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EICOSAPENTAENOIC ACID
SOURCES, HEALTH EFFECTS AND ROLE
IN DISEASE PREVENTION
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EICOSAPENTAENOIC ACID
SOURCES, HEALTH EFFECTS AND ROLE
IN DISEASE PREVENTION
THEODORE
G. BRADLEY
Al'\TD
FRANCISCO P. V ARGAS
EDITORS
New York
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CONTENT S
vii
Preface
Cha pter I
Cha pter 2
47
Cha pter 3
75
Cha pter 4
99
Cha pter 5
117
Chapter 6
129
Cha pter 7
143
Cha pter 8
155
VI
Contents
Chapter 9
165
Chapter 10
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
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
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
Chapter 1
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)
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
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.
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 ).
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).
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).
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
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.
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
tt,C
'''''' ,1
H, C
COOH
l
Docosah exaenoic acid rDHAl C22:6 n-3
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
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 ).
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.
2-rtes
pro..MIIdt
e.s.
PGf,
.(.series
kllllolllf~tt~es
v.g.
IOO{e ~nflammc;~toty
nu.
l
,_rillS
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
14
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.
15
16
or
~Popula ti on
subj ects
15 patiems/ 5
age/se.x.matched
COIHI'O!S
Case
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
Case.
comrol
comrols
... - 9 PD
cases m
. totat (thousands of
Prospe.cuve b'
- . )
PO at balo e1m..
51 PO cases in
Prospective total (5 .289 with
no PO
at baseline)
Dietary measures
Outcome
Citation
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
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
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
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 ).
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).
7.1. 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.
Model:
Citation
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
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
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
21
M odel:
Citation
DHA
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
Animal!cell
SH-SY5Y
cells and
pl'imary
midbrain
neurons
Model:
.
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,
si muh aneous i~
'I" IPP+.
\Vith
Citation
l uchtman
et al. ( nm
published
to date)
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.
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.
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.
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).
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.
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.
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) .
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.
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Chapter 2
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.
48
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
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.
50
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.
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
9 weeks
2 months
pre-OVX and
16 weeks
post-OVX
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
calcium-dcllcicnl
or c.aJciumadequate die t
1: I ratio of G LA
and EPA (diester)
5 weeks
14 weeks
~ e ffect on bo ne
mass
54
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].
55
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
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].
59
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]
I.
2.
3.
4.
5.
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
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
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.
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.
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].
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.
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
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.
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Chapter 3
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)
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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.
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.
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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).
79
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Carlos Horacio Laino, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
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 ).
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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).
83
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.
84
Carlos Horac io Laino, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
85
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Carlos Horac io Laino, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
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
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
87
in omega-3 fauy acid concentration and/ or invo lved mechan isms could vary as a
consequence of the medi cation.
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double-blind,
placebo-controlled
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98
Carlos Horac io Laino, Martin Gabriel Codagnone, lvlaria Femanda Podesta et al.
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
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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)
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
sunOowtr- oil
C ) \'ltW.\)
P1osragbmdint
PGD,
PCE,
ga.uuoa,.J.iuoltulc 01dd
(18:3n-6)
elong..~
v
c~
Unolnle :rodd
~0 :3
Prosm:laodiue
t-:;:s)
ror..
TXAa
.:15 dt>saturast
dihomo-;tUntllll-
A.racWdonic add
n 6)
(10 1 6)
L~
Ltucocrit:nt
I'-
l."" ll't'
t~Ot$
LT.-\,
LTC,
~clooxy;tnas~
l.TD,
PGD!
l'GE:
PGF,
PGJ,
TXA,
Lipu~~gortt:lt\11"
~~
~ftl''IO.l".$
Pro.sraglandint
ttu)"lnf''S
Lurotritnt
LT..I.;
LTB,
LTC,
LTD,
LIE,
pc;.n,
cox
.l S d ts.a cuaut
[kos-aitfrlltnok :add
(lO:~n-3)
PCE,
l'GI',
PCil.s
IX~
Elcos~ptnhtiiOi('
TXR,
add
(lO:Sn-3)
L.ipoxygenn<>t
nzrs
Stu.ridonic :tdd
(18: ~013)
alpha~llnoltnlc
arid
LOX
Leuco tritnt
LTA,
LTB,
LTC..
L TD;
LT,.
(18:3 n-3)
Co11nol;\. fiAXSH'd
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.
102
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.
103
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.
Cancer type
I. Acute lymphobla-stic
leukemia (A LL)
2 Acute m ye! oid
leukemia(AM L)
Bladder
Zuijdgeest-van
Leeuwen, 2002 [55]
adults
I. Pancreatic
2. Lung
3. Oesophagial
..
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%)
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
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 .
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
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].
107
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.
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11 6
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Chapter 5
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
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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).
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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.
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
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.
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
Competing I nterests
The authors declare that they have no compet ing interests.
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Chapter 6
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
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.
13 1
132
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
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
''
-.
Energy (kJ)
Fibr,: (g)
Chuleiiterul {mg)
Carboh~drate (%)
d iehl r~
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 ~
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
br P<0.05.
138
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.
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 anti- arrhythmic
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
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.
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and fish oi l n-3 long chain polyunsaturated fatty ac ids on the p lasma lipid profil e of
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reaction . .I Lipid Res. 1986;27 (1 ): 11 4-20.
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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.
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cereal. Eur .I C/in Nutr. 2004;58:503-9.
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[25] Buck ley R, Shewring B, Turner R, Yaqoob P, lvlin ihane AM . C ircu lating triacylglycerol
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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.
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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
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(31] Mart in LJ, Connell y PW, Nancoo D, Wood N, Zhang ZJ, Maguire G, et al. Cholesieryl
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(32] Schaefer EJ, Licbtenstein AH, Lamon-Fava S, Contois HH , Li Z, Rasmussen HE, et al.
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Vase Bioi. 1995;1 5: I 079-85.
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Chapter 7
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
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).
ro-3 PUFA due to the ir antithrombotic, ant i-i nflammatory and vasodilat ion propert ies
(Calzolar i et al., 2009).
145
146
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).
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.
148
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).
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.
Redu-.;;ing triglyoerides
levels
Anti-thrombo tic effect
Anti-arrhrthmatic effL'>
Anti-atherogenic eff~;_
Remarks
~1arine w-3
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)
tv1orris et at ( 1993)
Appd <tal., ( 1993)
B>nna e tal., ( 1990)
Ch risten s~n et al..
(l996)
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
Anlih)'Pertett.sivt
<ffttt
1
Anri.cllerogmic tfl"ea
11
,;\nli.Mtbythm..aric cfrec:t
1/
.....------:-------,
Cardio prottc:don
..
!5 1
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Chapter 8
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
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.
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."
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.
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
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)
2.03+0. 11 (')
1.880. 15 (NS)
3 1.03.5 (ns)
26.5+1.8 ( )
..
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)
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
..
(%)
4.68+0.35
4.82+0.26 (ns)
5.8 10.62 ( )
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)
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
..
..
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
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[5] Roche H. M., Gi bney M. J. (2000) . Effects of long-chain n-3 pol yunsaturated fatty acids
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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.
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
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[! 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
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[1 2] Japanese Patent: Registration Number JP, 3 11 8556,B.
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[ 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,
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[ 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
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Chapter 9
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.
E-mail address: j ong957 1@ korea.kr; T el: +82 31 299 1702, Fax: +82 31 299 1672. (C01Tesponding author)
166
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
!.c.
desaturase
18:1 oleic acid
A 12 desaturase
w3 desaturase
----------,1
(w6= n6)
~.c. 6 desaturase
(w3=n3)
' A 6 desaturase
1FA elongase
~ FA elongase
1A desaturase
20:5 eicosapentaenoic acid (EPA)
1 elongase
22:5 docosapentaenoic acid
1 elongase
5
FA
FA
f.c.desaturase
24:6 tetracosahexaenoic acid
1Peroxisomal ~-oxidation
Mammals
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.
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
[>rimer
Set
Primer Name
AsD6D X11ol-R
AsELOJIL5 Bam i-U-F
pY3-FAE
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
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
)),
extra fatty acid peak compared with the pYES2/pYES3-expressing control cells.
Standard
18:0
E.PA
- J.l
JlJJ.
STA
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
...
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
6 9 12
(" ' ' ))
14
))
~ 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 -'))
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
(" )
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
Identi ficat ion and Characterization ofn-6 and n-3 Fatty Acid Desaturase.. .
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
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-
Identi ficat ion and Characterization ofn-6 and n-3 Fatty Acid Desaturase.. .
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.
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.
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
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
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
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.
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
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
R1COOCH 2
~COOCH
Catalyst
+ 3CH3CHpH
HOCH2
HOCH
R3 COOCH2
HOCH2
Triglyceride
Glycerol
R, COOCH2CH3
R 2COOCH 2CH 3
~COOCH 2CH3
Ethyl esters
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.
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.
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
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
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].
185
Roller-wiper
Condenser
Heating
media -
Distillate
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
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;
187
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
component.
188
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
3.1.
From a mass balance for i and j components, the equations system is:
di
Ij
2::: Ik
dy
v=o.
(I I)
y = 0,
(12)
k= l
f}
j =
( 13)
j '
(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)
I; )!/<>;.
~J = ( I9
(17)
189
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)
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)
190
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
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
(26)
j=l
It is expressed as B =
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)
li
B = 0,
i; =
1.
(31 )
191
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
where:
v is the average speed of the fluid;
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
192
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)
op
- + \i'pv = 0.
ot
(37)
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)
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)
(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)
193
(44)
where,
D, W and F are the molar flows of distillate, res idue and feed respect ively,
c;
Pr =
1\!!M
L'"'fj
Pvi
X;
. I
=
i'vf
(46)
- '
wl1ere,
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.
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
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.,
195
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.
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
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
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.
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
4.2.
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.
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
Pablo Rossi, Nelson Ruben Grosso, Maria del Cannen Pramparo et al.
LD
M O~ I
Fl
/-,
,..
....
lXXX
01
"
./
w
(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
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.
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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,
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
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
community, 3 1, 89
comorbidity, 95, 122
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
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
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
,-------G
. ______. . I.
I
gait, 20
gallbladder, I I 5
g!l, I I , 162, 163, 178
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
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
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
11
kinase activity, 80
2 12
Lndex
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
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
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
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
111)1cc.ular mass, 11
rrol cc.ular structure, 86
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
l..________.o
._________..!l
Lndex
2 15
216
Lndex
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
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
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
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
36
TCC, 168
tau,
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
218
Lndex
11
vesicle, 14 82, 9 7
1
Ill!
young women, 88
11
zinc, 61