134
body weigh~ 16, 17, 24, 30, 52, 53, 59, 64, 132, 159
cardiovascular risk, ix, 129, 130, 138
207
Lndex
cardiovasc.ular system, 144
carotcnoids, 20 I
cartilaginous, 60
case study, I 19
casein, 156. 158

c.a spases, 7
catabolism, 62
catabolized, 78
catalysis, 202
catalyst, xii, 178, 181 , 182, 183, 184, 198
catalytic ac.tivity, 172
catatonic, 12 1
catccholamincs, 3
causality, 15
causatioo, I 18
cDNA, 167, 170, 174
cell culture, x, 26, 45, 51 , 143
cell cycle, 61
cell dcath, 7, 29, 45,8 1, 93, 109, 144
cell division, 61
cell line, 5, 26, 38, 45, 51 , 61 , 10 7, 109
celllines, 5, 51 , 6 1, 107, 109
cell membranes, x, 12, 89, 94, I 00, 118, I 19, 124,
126, 143, 147, 148
cell metabo lism, viii, 4 7
cell signaling, x, 6 1, 143
cell size, 3 I
rentral nervous system, viii, 75

cerebral blood Jlow, 9 1
cerebral cortex, 3
cerebral ll1nction, 88
cervical cancer, I 03, I 13
cervical intraepithelial neoplasia, I 15
cervix, I 03
chemical, 9, 70, 178, 180, 184, 200, 203
chemoprcvcntive agents, I I 0
chemotherapy, ix, 99, I 00, I 05, I 06, I I 0, I 14
chicken, 60
childhood, x, 76, 77, 93, 143
children, 39, 50, 56, 66, 69, 76, I 03, 112
China, L
chloro lbnm, 15 7
cholesterol, x, xi, 15, 16, 36, 65, 79, 82, 90, 94, 95,
97, 129, 130, 133, 138, 139, 140, 14 1, 142, 146,
155, 156, 15 7, 158, 159, 160, 16 1, 177, 178
cholic acid, x, 155, 156, 159
chopping, 162
chromatography, 163, 168, 17 1, 179, 195, 199
chromatography analysis, I 71
chronic d iseases, 50, 51 , 66, 96, I 00
chronic myelogenous, 60
cigarette smoking, I I I
circulation, 56
cl asses, 12 1, 14 1
cleaning. 13, 123
clinica l application, 28
clinical depression, I 19
clinical diagnosis, I 06
clinical symptoms, 4, 22, 87, 124
clinical trials, 15, 22, 76, 77, 95, 96, I 05, 127, 138,
139, 140, 151
cloning, 167
clothing, 131
clozapine, 122
CNS. viii. 11, 75, 78, 83
c.oconut oil, 54, 200
c.oding, 167
cognition, ix, 17, 36, I 17
rognitivc Jltnction, 51 , 127
rognitivc impairment, vii, I , 2, 12, 15, 23, 38~ 125

collagen, 60
colon, 60, 7 1, I 00, I 07, I I I , I 15, I 16
colon cancer, 60, 71, 107, I l l , 115, 116
calor, 178
colorcctal c.anc.cr, 102, 10 7, I l l , 114, 116
combi ned effect, 138
commercial, xi, 50, 140, 168, 177, 179
community, 3 1, 89
comorbidity, 95, 122
comparative analysis, 133

competing interests, 124
competition, I 00, I 07, 123, 132
complexity, xi, 3 1, 177, 178

comp1iancc, 133, 146, 153, 157
complications, 18, I 09
composition, viii, I 0, 11, 12, 19, 34, 35, 47, 52, 56,
66, 67, 68, 77, 79, 80, 8 1, 87, 88, 97, 102, 104,
11 2, 113, 116, 119, 126, 130, 142, 156, 157, 158,
175, 180, 18 1, 186, 19 1, 195, 196, 199
compounds, viii, xi, 3, 7, 29, 31 , 75, 80, 84, 85, 86,
177, 178, 179, 180, 182, 183, 185, 186, 187, 188,
19 1, 195, 196, 199
computer, 168
computer so flwarc, 168
conception, 140
condensation, xi, 177, 179, 180, 186, 200
conductance, 82, 86
conductivity, 197
con llict, 140
con llic.t o f interest, 140
consensus, 31
COn SC.nl, 131
conservation, 49
constituC-nls> viii, xi, 51 , 75> 144, I 77, 178
construclion, 168
consumption, viii. ix, xi, 16, 4 7, 50, 51 , 52, 63, 65>
76, 93, 96, 99, 102, 106, 108, 109, Il l , 112, 113,
125, 127, 130, 131 , 132, 133, 135, 137, 138, 141,
144,145, 146, 147, 149, 152, 155, 156, 163, 178,
18 1
conlamination, 173
control group, 19, 54, 56, I 03
controlled trials, ix, 117, 119, 126, 153, 174
controversial, 25, 26, 62, 146
conlrovcrsics, 88
convergence, 196
cooking, x, 131 , 155, 156, 163
coordination, 140
208
Lndex
derivatives, I 18
destruc-tion, 4
detachment, 13
detectable, 53
detection, 132, 157, 163
developing brain, 95
diabetes, 48, 6 1, 62, 65, 72, 125, 131 , 144, 153, 163,
cortic.al bone, viii, 47, 53, 58, 59, 63, 64, 65
173
cosmetic, 18 1
diabetic patients, 62, 9 1, 162
cost, 179, 18 1, 182
diacylglycerol, 97
cotton, 56
diagnostic <:rilcria, 12 1
c-reactive protein, vii, x, 129, 130, 132, 134, 137,
diastolic blood pressure, 134, 14 7, 149
diet, vii, viii, x, xi~ I 0, 12, 15, 19, 24, 25, 28, 29, 34,
139
35, 36, 47, 48, 50, 52, 53, 54, 55, 56, 57, 58, 59,
crops, xi, 165, 167, 174
60, 6 1, 62, 63, 64 , 66, 67, 75, 78, 79, 80, 89, 94,
cross-sectional study, I 02, 112
crude oil, 121
100, 104, 107, I l l , 11 4, 115, 124, 130, 14 1, 142,
143, 144, 145, 149, 151 , 155, 156, 158, 159, 160,
cuhure, 22, 28, 64 , 73, 86
<:uhurc media, 22
16 1, 177, 178
cure, 7
dietary fat, viii, 47, 52, 5 7, 58, 59, 65, 66, 104, 110,
current limit, viii, 2
11 2, 113, 135
CV D, ix, 48, 129, 130
dietary habits, 15, 16
dietary intake, viii, ix, 8, 11, 15, 16, 17, 24, 4 7, 49,
cycling, 9 1, 125
50, 54, 76, 99, 100, 102, I l l , 117, 144
cyclooxygenase, 37, 63, 83, 85, I 00, I 0 I , I 18, 146
.
, _' 7 , 62 , 7 -? 77 , 8 _..,
< 9 .:>,
'
'
cyclo-oxygenase 2 (COX-2), ix, 99, I 02
di ctary suppI cmcntauon,
x, )- .:>,
129, 147, 151
cyclophosphamide, I 05
dietary suppl)', 84
cytochrome, I IS, 169
cytokines, 6, 12, 13, 20, 28, 4 1, 80, 82, 85, 86, I 07,
di Oercntial equations, 187, 19 1
11 8, 12 1, 125, 126, 130, 140
di nTaction, 95
digestion,
cytoplasm, 25
167
disability, 93, 118, 119, 126
cytoskcle!on, Si
disease model, x, 36, 46, 155, 159
cytotoxicity, 94, 97
diseases, vii, x, I, 8, 40, 50, 51 , 65, I Oil, 102, I 09,
143, 155, 163
._I;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;iD
i;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;ll disorder, vii, 1, 2, 3, 7 6, 77, 9 3, 9 6, 9 7, 12 12 2,
..
123, 125, 126, 127
danger, 18 1
distillation, vii, xi, xii, 177, 178, 179, ISO, 18 1, 182,
DART, 15 1
183, 185, 186, 189, 196, 197, 198, 200, 20 1, 202
data collection, 14 0
distress, 123
database, 13 1, 132
distribution, I 0, 39, 4 8, 49, I 12, 113, 180, 185
DBP, 132, 133, 134
DNA, 37
deaths, I 00
DNA damage, 37
dcc.ay, 96
docosahexaenoic acid, viii, ix, x, xi, 8, 17, 22, 32, 33,
declarative memory, 23
34, 35, 36, 4 1' 43, 44, 46, 50, 66, 68, 7 1' 75, 76,
delicicncy, 14, 23, 32, 34, 38, 4 1, 43, 57, 67, 8 1, 84,
78, 87, 88, 89, 90, 92, 93, 94, 95, 96, 97, 99, 100,
89,90,9 1, 94, 95, 97,98
11 0, 115, 11 6, 11 7, 11 8, 127, 131 , 14 1, 142, 151 ,
deilcit, I 18
152, 153, 155, 158, 162, 165, 166, 175, 199, 202
degradation, viii, I 0, 4 7, 58, 65, I 06
doctors, viii, 2
Delta, 7 1, 165
dogs, 147, 163
delusions, 120, 12 1
dopamine, 2, 3, 4, 22, 3 7, 38, 39, 40, 42, 45, 46, 79,
dementia, 2, 34, 35, 38
83, 84, 9 1,97, 98, 11 8
dendrites, 25
dopamine agonist, 42
dentin, 58
dopaminc-rgic, vii, I , 2, 3, 6, 7, 11, 12, 19, 23, 24, 25,
depolarization, 14 7
26, 28, 30, 3 1' 32, 34, 35, 36, 38, 40, 4 1' 42, 43,
deposits, 185
45, 79, 83, 84, 89
depression, viii, ix, 2, 8, 15, 30, 3 1, 6 1, 75, 76, 77,
dosage, 97, 184
83, 84, 85, 86, 87, 89, 90, 9 1' 93, 94, 95, 96, 118,
dosing, 7, 19, 20, 24, 26
11 9, 120, 12 1, 123, 124, 12 5, 126, 127
doubl e bonds, 6, I I, 8 1, 166, 178, 194
depressive symptoms, 76, 77, 86, I 18
drug interaction, viii, 75
deprivation, 12, 27, 32, 35, 42, 84, 94
coronary artery bypass gra ft, 148, 149
coronary artery disease, 144, 152
coronary heart disease, vii, x, 50~ 71, 1 30~ 143, 145,
15 1, 152, 153, 163
correlation, 56, 6 1, 94, I 02
correlations, 194
cortex, 5, 17, 19, 84, 97
o,
Lndex
209
dmgs, viii, x, 2, 6, 7, I 0, 31 , 45, 94, 122, 126, 143,

153
drying, 15 7
DSC, 95
dyslipidemia, 140, 144
ethanol, xii, 178, 18 1, 183, 184, 195, 198

ethnicity, 69
ctiology, 76, I I I, 144
European Community, 113
euthanasia, 2 1
evaporation, xi, 177, 179, 180, 185, 186, 187, 189,
r-------~------.,
19 1, 193, 196, 197
r;l;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;iEi;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;lll cv i dencc, vii, viii, ix, I , 2, 6~ 7, 8, 15, 25, 26, 29, 30,
37, 42, 47, 50, 51 , 52, 55, 61 , 62, 63, 70, 76, 77,
cicosapcnlacnoic acid, vii, viii, ix, x, xi, 2, 8, 17, 22,
84, 86, 94, 99, 101 , 102, 105, 110, 11 7, 118, 119,
33, 39, 40, 4 1, 44, 63, 66, 67, 68, 7 1, 73, 76, 88,
12 1, 123, 125, 126, 130, 139, 151
91, 93, 95, 96, 99, 100, I l l, 114, 116, 117, 124,
excitabilit)', 82, 97
125, 126, 127, 135, 141 , 143, 144, 152, 153, 155,
exclusion, 89
158, 165, 166, 199
excrctioo, 19, 55, 56, 5 7, 61 , 63, 68, 130
aJSA, 132
exercise, 54
elongation, 35, 78, 166, 17 1, 172, 173, 174
cxocytosis, 34, 82, 93
emulsions, 178
experimental c.o ndition, 183
enc.oding, xi, 165, 167, 170, 173, 174
c.xpcrimcnlal design, 24
endocrine, I 15
exposure, xi, 27, 38, 55, 62, 177
endothelial cells, 85, 88, 115, 148
extraction, 95, 167, 168, 179
endothelial dys lc~nction, 130
endothelium, 148
energy, 16, 17, 30, 49, 58, 59, 65, 78, 80, 8 1, I 00,
F
11
11 1, 112, 114, 118, 119, 120, 133, 189, 193
energy consumption, 80, 133
fasting, 131 , 139, 145, 162
energy expenditure, 17
J3l, 15, 16, 17. 33, 35, 40, 48, 53, 58, 60, 65, 69, 87,
engineering, 173, 174, 202
89, 100, I l l , 112, 126, 132, 133, 135, 151 , 199
en\i ronment, 7, 27, 28, 62
Jat intake, 15, 16, 17,40, 11 2, 126
enzymatic activity, 87
Jatal arrhythmia, 152
Jlmy acids, \ii, viii, x, xi, xii, 2, 7, 8, I0, 12, 13, 15,
enzyme, 3, 13, 58, 82, 83, 89, I 00, I 02, I 07, I 08,
167, 168, 174
16, 25, 31 > 32, 33, 34, 35, 39, 4 11 43, 44, 46, 4 7
enzyme-linked immunosorbcnl assay, 132
48, 49, 50, 51 ' 52, 54, 55, 56, 5 7, 6 1, 62, 63, 64,
enzymes, x~ 5, 6, 12, 18, 26, 39, 45, 80, 83, 94, I08,
65, 66, 67, 68, 69, 70, 7 1' 72, 75, 76, 79, 8 1' 83,
84, 86, 87, 88, 89, 90, 9 1, 92, 93, 94, 96, 97, I 00,
11 6, 145, 165, 166, 167, 173, 174
EPA, vii, viii, ix, x, xi, 21 S, 9, 10, 12, 13, 14, 15, 17,
104, 109, 110, Il l, 112, 113, 11 4, 115, 11 7, 124,
125, 126, 127, 129, 133, 137, 138, 140, 14 1, 145,
20, 21 , 22, 23, 24, 25, 26, 27. 28, 29, 30. 31 ' 38,
44, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 5 7, 58,
15 1, 152, 153, 154, 160, 162, 163, 165, 167, 168,
170, 17 1, 172, 173, 174, 175, 177, 178, 179, 180,
59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 7 1, 73,
75, 76, 77, 78, 79, 8 I, 82, 83, 85, 86, S7, 89, 90,
IS I, 182, 183, 185, 194, 195, 196, 198, 199
91, 93, 95, 96, 97, 99, 100, 101 , 102, 103, 104,
FDA, 48, 50
105, 106, 107, 108, 109, 114 , 117, l iS, 11 9, 120,
fear, 123
fcces, 55
12 1, 122, 123, 125, 126, 127, 130, 131 , 133, 137,
138, 139, 14 1, 143, 144, 145, 146, 147, 14S, 149,
feelings, I 18
155, 156, 158, 160, 161, 162, 165, 166, 167, 174,
female rat, 54, 67, 70
fem ur, 53, 54, 58, 59, 61 , 63
177, 178, 183, 197
EPA-ric.h fish oil, vii, x, 129, 131 , 133, 137, 138, 139 fertility, 63
epidemic, I 00
fiber, 16, 65
episodic memory, 23
llbers, 2 1, 25
llbrinolysis, 146
epithelial ovarian cancer, 115
equality, 184
llbroblasts, 27, 88, 153
llbrosis,
144
equilibrium, 180
equipment, 179, 18 1, 183, 185, 19 1, 192, 197
Jilm thickness, 180, 186, 192
Hsh, vii, viii, ix, x, xi, 8, 15, 17, 2 1, 22, 24, 25, 26,
erythrocyte membranes, I 04, I 13
30, 32, 33, 47, 50, 51 , 52, 53, 54, 55, 56, 57, 58,
crythrOC)'tes, I 00, I 03, I 04, 152
csophageal cancer, 114
59, 60, 62, 63, 64, 65, 67, 68, 70, 72, 75, 76, 77,
essential fatty acids, 43, 44, 46, 55, 68, 94
81, 86, 88, 93, 94, 96, 97, 101 , 102, 105, 106, 110,
ester, 38,46, 53, 132, 14 1, 142, 157, 160, 196, 197
Il l , 11 2, 113, 114 , 126, 127, 129, 130, 131 , 132,
133,
137, 138, 139, 14 1, 143, 144, 145, 146, 147,
estrogcn, I 07, I 08
ETA, xi, 165, 167, 17 1, 172, 173, 174
2 10
Lndex
148, 149, 151 ' 152, 153, 155, 156, 160, 162, 163,
165, 169, 170, 173, 175, 199
lish oil, vii. viii, ix, x, 8, 15, 17, 2 1, 22, 24, 25, 26,
30, 33, 47' 50, 52, 53, 54, 55, 56, 57, 58, 59, 60,
62, 63, 64, 65, 67, 68, 70, 72, 75, 76, 77, 8 1' 86,
88, 93, 94, 96, 105, 106, 110, 113, 11 4, 127, 129,
130, 13 1, 132, 133, 137, 138, 139, 14 1, 143, 145,
146, 147, 148, 149, 151 , 152, 153, 155, 156, 160,
162, 163, 165, 173, 199
llame, 157
llank, I 07
Oexibility, 146
Ouid, 19 1, 192, 197
Ouoxetine, 78, 92, 94, 120, 125
JOiate, 16
JOod, viii, ix, xi, 16, 22, 32, 33, 37, 51 , 75, 99, 102,
112, 130, 131 , 132, 133, 135, 155, 159, 162, 167,
18 1
lood industry, 18 1
lood intake, 1 12, 159
glycerol, xii, 178, 179, 18 1, 182, 183, 184, 198

goal-directed bchavior, I2 I
graduate students, viii, 2
grants, I 74
gravitational force, 62
gravity, I 92
growth, x, 33, 4 0, 48, 50, 54, 57, 58, 59, 60, 6 1, 62,
8 1, 84, 86, 88, 93, 100, 106, 107, 109, I 15, I 16,
143, 17 1, 174
growth arrest, 6 I
growth factor, 40, 48, 58, I 07, I 09, I I 5, I I 6
guidelines, 50, I 14
guilt, I I 9
I!
hallucinations, 120, I 2 I
healing, 6 1, 69
health, vii, x, 12,46,50, 51 , 52, 53, 5 7, 64, 65, 66,
lood products, xi
lootball, 1 10, 1 12
lorc.c, 60
Jorcbrain, 4, 21, 22
JOrmation, 6, 25, 26, 27, 28, 29, 30, 33, 37, 43, 44,
55, 56, 57, 63, 97, I 00, I 07, I 08, 144, 148, 178,
186
lormuta, 172
~actures, 55, 61
~agility, 55, 6 1
~agments, 167
France, 65
~cczing, 2
~cshwater, 17 5
~on tal cortex, 4, 14, 17, 2 1, 23, 35, 42, 46, 84, 89,
94 97
'
~uits, 130
llmctional lood, I 30, I 38, 155, 156

Jlmding, I 40
lc1sion, 79, 82, 96
,-------G
. ______. . I.
I
gait, 20
gallbladder, I I 5
g!l, I I , 162, 163, 178
gene expression, x, I I, I2, 27, 38, 83, 84, 87, 90, 9 1,

92, 116, 130, 143, 153
genes, viii, xi, 14, 38, 4 7, 8 I , 83, 84, I 0 7, I I 6, 125,
165, 167, 170, 173, 174
genetic factors, I00
gcnctic.s. 144
genotype, I42
geometry, 189
glial cells, 85, 90
glucose, 62, 72, 8 1, 83, 94, 141, 154, 163, 168
~utamate, SO, 87
gjutatbione, 6, 22, 26, 46
I_
67, 88, 96, 121 , 129, 130, 139, 143, 145, 151 , 152,
156, 163, 166, 173, 174, 178, 18 1
health eO'eets, vii, I 45, I 66
health promotion, 50
heart attack, 14 7
heart disease, 48, 65, 66, 144
heart failure, I 44, 148
heart rate, 132, 133, 147, 149, 151 , 153
heat capacity, I 93
heat transler, 180
height, 13 1
heme, 82, 92
heme oxygenase, 82, 92
hemodialysis, I I 0
hemostasis, 152
hepatitis, 90
hepatocellular carcinoma, I 06
hetc.rogcnciry, 23, 38
hcxanc., 15 7
high density lipoprotein, 142
high Jllt, 60
hippocampus, 3, 4 , 14, 23, 3 1, 32, 34, 39, 40, 86, 87,
9o. 9 t, 92, 94, 97, 126
histidine, 169, 170

histology, 60
homc.ostasis, 7, I 3, 14, 49, 6 1, 72, 94
homocysteine, I 24
hormone, 45, 54, 58
hormones~ xi, 177, 178
host, 167
HPA axis, 92
human, vii, viii, x, xi, I, 5, 15, 19, 26, 27, 29, 39, 4 1,
42, 45, 47, 5 1, 54, 55, 6 1, 62, 65, 7 1, 85, 88, 89,
90, 9 1, 94, 100, 107, 108, 109, 115, 116, 139, 140,
14 1, 143, 145, 146, 152, 153, 162, 163, 165, 166,
169, 174, 175, 177, 178, 18 1
human body, xi, 177, 178
human health, xi, I00, 165
human organisms, I78
2 11
Lndex
human subj ects, 94, 14 1, 162
Hunter, I I I, 131 , 132
hybrid, 19 1
hydrogen, 15 7
hydrOI)SiS, 18 1
hydroperoxidcs, 26
hydroxide, 18 1, 198
hydroxyl, 90
hyperactivity, 20, 92
hyperc.holcsterolemia, x, 155, 156, 159, 160
hyperglycemia, 144
hyperlipidcmia, vii, ix, x, 129, 130, 138, 139, 140,
14 1, 155, 160
hypersensitivity, 142
hypertension, 131 , 144, 147, 151 , 162
hypcrttiglyc.eridemia, 151 , 154
hypotensive, 130
hypothesis, ix, 35, 75, 84, I 18, 125
89, 100, 107, 109, 118, 124, 127, 142, 144, 146,
148, 153, 163
inllammatory disease, 60, 72, 124
inllammatory mediators, 9 1, 13 7
inllammatory responses, I 06
ingest, 78
ingestion, 57, 79,9 1
ingredients, 138
inhibition, ix, 6, 7, 25, 31 , 35, 38, 43, 63, 99, I 06,
107, 108, 130, 146, 151 , 154
inhibitor, ix, 45, 99, 122
initiation, ix, I 00, I 0 I, I I 0
injections, 19, 20, 2 1, 24
injuries, 147
injury, 45
institutions, 50
insulin, 62, 72, 97, 14 1, 163
insulin sensitivity, 62
integration, 11 , 152
integrity, 3, 11 , 14, 12 1, 147
I,~iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii&iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiil11 interaction
intcracrion c flCct, I32, I37, 139
eflects, 132, 139
ideal, 180, 193
interfac.e, 180
idcntitication, vii
interference, x, 143
interferon, 6, 80, 9 1
identity, 169, 170

idiopathic, 4
IF:'-!, 6, 28
inagc, 122
imbalances, 15
inmune Junction, I 00, I 18
inmune response, 63, 83, 125, 152, 153
inmunity, 12, 14
inmunodeficicncy, 115
interpersonal relations, 122

interpersonal relationships, 122
intervention, ix, x, 39, 51, 57, 59, 99, I 05, 11 4, 12 1,

131 , 132, 133, 134, 136, 137, 143, 146, 147, 148,
151
intestine, 58, 160
intima, 148
intoxic.ation, 4, 6
investmCnlS, 7
iodine, 194
ion channels, 11 , 80, 92, 147
ionization, 157
ions, 79
IRC, 21 , 24, 26
iron, 16, 36, 39
irritability, I 18
ischemia, 32
isolation, 163, 167, 173, 199
isomers, I 78
isotope, 96
issues, viii, 75, 77
Italy, I I I
inmunohistoc.hcmistry, 3, 25
inmunorc.activity, 21
inpairmcnts, 18, 39
inprovcments, 23, 121 , 122, 198
inpulsivity, 120
i1 transition, I 07
i1 virm, 45, 51 , 58, 61 , 62, 63, 85, 9 1, 94, 96, 10 7,
115, 163
i1 vivo, 34, 3 7,44, 51 , 58, 60, 62, 63, 71, 78, 79, 9 1,
96, 107, 115, 11 8, 125, 163
incidence, 76, I 09, 112, 148
indecisiveness, 11 8
independent variable, 132
ildirect measure, 23
ildividuals, ix, x, 7, 17, 22, 50, 78, 99, I 02, I 04,
129, 130, 138, 142, 145
ilduction, 12, 37l 38, 4 1 ~ 115, 153
industries, 18 1
industry, 20 I
infancy, 15
infants, 50, 156, 162
inl1lr<:lion, 151
infection, I 15, 173
inflammation, vii, I, 6, 7, 11 , 12, 13, 14, 18, 19, 27,
28. 30, 33, 35, 39, 43~ 44, 45, 54, 82, 83~ 85, 86.
11
Japan, 40, 50, 11 7, 126, 155, 156, 157, 158, 162

jaundice, I 06
jurisdiction, 157
11
kinase activity, 80
2 12
Lndex
kinetics, 88, 94, 140, 182

KOH, 15 7, 18 1, 184, 194
Korea, 165, 167, 174
11
macronutricnts, 16, 49
macular degeneration, 32
~,1iiiiiiiiiiiiiiiiiiiiiiiioiLi.iiiiiiiiiiiiiiiiiiiii-!111- magnitude,
120, 185, I 9 I
maj or depression, 15, 77, 92, 95, 119, 125, 126
labcling, 44
lactation, 86, I I 0
laminar, 180
LD L, 130, 133, 138, 139, 14 1, 145, 146, 178
lead, viii, 6, 12, 27, 47, 51 , 5 7, 58, 65
leaks, 90
lean bo dy mass, 4 1, I 06, 11 4
learned helplessness, 90
learning, x, 11, 36, 40, 44, 45, 87, 143
learning task, 87
legume, 16
leptin, 58
lesions, 4, 32, 34, 40, I 07, 144, 148
leukemia, 60, 61 , 7 1, I 02, I 03
lcukotricncs, viii, 12, 13, 47, 83, 85, 146
li lcstylc changes, 55
li JCtimc, I I S
i gand, 49, 62, 89
i ght, ix, xii, I 0, 76, I 02, I 3 I , 156, 178, 196, 198
i noleic acid, xi, 8, 9, 35, 56, 68, I 00, I I 0, I I I , I 65,
166, 17 1, 199
i pid metabolism, 72, 84, I 30
i pid peroxidation, I6, I7, 18, 26, 27, 30
i pid peroxides, 24, 26
i pids, 8, I 7, 18, 24, 25, 39, 40, 60, 70, 82, 9 I , 96,
97, 102, 103, I 13, 130, 132, 138, 14 1, 146, 157,
162, 163, 166, 17 1, 182
i poproteins, 10, 14 1, 145, 146, 152, 162
i quid c.hromatog,aphy, 157, I 63
liquid phase, I 80, 18 I , I 92, I 93
i thium, I 68, I 7 I
liver, I 0, 42, 56, 69, 94, 95, 96, 113, I 3 I , I 54, I 56,
157, 159, 160, 161 , 167
iver damage, I0
localization, 45, 89, I 09
locomotor, 20, 22, 33
loc us, 3, 40
bw temperatures, xii, 178
bw-dcnsity lipoprotein, I 30
bwcr lip, 60
LTB4, I 3, 85, I 32, 134, 139
lubricants, I 8 I
l 1mbar spine, 54
l 111g cancer, I 04, I 06, I I 0, I I 2, I I 4
lutein, 16
lycopcne, 7 I
lymphocytes, 85, 88, 89, I 00, I 03
lymphoma, ix, 48, 6 I , 99, I 02, I 03, I 04, I 05, I I I ,
113
maj or depressive disorder, 76, 119, 125, 127

maj ority, I 0, 51 , 52, I 00
malignancy, 59, I 06, 115
mammalian brain, 8
mammals, I I , 167, I 70, 174
man, 36, 64
management, 42, 68, 140, 152
mania, 95, 120, 12 1
manic, 120, 12 1
manipulation, 68, 167, 173
marine ilsh, xi, 72, 165, 167
marrow. 48, 58
mass, 16, 44, 51 , 53, 59, 67, 101 , 105, 11 4, 131 , 132,
133, 134, 180, 18 1, 184, 185, 186, 187, 188, 189,
192, 195, 196, 197, 200, 202
mass loss, I 0 I
mass s:pcctromctry, 44
matrix, 56, 5 7, 169
matter, I 0, 11, 35, 89
mcasurcmcnl, 52
measurements, 132
meat, x, 8, 16, I l l , 112, 155, 156
mechanical loadings, 62
mechanical properties, 53, 58, 59, 63, 64, 65
media, 26, 52, 79, 131
median, 22, 14 7
medical, 95, 131
medical history, 131
medication, 17, 84, 87, 93, 122, 126
melanoma, 9 1
mellitus, 6 1
membrane permeability, 79, 86, I 18
membranes, I 0, 11 , 12, 27, 30, 31 , 58, 70, 79, 80, 83,
87, 90, 93, 96, 97, 117, 118
memory, 11, 15, 20, 23, 28, 34, 36, 44, 45
memory pcrfbrmance, 44
menopause, 42, 53, 55, 57, 59, 64, 69
mental disorder, ix, I 17, 124, 126
mental health, vii, ix, 76, 96, I 17, I 18, 123, 127
mental illness, 127
mesencephalon, vii~ I , 25
mcta analysis, I 05, 147
mcta-analysis, 76, 77, 87, I 02, I I I , I 12, 123, 124,
138, 147, 151, 153
Metabolic, 48, 69
metabolic changes, I 06
metabolic. pathways, 146
metabolic syndrome, 163
metabolism, vii, viii, h, I, 2. 3, 6, 8, 9, 11, 13, 14,

18, 24, 27, 42, 46, 47, 49, 52, 53, 54, 57, 58, 59,
61 , 64, 65, 66, 68, 69, 70, 80, S I, 84, 88, 91 , 93,
Lndex
2 13
94, 97, 99, 101 , 102, 124, 125, 139, 148, 152, 162, muscle mass, 104, 106
174
muscles, 19, I 06
rretabolites, viii, 3. 9. I I, 18, 2 1, 23, 24, 37, 4 7, 64:
mutation, 25
84, 85, 97, I00, 11 8, 146
mutations, 4
rrctabolizcd, 63, 78, 85
myocardia! infarction, 144, 151, 163
metastasis, 96, I 0 I
myocarditis, 144
rrcthanol, 15 7, 18 1
myoc.yte, 147
rrcthodology, 3 I
rrcthylprednisolone, 67
11'--------~-------MHC, 6
11
N
rrice, x, xi, 4 , 6, 7 ~ 12, 18, 19, 20, 2 1, 22, 23, 24, 25 ,
26, 27, 28, 29, 33, 35, 37, 38, 40, 4 1, 42, 43, 46,
NaCI, 15 7
53, 58, 60, 62, 63, 67, 68, 70, 7 1, 93, 107, 114,
National lnstitutcofMcntal Health, 119, 126
116, 142, 152, 155, 156, 158, 159, 160, 161, 162,
natural killer c.ell, 9 1
163
necrosis, 35, 39, 49, 6 1, 85
nicrodialysis, 46, 97
negative cffec.ts, viii, 47, 55, 64
nicroorganisms, 13, 4 1
neoc<lrtex, 3, I 7, 34
nicrostructurc, 58, 63, 67, 70, 119
ncphrocalc.inosis, 55
nidbrain, 20, 22, 28, 29
nephrolithiasis, 68
nigration, I 07
nerve, xi, 3, 28, 38, 40, 44, 86, 87, 90, 93, 177, 178
nincralization, 55, 56, 69
nerve growth factor, 28, 40, 44, 86, 9 0
Minncapo1is, 132
nervous system, 78, 95
nitogens, 94
:-.Jctherlands, I I I, I 12
nixing, 156, 180
neural network, 20 I, 202
model system, 27, 42
neuroblastoma, 26, 45, 92
modelling, 115
neurodcgencration, 7, 18, 28, 30, 37, 4 1, 43, 44, 45
...I
rrDdcls, viii, xii, 2, 4, 27, 28, 30, 31 , 32, 34, 36, 3 7,
neurodcgcncrativc diseases, I 0, I I , 32, 39, 44,45
40, 45, 51, 52, 55, 57, 60, 64, 77, 86, 105, 107,
139, 147, 178, 202
modilications, 11, 90, 157
ITl)iSture, xi, 177, 178
mole, 184, 187, 190, 194
molecular distillation, vii, xi, xii, 177, 178, 179, 180,
18 1, 182, 183, 189, 196, 198, 200, 20 1, 202
neurodcgencrative disorders, x, 8, 143

ncurolilamcnts, 4
ncurogcncsis, 11, 12, 35, 84, 87, 89,9 1
neuroinllammation, 32, 85
neurological disease, 89
neuronal apoptosis, 35, 4 1, 11 8
neuronal cells, 32
111)1cc.ular mass, 11
rrol cc.ular structure, 86
neurons, vii, I , 2, 3, 4, 5, 6, 7, 12, 20, 2 1, 22, 25, 26,

27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 4 1, 43,
molecular weight, 44, 184, 185, 187, 193, 194

mJiec.ulcs, xi, 6, 12, 13, S2, S3, S5, 95, 10 7, 109,
130, 148, 151 , 177, 178, 179, 180, 18 1, 185, 186
ITl)tnCntum, 19 1
monounsaturatcd lauy acids, 15, 16, 95, 173
ITl)Od disorder, vii, 1, 2, 90, 11 8
mood swings, 120
Moon, 33, S2, SS, 93~ 175
morbidity, ix, 12 1, 129
morphology, 39, 58, 59, 64, 86
IT1)rtalit-y, ix, 50, 93, 126, 129, 130, 144, 152
mortality rate, 144
Moses, 111
motil; 170
motor behavior, 18, 19
motor control, 36
MI'TI' model, vii, 2, 4, 18, 19, 23, 31
MRJ, 95
mR:-.IA, 20, 2 1, 22, 25, 26, 27,28, 29, 43, 95, 107,
115
m.tltidimcnsional, 196
m.tltiplc myeloma, I I I
muscle atrophy, viii, 47
45, 78, 8 1, 86, 88, 92, 95, 97

neuropathology, vii, I, 2, 4, S. 19, 23, 25,29, 30, 3 1,
45
neuroprotcetion, 12, 14, 15, 32, 3 7, 39, 43, 92
ncuropsyc.hiatry, 38
neurotoxicity, 6, 7. IS, 24, 30, 33, 35, 36, 3S, 40, 4 1,
42, 46, 80
neurotransmission, 12, 33, 34, 79, S3, 84, SS, S9, 9 1,
98, 124
neurotransmitter, vii, I, 14, 24, 8 1, 83
neurotransmittcrs, 19, 24, 39, S3
neurotrophic factors, 28
neutrophils, 148
New England, 167
New Zealand, 50, 59, 65
nigrostriatal, 2, 3, 4, 18, 19, 20, 2 1, 23, 24, 29, 30,
32, 44
nilric oxide, ix, 4 1, S2, 99
nitric oxide synthase, 4 1
nitrogen, 167
NM R, 90, 95
non-linear equations, 19 1
nonlincar systems, IS2
214
norepinephrine, 90, 11S
notmal development, 117
North Americ.a, 144, 174
North Atlantic Treaty Organization, 49, 50, 66
Norway, 131
NSA IDs, 6, 4 1
nuclear magnetic rc.sonancc, 90
nuclear reccptors, 35, 89
nuclei, 3
nucleus, 14, 39, 84, 98
nutraccutical, 178
nutrient, x, 37, 106, 155, 156
nutricnlS, 37, 70
nutrition, 32, 66, I 00, I 06, I ll , 114, 175
nutritional status, 35, 67, 113, 133
Lndex
oxidation products, 27
oxidative damage, 6, 27
oxidative stress, vii, I, 6, 7, 11, 18, 20, 2 1, 24, 25,
26, 27, 31 ' 34, 43, 45, 82, 83, 84, 86, 87, 130, 148
Ox)gen, 58, 83, 197
11
Paci lic, 162

pain, xi, 177, I 78
palm oil, 19, 20, 2 1, 20 I
panc.reas, I 04
pancreatic cancer, I 06, 11 4, 115
parallel, 3, 97
.
.
parkmsomsm,
-) 4
f=================="iJ participants, 120, 131 , 132, 137
pathogcnesis, vii, I, 2, 6, 8, 25, 27, 40, 68, 93, 127
pathology, 33, 36, 44
obesity, 144
pathophysiological, 85, 110
OCD, 123, 125
pathophysiology, 84, 11 8, 126, 153
oesophageal, I 03, 113
pathways, 2, 4 1, 6 1, 63, 82, 88, I 05, I 06, 130, 145,
oil, x, xi, 17, 21, 22, 26, 48, 49, 50, 52, 53, 54 , 55,
166, 167, 173
56, 57, 58, 59, 60, 63, 69, 72, 76, 86, 9 1, I l l, 122, per lusion, 10,4 1
125, 129, 130, 131 , 133, 137, 138, 140, 145, 146,
perinatal, 38, 4 1
147, 149, 151 , 152, 153, 155, 177, 178, 180, 18 1,
permeability, 79, 82, 90
183, 184, 194, 195, 196, 199, 200, 20 I, 202
permission, 157
oilsced, xi, 165, 167
peroxidation, 26, 30, 38, 60, 116
Oklahoma, 47
peroxide, 27, 127
old age, 39
personality, 122, 127
oleic acid, 153, 163
personality disorder, 122, 127
oligomerization, 25
phagocytosis, 29
oligomers, 25, 30, 35, 44
pharmacology, 153
olive oil, 57, 72, I 09, 20 I
pharmac.othcrapy, 7
omcga3, vii, viii, ix, xi, xii, I, 2, 3, 8, 9, 32, 34, 35,
phenomenology, 36
38, 39, 43, 44, 65, 66, 67, 70, 71, 72, 75, 76, 77,
phenotype, 25, 27,4 1, 14 1, 142
78, 84, 85, 86, 87, 88, 91, 92, 93, 94, 96, 97, I 09,
phenylketonuria, 56, 69
110, I l l, 112, 113, 114, 115, 116, 125, 126, 127,
pheochromOC)tOma, 93
129, 130, 135, 138, 139, 140, 14 1, 151, 152, 153,
Philadelphia, 70, 72
163, 174, 177, 178, 179, 182, 183, 186, 195, 196,
phosphate, 61
197, 198, 201
phosphatidylc.holine, 94, 96, 104
Omega-3 polyunsaturated fat, viii, 43, 75, 89, 124
phosphatidylethanolamine, 78
operating costs, 197
phosphatidylscrine, 78
operations, 180
phospholipids,ix, 8, 10, 12, 13, 33, 35, 76, 78, 79,
optimization, 182, 196
SO, 85, 87, 92, 93, 99, 102, 103, 104, 105, 106,
optimization method, 196
110, 11 2, 113, 114, 11 7, 126, 146
organ, 49, 64, 73
phosphorus, 69
organic compounds, 180
phosphorylation, 93, I 09, 118
organism, 85, 178
physical activity, I 06, 132, 133
organs, 96, 161
physical health, 118
osteoarthntis, 67
physical properties, 11
ostcoclaswgenesis, 63, 68
physic.ochemic.al properties, xi, 177, 182
osteoporosis, 5 1, 52, 54, 55, 61 , 64, 67, 68, 70, 125
Physiological, 145, 14 7, 151 , 153
outpatients, 122
physiology, 166
ovarian cancer, I 02, I l l
phytoplankton, 145
ovariectomized (OVX), viii, 47
phytosterols, vii, ix, x, 129, 130, 13 1, 132, 133, 137,
ovariectomy, 52, 55, 64, 67, 72, 73
138, 139, 140, 14 1
oxalate, 68
pilot study, 15, 34, 53, 93, I 05, I 06, 124, 127
oxidation, 6, 22, 24, 27, 30, 88, 97
pioglitazone, 12, 33
l..________.o
._________..!l
Lndex
2 15
placebo, x, 17, 52, 76, 77, 105, 11 9, 120, 12 1, 122,

promoter, I 54, 170
123, 126, 129, 131 , 133, 137, 138, 146, 148
prostacyclins, 12
prostaglandins, viii, 6, 12, 13, 47, 80, 83, 85, I07,
plant sterols, 140, 14 1, 142
11 6, 11 8, 173
plants, 173, 174, 175
plaque, 144
prostate cancer, ix, 99, I02, I04, 11 2, I I3, 174
plasma levels, 90
prostate carcinoma, I l 2
plasma lipid prolile, vii, x, 129, 138, 139, 14 1
protection, 20, 22, 24, 25, 26, 29, 30, 32, 36, 148
plasma membrane, 44, 79, 82, 88
protective role, 14, 68
plasma proteins, I0
protein kinase C, I 07
plasmid, 172
protein sequence, 169
proteins, 6, 7, 10, 11 , 14, 37, 38, 44, 79, 88, 145,
plasticity, 32
platelet aggregation, 146
152, 162, 163, 170
Platelet- Derived Growth Factor ( PDGF), ix, 99
proteolysis, I 06, I 14
protcomc, 44
platelets, I 00, I IS
psyc.hiatric disorders, 3 1, 4 1, 123, 124
polych!orinated b.iphenyl, 145
polymerization, 25
psychiatry, 2, 94, 125
polymers, 25
psychopathology, 93
polymorphism, I 02, I 12
psychosis, 120, 124
polyps, I 16
pubcrt)', 54
polyunsaturated lln, vii, viii, ix, xi. I , I 0, 32, 33, 35,
public health, 5 1, I 09
36, 38, 39, 4 1, 42, 43, 44, 46, 65, 66, 67. 70, 72,
puri lk ation, 90, 163, 167, 179
75, 76, 78, 87, 88, 89, 90, 9 1, 92, 93, 94, 95, 96,
purity, xii, I 06, 178, 182, 19 1, 196, 198, 199
97, 98, 99, 100, 110, 11 4, 11 5, 11 6, 11 7, 124, 126,
14 1, 142, 152, 153, 155, 158, 162, 166, 167, 173,
Q
174, 175, 177, 178, 179, 199
11
polyunsaturated lluty acids, vii, viii, ix, xi, I0, 33,
qualit)' o f li fe, 6 1, 106
35, 38, 39, 4 1, 43, 44, 65, 70, 72, 75, 76, 78, 87,
quartile, I 04
88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 100, 110,
questioning, 29
11 4, li S, 116, 11 7, 124, 126, 14 1, 142, 152, 153,
questionnaire, 16, 3 7, 131
155, 162, 166, 167, 173, 174, 177, 178, 179, 199
Polyunsaturated fatty acids ( PUFAs), viii, xi, 7, 47,
165, 173
R
population, viii, ix, 2, I S, 50, 5 1, 65, 96, 99, I 03,
11
106, 139, 140, 162, 163
radiation, I 05
population group, I 03
radius, 186, 187, 192
positive relationship, 54
reaction rate, 18 1
postpartum depression, 126
reaction temperature, 184
potassium, 88, 18 1, 198
reaction time, 184
potato, 131
reactions, 6, 148, 166, 173
poverty, 12 1
reactive oxygen, 35
prefrontal cortex, 84
reactivity, 85, 144
pregnancy, 86, I I 0
reagents, 132, 183
preparation, xi, 4, 19, 56, 105, 177, 18 1
rec.alt, 17, 132, 133, 135
preservation, I 06
reccptors, 4 , I0, 11 , 12, 32, 38, 45, 79, 84, 85
pretcrm in fants, 174
recommendations, 3 1, 50, 51 , 52, 65, 145
prevention, vii, x, 29, 43, 50, 52, 60, 61 . 66, 7 1, 93,
recovery, xii, 18, 19, 31 , 178, 195, 196, 198, 202
11 0, l iS, 11 9, 140, 143, 144, 148, 15 1, 152, 153
rectum, I 04
primary cells, 5
recycling, 14 , 95, 197
primary pathogenic !actors, vii, I
regression) 152
primate, 5, I 9, 36, 37, 40
regression analysis, 152
proc.edural mcmOI)', 23
reinfOrcement, 44
process control, I 82
relatives, 12 1
prodrugs, 44
relevance, 18, 25, 29, 37, 4 1, 46, 58, 11 8
pro li tability, 197
remission, 85, 90, 94, I 05, 127
prognosis, I 07
repetitive bchavior. 123
pro-inllammatory, 6, I 2, I 3, 20, 28, 83, 86, 93, I 0 7,
requirements, 59, 15 1
12 1, 130
researchers, 123
proj ect, I I 4
residucs, 169
proli feration, 6 I, 7 1, 84, 93, I 06, I 07, I 09, I 15
216
Lndex
resistance, 87, 180

resolution, 7, 14, 95, 191
resources, 173
respiration, 58
response, I I , 13, 19, 20, 49, 51 , 54, 61 , 84, 85, 92,
94, 105, 109, 113, 120, 138, 139, 14 1, 152, 154,
162, 196, 202
retardation, I 19
rctic.utum, 78
retina, 8, 78, 87, 95

retinoblastoma, 97
mcumatoid arthritis, 64, 72
rickets, 69
risk, ix, 6, 7, I I , 15, I 6, I 7, 29, 30, 33, 34, 37, 39,
40, 50, 51 , 52, 55, 56, 66, 76, 99, 100, 101 , 102,
104, 10 7, 110, I l l , 112, 113, 119, 12 1, 130, 138,
144, 145, 148, 151 , 152, 163, 166, 197
risk factCirs, 130, 138, 144, 148
risks, 7, 41, 65, 11 0, 185
nspcridone, 122
R:'\IA, 167
rodents, 3, 6, 11, 14, 22, 40, 9 1
rods, 157
room temperature, 156
routes, 27
1________.s..,_______.,.!I
111
safety, 140
salmon, 60, 169
sarcopenia, 17, 3 0
saturated Jlu, 15, 16, 25, 52
saturated fatty acids, 16, 25
~turation, 16
~hizophrenia, 8, 89, 90, 118, 12 1, 122, 124, 125,
126
~hizophrcnic patients, 12 1, 122
~iencc, 38, I 15
, I?
~o pe., o , seafood, x, 50, 93, 155, 162
secretion, 85, 11 8, 145
seed, 56
se! k stcem, I I 9
~nsitivity, 6, 19, 36, 62, 72
scptum, 4
Serbia, 99, I 09
serotonin, 4, 5, 22, 45, 79, 83, 89, 118
scrum, x, xi, 57, 60, 76, 77, 92, 95, I 03, 113, 115,
11 9, 126, 138, 14 1, 145, 146, 148, 149, 152, 155,
156, 159, 160, 162
shape, 90
shear, 60
shell lish, 50, 145, 153
showing, viii, 24, 26, 47, 55, 58, 59, 76, 86, I 05,
I 07, 138, 160, 169
shrimp, 50
sidecflbcts, vii, viii, 1, 7, 18, 75, 105, 120
signal transduction, 81, 83, 97, I 07
signaling pathway, I I
signals, I 15
signs, 40, 4 1
silver, 179, 199
simulation, 182, 200
Singapore, I 02, I I I
skeletal muscle, I 06, I 14
skeleton, viii, 47, 56, 58, 59, 62, 64
skin, 88
sleep disturbance, I 18
smoking, 100, 147
smooth muscle, I 07, I 15
smooth muscle cells, I 07, I 15
social phobia, I 19
society, 8, 19, 49
sodium, 82, 88, 90, 97, 147
soJt warc, 158, 200
sol id state, 95
solution, 24, 157, 187, 19 1
Spain, I 12, 200
spatial memory, 23
specks, 18, 27, 34, 35, 36, 39, 42, 50, 95, 145, 167,
175
speech, 120, 12 1
spinal cord, 19
spine, 54, 90
spleen, 60
Spraguc-Da" 1ey rats, 60
sprouting, 4, 36
squamous cell, 114
stability, 106, 118
stabilization, 83, 85
stabilizers, 127
standard deviation, 123, 158, 159, 160, 161
standardization, 31
stal"
' 11' "') 10 7' 146 184' 186 19'~. 197
statistics, 126
stc n cells 48 ' 9 60 7 1
I
' " '
steroids, 108
sterols, 90, 140
stomach, 102
storage, 84
stress, 5, 14, 22, 26, 31 , 59, 92, 95, 147
striatum, vii, I , 3, 4, 5, 14, 21 , 23, 25, 26, 28, 29~ 35,
"'
38, 43
stroke, 130, 148

stn iCturc, 9, I 0, I I , 14, 55, 62, 64, 70, 79, 8 1, 92, 96,
100, 106, 11 7, 167
subac.utc, 42
substratc, 27, 83, 85, 145, 168, 170, 171, 172
substratcs, xi, 165, 167, 172, 173
suicidal ideation, 11 8, I 19
suicide, 76, 85, 87
Sun, 68, 72, Ill, I 12, 165, 166, 168, 170, 172, 174,
20 1
supplementation~ viii, ix, x, 14, 15, 2 1, 30, 34, 41,
47, 52, 53, 54, 56, 5 7, 58, 59, 62, 64, 65, 67, 68,
70, 7 1, 72, 73, 75, 76, 77, 8 1, 86, 92, 93, 100, 105,
106, 107, 109, 110, 113, 114, 115, 11 9, 12 1, 123,
2 17
Lndex
124, 125, 126, 129, 130, 133, 138, 139, 14 1, 146,
147, 148, 149, 151 , 152, 153, 162, 174
suppression, 86, 153
survival, ix, 11, 14, 28, 31 , 32, 46, 60, 86, 87, 99,
106, I 12, I 14, I 15
survivors, 147, 149, 151
susceptibility, 18, 26, 38, I l l
S\\'C8l , 55
Sweden, 112, 113
Switzerland, 132
symptoms, 2, 7, 16, 17, 18, 76, 106, 119, 120, 12 1,
122, 123, 125, 126, 159
synapsc, 14, 86
synaptic plasticity, I I , 12, 81, 89, 9 1
synaptic transmission, I I , 14
syndrome, 4, 39, 106
syncrgistic. effcc~ 14
synthesis, xi, 3, 13, 78, 83, 85, 94, 97, I 00, 107, I 16,
11 8, 130,145, 146, 165, 167, 169, 173, 174,20 1
systolic blood pressure, 134, 147
toxic eflbct, 27, 30, I 00

toxicity, 6, 25, 26, 28, 29, 30, 34, 35, 39, 43, 83
toxin, 42, 43.45
trafficking. 14, 8 1, 85
training, 54
ll
trajectory, I 09
transcription, 11, 12, 14, 84, 85, 91 , 94, 107, 110
transcription factors, I I , I I 0
transduction, 58, I 06, 119
transesterillcatioo, xii, 141 , 178, 18 1, 183, 195, 198,
20 1
transference, 180
transformation, ix, I 00, I 07
transmembrane region, 169, 170
transmission, xi, I I S~ 177, 178
transport, I 0, 83, 87, 97, 186
lrCatmCnl, viii, ix, 2, 7, I0, 17, 18, 20, 2 I, 22, 23, 24,
25, 26, 28, 29, 30, 3 1, 39, 4 1, 42, 43, 44, 55, 6 1,
62, 75, 76, 77, 78, 86, 87, 88, 89, 9 1, 92, 93, 94,
96, 102, 105, 106, I ll , 114, 118, 119, 120, 12 1,
U2, 123, 124, 125, 126, 127, 129, 130, 132, 133,
138, 13;. 1 54, 1 ~6, 1 74, 1 78, 1 83
I
Ill
tremor, 2, _ I, 22, 4'
trial, 52, 53, 77, 92, 96, 110, I l l , 114, 115, 119, 120,
tachycardia, 149
tardive dyskinesia, 122
target, 3, 6, 23, 32, 50, 83, 125
tartrate-resistant acid phosphatase, 63
121 , 122, 123, 125, 126, 127, 131 , 144, 151

lriggcrs, 7, 4 1
triglyccrides, x, 129, 133, 138, 139, 146, 149, 160,
163, 178, 182, 184, 199
tryptophan, 168
Tuke)' HSD, 132
tumor, ix, 32, 35, 62, 80, 82, 85, 96, 99, I 06, I 07,
11 0, 115, 118, 130, 134
tumor cells, 96
tumor growth, I 0 7, I 15
tumor necrosis Ja ctor, 32, 62, 80, 82, I 07, I 18, 130,
134
tumorigenesis, I 14, I 15
tumors, 107
turnover, 3, 4, 20, 2 1, 23, 24, 51 , 54, 57, 63, 94, 127
type 2 diabetes, 72, 146, 149
tyrosine, 3, 22, 28
T)TOSinc, 3
tyrosme
' hd
y roxyI asc, .),
' __
n
36
TCC, 168
tau,
technica l assistanc.c, 162

techni ques, 163, 184, 200
technologies, 18 1
technology, 179, ISO, 200
teeth, 55
telephone, 13 1
temperature, 13 1, 15 7, ISO, 18 1, 184, 186, 187, 189,
19 1, 193, 194, 195, 196, 197, 198, 200
temporal lobe, 23
tcnsions, 80
tc1111inals, 3, 82
lCSting. vii, I , 19, 3 1, 132
thalamus, 37
Ultrapcutic. benellts, 199
U1crapcutic eHbcts, 120, 125
l.herapeutic.s, vii, I , 23, 85
therapy, vii, viii, I , 2, 6, 7, 8, 48, 54, 55. 75, 76, 90,
97, 100, 105, 106, 109, 114, 120, 14 1
them1al decomposition, I SO
lhoughts, 121 , 122, 123
lhrombosis, 144, 146, 152, 155
lhromboxancs, SO, 83, 85
tissue, 6, 7, 11, 23, 51 , 55, 58, 59, 67, 78, 84, I 04,
106, I l l, 113, 146
DIF, 6, 62
DIF -alpha, 35, I 18, 127
klOth, 52, 66
mal cholesterol, 146, 154, 157, 162
mal energy, I 06
11
Ldcerative colitis, I I 0
underlying mechanisms, ix, 65, 117, 130

unhappiness, I IS
United, 50, 144
United Kingdom, 50
United States, 50, 144
L~ca. 199
urine, 55, 56
US Department of Health and Human Services, 65
USA, 38, 44, 45, 117, 132, 152, 154, 157, 199, 200,
202
218
Lndex
vac.u um,xi, 177, 179, 18 1, 197, 199

validation, 42
vapor, 179, I SO, 18 1, 185, 186, 187, 19 1, 193, 194,
195, 200
\ariables, xii, 20, 2 1, 22, 86, 89, 140, 178, 180, 18 1,
182, 184, 186, 189, 190, 19 1, 196, 199
vascular cell adhesion molecule, 148
vasoconstriction, 83, 146
vasodilation, 144
vector, 170, 17 1
vegetable oil, 12, 130, I 78
vegetables, 130, 13 1
VEGF expression, I 07, I 15
' cin, I 18, 148, 149
venipunclurc, 132
-.cntrieular tibri!Jation, 14 7
-.cntrieular tachyc.ardia, 153, 174
\Crtcbrac, 54, 58, 63
wall temperature, 197

Washington, 5 1, 65, 68, 124
water, 20, 23, 40, 110, 156, 15 7, 168
weight gain, viii, ix, 75, 99, I 18
weight loss, ix, 67, 99, I 05, I 06, I 14
well-being, 64
Western countries, I 00, I 02
wood, 162
working condilions, ISO

working memory, 40
World Health Organization (W HO), 49, 50, 66, I 09,
11 9, 145
\\'Orld"i de, 50, I 00, I 09, 118, I 19
worry, 123
11
yeast, xi, 165, 168, 170, 171, 172, 173, 174

yield, 182, 197, 20 I
vesicle, 14 82, 9 7
1
viscosity, 180, 192, 193, 197

vitamin C, 16
vitamin D, 52, 69
vitamin E, 16, 58, 59, 64, 115
\itamins, 16
VLDL, 138, 145, 146
'olatility, xii, 178, 180, 185, 186, 195, 198
' ulncrability, 37, 40
Ill!
young women, 88
11
zinc, 61

Eicosapentaenoic Acid: Sources, Health Effects and Role in Disease Prevention

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JSB N: 978- 1-62257-500-8 (eBook)

Published by Nova Science Publishers, Inc. !'New York

Omega-3 Po lyunsaturated Fatty Acid T reatment in Park inson's

Eicosapentaenoic Acid (EPA) and Bone Metabo li sm

EPA and DHA Antidepressant Eirect : Alone or Togethe r?

Eicosapentaenoic Acid: The Role in lvlalignant Diseases

Role o f Eicosapentaeno ic Acid in Menial Health

Combined Supp lementa tion with EPA-Ricb Fi sh Oil

Physio logical Role o f Eicosapentaenoic Acid (EPA) in Reduc ing

Hypocholestero lemic and Hypolipidem ic Effects of Emu lsified

ldenti tication and Characterization of n-6 and n-3 Fatty Acid

Fractionation and Concentration ofOmega-3 by lvlolecu lar

T heodore G . Bradley and Francisco P. Vargas

Theodore G. Bradley and Francisco P. Vargas

hyperlipidemia. Fi fty-eight healthy hyperlipidemic adults, were randomly assigned to

Theodore G. Bradley and Francisco P. Vargas

It is important to highlight that for obtaining a concentrate of omega-3, it is first

In: Eicosapentaeno ic Acid

ISBN: 978- 1-62257-480-3

O MEGA-3 POLY UNSATURATED FATTY A CID

Department of Pharmacology, Univers ity of Alberta, Edmonton, Canada;

Di rk W. Luchtman, Jeflrey A . Zidichouski and Cai Song

Omega-3 Pol yunsaturated Fatty Acid Treatment in Park inson's Disease.. .

2.2. 1\on-Dopamin ergic Ne uropathology

Di rk W. Luchtman, Jeflrey A . Zidichouski and Cai Song

2.3. Lewy Bodies

3. The MPTP Model of Parkinson's Disease

Omega-3 Pol yunsaturated Fatty Acid Treatment in Park inson's Disease...

3.1. M l'Tl' Mechanisms of Action

3.1.1. Oxidative Stress

Di rk W. Luchtman, Jeflrey A . Zidichouski and Cai Song

Omega-3 Pol yunsaturated Fatty Acid Treatment in Parkinson's Disease. ..

Dirk W. Luchtman, Jeflrey A . Zidichousk i and Cai Song

Omega-3 Pol yunsaturated Fatty Acid Treatment in Parkinson's Disease...

Brain lipid profile

Linoleic acid (LA) C18:2 n-6

A lo ha-linolenic acid (ALA) C16:3n-3

Stearido nic acid (SDA) c 18:4n -3

Gamma-Linolenic acid (GLA)

Eicosatelraer.o ic acid C20:4 n-3

Oihomo-gamma linolen ic acid (D G LA) C20:3n-6

E icosapent aenoic a-cid (EPA ) C20:5 n-3

A rachidonic acid (AA) C20:4n-6

Oocosalelraenoic acid C22:4 n-6

Oocosapentaeooic acid (OPA ) C22:5 n-3

Oocosapen taenoic acid (DPA) C22:5n--6

Dirk W. Luchtman, Jeflrey A . Zidichousk i and Cai So ng

5.1. Uptake and MetaboHsm of Omega-3 PUFAs

5.2. Function of Omega-3 PUFAs

Omega-3 Po lyunsaturated Fatty Acid Treatment in Park inson's Disease.. .

5.2.1. Mem brane Fluidity

5.2.2. Gene Expression

Dirk W. Luchtman, Jeflrey A . Zidichousk i and Cai So ng

5.2.3. Eicosanoid Production and Inflammatio n

Omega-3 Pol yunsaturated Fatty Acid Treatment in Parkinson's Disease. ..