The role of n-3 dietary polyunsaturated fatty acids in brain function

and ameliorating Alzheimers disease: Opportunities for biotechnology

in the development of nutraceuticals
Agnieszka M. Falinska
a,b
, Ce cile Bascoul-Colombo
a,b
, Irina A. Guschina
a
,
Mark Good
b
, John L. Harwood
a,n
a
School of Biosciences, Cardiff University, Cardiff CF10 3AX, Wales, UK
b
School of Psychology, Cardiff University, Cardiff CF10 3AT, Wales, UK
a r t i c l e i n f o
Article history:
Received 3 June 2011
Received in revised form
19 August 2011
Accepted 2 September 2011
Available online 17 September 2011
Keywords:
Dietary docosahexaenoic acid (DHA)
Alzheimers disease
n-3 Dietary polyunsaturated fatty acids
a b s t r a c t
Prospective epidemiological surveys and numerous animal studies have shown an important role for
dietary docosahexaenoic acid (DHA) in healthy brain function and reducing the risk of dementia. The
evidence for this is summarised and some further experiments of our own are described. For the
experimentation we have used the Tg2576 mouse, which is a well known model of amyloid pathology
and cognitive impairment as seen in Alzheimers disease. We found that, while brain levels of DHA
showed a positive correlation with behaviour and a negative correlation with insoluble b-amyloid
(1-40), the general benets of DHA-enriched diets were not as great as have sometimes been reported
in the literature. This may be due to either the age of the animals we tested and/or the fact that we
examined the effects of DHA supplementation against a normal healthy control diet condition, unlike
previous studies, in order to mimic dietary supplement use in the human population.
In addition, we point to some ways in which biotechnology could be used to supplement the worlds
supply of omega-3 PUFAs (especially DHA) since there is an increasing shortage of such compounds for
dietary consumption.
& 2011 Elsevier Ltd. All rights reserved.
1. Dietary polyunsaturated fatty acids
Polyunsaturated fatty acids (PUFAs) of both the n-3 and the
n-6 series are essential dietary components for humans. This is
because the D12- and D15-desaturases required for their forma-
tion from 18C fatty acid precursors are absent in humans (Gurr
et al., 2002). Over the last few decades it has also been realised
that greater quantities of PUFAs (than are needed merely to
prevent essential fatty acid deciency) are necessary for good
health (Gunstone et al., 2007; Watson, 2009). In recent years,
particular emphasis has been placed on research into the possible
benecial effects of n-3 PUFAs and also on the ratio of n-6/n-3
PUFAs in the diet. The latter is because trends in average diets in
North America and Europe have dramatically raised the ratio to as
high as 20:1 (Simopoulos, 2001; Staessen et al., 1998) when the
optimal rate is usually considered about 4:1 (Barcelo-Coblijn
et al., 2003; Yehuda and Carasso, 1993; Yehuda, 2009).
The importance of the ratio of n-6 and n-3 PUFAs in the diet
derives from two facts:, rst, these two classes of PUFAs are
metabolised by the same enzymes and, therefore, compete for the
enzymic active sites; and, second, that (as a generalisation) n-6
PUFAs (arachidonic acid, in particular) give rise to pro-inamma-
tory metabolites (eicosanoids) while n-3 PUFAs produce eicosa-
noids, which are only mildly or anti-inammatory (Gunstone
et al., 2007; Funk, 2001; Schmitz and Ecker, 2008; Tapiero et al.,
2002). Furthermore, over the last decade it has been discovered
that n-3 PUFAs (eicosapentaenoic (EPA) and docosahexaenoic
(DHA) acids) can give rise to new classes of signalling lipids that
have acute anti-inammatory properties. Thus, EPA and DHA can
give rise to resolvins (that resolve acute inammatory episodes
(Serhan et al., 2008)), and maresins (that are also powerful anti-
inammatory and pro-resolving compounds) while DHA can also
be metabolised to docosatriene (or neuroprotectin D
1
), which, as
the name implies, is neuroprotective (Bazan, 2006).
2. n-3 PUFAs and human health
As summarised by Harwood and Caterson (2006), many of the
diseases where dietary n-3 PUFAs appear to have benet, are ones
with which chronic inammation is associated. This applies not
only to typical inammatory diseases such as Type I diabetes,
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1878-8181/$ - see front matter & 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bcab.2011.09.001
n
Corresponding author.
E-mail addresses: Harwood@cardiff.ac.uk, Harwood@cf.ac.uk (J.L. Harwood).
Biocatalysis and Agricultural Biotechnology 1 (2012) 159166
inammatory bowel diseases or rheumatoid arthritis (Sijben
et al., 2009) but also cardiovascular disease (CVD) (Harris,
2004), cancer (Rose and Connolly, 1999) and cognitive impair-
ment (Cunnane et al., 2009). Dietary n-3 PUFAs are also important
for neonatal health and development (Horrocks and Yeo, 1999;
Innis, 2000; Voigt et al., 2000) and the generation of a normal
immune system (Sijben et al., 2009).
While the bulk of dietary PUFAs are 18C fatty acids produced
by plants, it is the very long chain PUFAs (20 or 22C) that give rise
directly to active metabolites such as the eicosanoids. Humans
are not particularly efcient at converting dietary linoleic and
a-linolenic acids into tissue VLC-PUFAs and, in particular, the
production of DHA via the Sprecher pathway is inefcient with
estimates of 1% or so from a-linolenic acid (Barcelo-Coblijn and
Murphy, 2009). Whether dietary a-linolenic acid on its own can
supply all the n-3 PUFA requirement for humans is controversial,
although estimates of EPA and DHA production (particularly by
the liver) suggest that it could. This aspect has been discussed
thoroughly in a recent review (Barcelo-Coblijn and Murphy,
2009). Nevertheless, because many human diets have a large
excess of n-6 over n-3 PUFA and because it is EPA and DHA that
are needed for good health, most dietary guidelines now specify
the latter components as part of healthy diet and, particularly, for
the treatment of patients, such as those suffering from CVD
(Barcelo-Coblijn and Murphy, 2009). In fact, because DHA can
be retro-converted to EPA (Fischer et al., 1987), it has been argued
that DHA can be considered more important (Barclay et al., 2005).
Nevertheless, because we do not always know the efcacy of
particular n-3 PUFAs towards individual diseases, nor the speed at
which EPA and DHA can be interconverted, then a mixture of the
two would seem sensible as, indeed, is usually the case in practice
when sh or sh oils form the main source of the acids.
3. n-3 PUFAs and neuronal function
After adipose tissue, the brain has the next highest lipid
content (around 60% of the dry wt.) of mammalian tissues.
Furthermore, there is usually little triaclyglycerol with the brain
lipids consisting predominately of membrane constituents.
Because of myelination, derived from the plasma membrane of
the Schwann cell, brain lipids tend to be enriched in cholesterol,
sphingomyelin, phosphatidylserine, etc. Of particular note are the
relatively high percentages of the PUFAs, arachidonate (ARA)
(about 6% dry wt.) and DHA (about 8% dry wt.) in human brain.
n-3 PUFAs are implicated in neuronal function in the context
of pre-term or neonatal development to late-onset dementias.
In mammals, the period for ARA and DHA accumulation differs
depending on species. For humans it is the last trimester and the
rst 610 months after birth (Clandinin et al., 1980a; Clandinin
et al., 1980b). The accumulation of DHA reects a number of
crucial functions that it plays for normal neurological and visual
development (Barcelo-Coblijn and Murphy, 2009; Innis, 2007). In
that regard, it should be noted that, while breast milk appears to
provide sufcient DHA (or its precursors) for normal brain
development, the same may not be true for pre-term or infants
fed formula diets. Indeed, several studies showed that the lack of
DHA in some infant formulae leads to lower DHA levels in the
brain and other tissues (Barcelo-Coblijn and Murphy, 2009). For
this reason (and also as a result of animal experiments) many
manufacturers now include ARA and DHA in their milk powders
(Blank et al., 2002).
Large reductions in brain or retinal DHA are associated with
impaired cognitive and visual function in mammalian species in
general (Barcelo-Coblijn and Murphy, 2009). Moreover, n-3 de-
ciency leads to reduced brain DHA and increased n-6 docosapen-
taenoate, altered enzyme activities, depressed learning ability
(Wainwright et al., 1991), disturbed behaviour (Reisbick et al.,
1994) and water intake (Voigt et al., 2000). In addition, three
cross-sectional studies showed that regular consumption of sh
resulted in better cognitive performance on various neuropsy-
chological tests for middle-aged or older persons (Kalmijn et al.,
2004; Barberger-Gateau et al., 2005; Nurk et al., 2007).
While n-3 PUFA are needed for normal brain development,
reduced levels of them are also implicated in a host of neurolo-
gical disorders and mental health. These are summarised in
Table 1. Of these complaints, we will concentrate on Alzheimers
disease for the remainder of this paper.
4. Dementia and Alzheimers disease
With increasing longevity, cognitive decline (especially with
Alzheimers disease, AD) is becoming more and more prevalent.
About 60% of dementia patients suffer from AD. Depressingly,
once diagnosed, there is little prospect of improving the prognosis
of AD (Blennow et al., 2006). Hence, there is an urgent need to see
how lifestyle behaviours can be altered to delay the onset and,
perhaps, the speed of progression of AD. Dementia is associated
with memory disorder and impairment in at least one other
Table 1
n-3 Polyunsaturated fatty acids and neurological dysfunction.
Complaint Notes
Poor infant brain development Associated with n-3 PUFA, particularly DHA, decient diets
Infant cognitive ability Associated with decient diets
Attention decit hyperactivity disorder (ADHD) Plasma EPA, ARA and DHA lower in ADHD patients. 5 double-blind intervention trials with both n-3 and n-6
PUFAs successful
Dyslexia, autism Imbalance of n-3 PUFAs and low DHA reported. Intervention trials show some improvements
Depression Associated with lower dietary intake and plasma/serum levels of n-3 PUFA, esp. DHA. Intervention trials
generally unsuccessful
Bipolar disorder Plasma ARA and DHA lower than matched controls. Intervention helpful in 3 out of 8 trials but details of n-3
PUFA administration very different
Suicide risk Lower erythrocyte EPA, DHA, total n-3 PUFA. 2.1 g/day of EPADHA improved suicide scores
Schizophrenia Diets and erythrocytes were low in n-3 PUFA. Intervention trails with sh oil (or EPADHA) mostly successful
Aggression, hostility Low n-3 PUFA states associated with some groups. Reduction reported in some intervention trials with sh oils
or various n-3 PUFAs
Dementia, Alzheimers disease Increased with low n-3 PUFA intake. Some studies show low brain DHA levels. Intervention benecial in animal
studies and prospective trials but not convincing for patients with diagnosed Alzheimers or vascular dementia
See Sinn et al. (2009) for further details.
A.M. Falinska et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 159166 160
cognitive domain, both of which interfere signicantly with social
function or activities of daily living (Dubois et al., 2007). AD
patients typically show a gradual reduction in episodic memory
on neuropsychological testing. The decit is associated with
atrophy in the medial temporal lobe and/or hippocampus and
can be seen as volume reduction on brain imaging scans and as
abnormal patterns of glucose uptake with positron emitting
tomography. Neurobrillary tangles and senile plaques in the
medial temporal lobe structures and cortical areas are cardinal
neurological hallmarks of AD (Cunnane et al., 2009). The plaques
contain b-amyloid (Ab) peptides that are produced continuously
but also do not accumulate in healthy individuals.
According to the amyloid cascade hypothesis (Hardy and Selkoe,
2002), increased Ab production in familial (early-onset) and
decreased clearance of Ab in sporadic (late-onset) forms of AD are
central to pathogenesis (Cunnane et al., 2009; Mawuenyega et al.,
2010). Soluble forms of the proteolytic products of amyloid catabo-
lism, Ab140 and Ab142, are believed to be key to the onset of
neurodegeneration (McLean et al., 1999). The amount of insoluble
Ab, which makes up the plaques, distinguishes AD individuals from
controls but does not correlate with disease severity. In contrast to
Ab formation, the neurobrillary tangles (containing hyperpho-
sphorylated tau protein) are normally considered a later event but
may lead to more marked neuronal and cognitive impairment
(Cunnane et al., 2009). Synaptic dysfunction and neuronal apoptosis
are also found in AD.
Age is the major risk factor for AD, with prevalence doubling for
each decade beyond 60 years of age. Prevalence of AD for people
aged 85 years is about 25% (Blennow et al., 2006). Apart from genetic
predisposition (for early onset AD), other risk factors include reduced
brain rescue capacity (Cunnane et al., 2009) and the same condi-
tions as for cardiovascular disease (e.g. hypertension, atherosclerosis,
smoking, obesity). Dietary factors such as anti-oxidants and n-3
PUFA (particularly DHA) are protective (Gillette Guyonnet et al.,
2007; Gomez-Pincilla, 2008; Luchsinger et al., 2004). Nevertheless, it
should be born in mind that increased dietary DHA only seems to be
of benet for healthy individuals or for persons with mild cognitive
impairment (Cunnane et al., 2009). Once Alzheimers has been
diagnosed then it seems too late for a benecial effect of n-3 PUFAs
(Cunnane et al., 2009) and this has been conrmed in animal models
(Arendash et al., 2007).
A number of prospective epidemiological studies have shown
good correlations between sh (and/or n-3 PUFA) intake and a
decreased risk of cognitive decline, dementia or AD in the elderly
(Cunnane et al., 2009). Three examples, from The Netherlands,
France and the USA are shown in Table 2. In each of these studies
there was a very similar quantitative outcome i.e. 6070% less risk
of developing AD if sh or n-3 PUFA consumption was appreciable
during the period of the trial. With one exception (Schaefer et al.,
2006), all other prospective studies (12 in total) showed benet
(Cunnane et al., 2009) and even in the case of the Framingham
heart study (Schaefer et al., 2006) there were confounders
(Cunnane et al., 2009). For further information on the connection
of low dietary intake of DHA and increased risk of AD and other
forms of cognitive decline please refer to (Cunnane et al., 2009;
Fotuhi et al., 2009; Maclean et al., 2005).
The positive correlation of sh or DHA intake with better
cognitive function and less risk of developing dementia (Cunnane
et al., 2009), would suggest that brain levels of the important
constituent, DHA, might be reduced in different types of demen-
tia, including AD.
Cunnane et al. (2009) report data from 9 studies. Of these,
the most consistent effects were reductions of 3050% for the
hippocampus in free DHA, or its levels in phosphatidylcholine and
phosphatidylethanolamine by 3050% following post-mortem
analysis of hippocampus from AD patients (Lukiw et al., 2005;
Soderberg et al., 1991; Prasad et al., 1998). However, even for the
hippocampus the data must be interpreted cautiously because
dissection techniques, analytical methods, expression units for
lipid levels, and diagnosis of AD may differ between studies. No
consistent reductions in DHA were found in any other brain area
examined (Cunnane et al., 2009).
Apart from DHA, arachidonic acid (ARA), an n-6 PUFA, is a
major component of brain lipids. Signicant alterations in ARA in
the parahippocampal cortex of AD patients have been reported
(Corrigan et al., 1998) and, in particular, in the phosphatidyletha-
nolamine fraction from this brain area as well as the hippocampus
(Prasad et al., 1998). Soderberg et al. (1991) also found decreases
in ARA (as well as DHA) in the phosphatidylethanolamine fraction
from the grey matter and hippocampus of AD patients.
Indeed dietary supplementation with ARA, as well as DHA,
improves cognitive dysfunction Kotani et al. (2006).
However, despite the evidence for an important role of PUFAs
in brain function and cognition, dietary intervention has not
proven particularly useful for patients exhibiting AD symptoms,
e.g. Quinn et al. (2010). In contrast, these are benecial effects of
dietary DHA in improving cognition in age-related cognitive
decline in healthy older adults (Yurko-Mauro et al., 2010).
The persuasive link between dietary n-3 PUFAs and lower
cognitive decline in epidemiological studies has led to further
experiments aimed at nding the molecular basis (or bases) for
benets. Apart from the known effects of DHA on neuronal
conduction (Hashimoto et al., 2006), in reducing amyloid-b
accumulation (Calon and Cole, 2007), for beneting cardiovascu-
lar function (Balk et al., 2007), and reducing inammation and
oxidative stress (Cunnane et al., 2009), DHA gives rise to lipid
signalling molecules apart from the well-known eicosanoids
formed from other PUFAs (e.g. ARA, EPA). For instance, DHA can
form resolvins RvD
1
RvD
4
and neuroprotectin D
1
, which are
potent modulators of inammation and preserve brain activity.
The discovery of such mediators over the last decade has opened
an exciting chapter in lipid biochemistry.
Moreover, n-3 PUFA are also known to be important for
normal brain development (Cunnane et al., 2009; Innis, 2007)
while DHA, itself, has been shown to support neuronal survival
(Akbar et al., 2005) and to protect cultured neuronal cells against
damage from Ab (Florent et al., 2006).
5. Animal models to help study Alzheimers disease
Animal models have been developed to study the aetiology
and evolution of Alzheimers disease and potential therapies. In
Table 2
Examples of prospective studies showing benets of dietary sh or n-3 PUFAs in
reducing the risk of dementia.
Study Comparison Relative risk
Dementia AD
Rotterdam study (Kalmijn
et al., 1997)
419 g sh/day 0.4 0.3
Dietary fat and risk of dementia vs. o3 g sh/day
n5386, age 455
Bordeaux study (Bargerger-Gateau
et al., 2002)
Daily sh 0.34 0.44
Fish, meat and dementia risk vs. oweekly
n1416, age 467
Chicago (Morris et al., 2003) Weekly or more
frequent sh
Fish, n-3 PUFA and AD vs. none 0.4
n815, age 464
A.M. Falinska et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 159166 161
their studies, Hashimoto et al. (2002, 2005a, 2005b, 2006) used
rats that were injected the Ab1-40 protein into the cerebral
ventricle, as a model of the amyloid pathology. Transgenic mouse
models reproducing the Ab pathology have mainly been gener-
ated with a mutated human APP gene. The PDAPP mouse expres-
sing a human APP with the Indiana mutation APP
V717F
was the
rst transgenic mouse presenting with Ab pathology (Games
et al., 1995). The Tg2576 mouse was generated shortly after
(Hsiao et al., 1996) and then, other APP transgenic mice including
the APP23 mouse and the TgCRND8 mouse were also developed
(McGowan et al., 2006). Transgenic mice carrying a mutant APP in
combination with one or two other transgenes such as a mutant
presenilin 1 (PS1) or a mutant microtubule-associated protein
tau (MAPT) have also been recently produced (McGowan et al.,
2006). For example, the APPswe/PS1dE6 mouse expresses
mutants of APP and PS1, and the 3xTg-AD mouse expresses
mutants of APP, PS1 and MAPT.
There are several excellent reviews, which describe animal
models for studying Alzheimers disease, especially transgenic
mouse models (McGowan et al., 2006; Elder et al., 2010; Philipson
et al., 2010; Wilcock, 2010; Zahs and Ashe, 2010).
The Tg2576 mouse is perhaps one of the most commonly used
APP model in AD research. This model of Ab pathology was
developed in 1996 by Karen Hsiao and colleagues at the University
of Minnesota (Hsiao et al., 1996). Tg2576 mice carry a transgene
coding for a mutated 695 amino acid isoform of the human APP
(HuAPP
695
. K670N-M671L). As the mutation was found in a
Swedish family, it is also known as the Swedish mutation
(APPswe). It is a double mutation occurring on the amino acids
670 (LysAsn) and 671 (MetLeu). The gene of the human APP
695
containing the double mutation was inserted into a hybrid back-
ground of C57BL/6SJL mice using a hamster prion protein
cosmid vector. The resultant transgenic mice express the APP
mutant under the control of the hamster prion protein (PrP)
promoter. To produce the animals used in experiments, hetero-
zygous male mice expressing the Swedish double mutation in the
hybrid background of C57BL/6SJL were mated with female
C57BL/6SJL F1.
The Swedish mutation is known to cause an increased produc-
tion of both Ab1-40 and Ab1-42 (Cai et al., 1993; Citron et al.,
1994; Scheuner, 1996). Previous work with Tg2576 mice has
shown that these mice display both types of Ab with an age-
dependent Ab plaque deposition (Harigaya et al., 2006; Irizarry
et al., 1997; Kawarabayashi et al., 2001). Changes begin at 67
months of age. Between 6 and 10 months of age, when SDS
insoluble Ab1-40 and Ab1-42 are easily detected in every animal,
histopathology is minimal; only isolated Ab plaques can be
identied. By 12 months of age, diffuse plaques are evident,
and from 12 to 23 months, Ab plaques increase to levels similar
to those observed in human AD brains. In addition to the
development of Ab histopathology, Tg2576 mice also develop
age-dependent spatial memory decits in different tasks such as
Y-maze, T-maze and Morris water maze procedures (Games et al.,
1995; Chapman et al., 1999; Lalonde et al., 2003; Westerman,
2002). Similar to AD patients, they also exhibit signs of neuroin-
ammation evidenced by high numbers of plaque-associated
microglia and astrocytes as well as expression of inammatory
cytokines, including interleukin-1b, tumour necrosis factor-a and
interleukin-6 (Irizarry et al., 1997; Benzing et al., 1999; Frautschy
et al., 1998; Mehlhorn et al., 2000), oxidative stress (Pappolla,
1998; Smith, 1998), neuronal abnormalities (Irizarry et al., 1997;
Chapman et al., 1999) and tau phosphorylation (Kawarabayashi
et al., 2004).
As the Tg2576 mouse model shows many features of the
human AD pathology, it provides an opportunity to study the
effect of drug treatments on the Ab pathology in isolation.
However, Tg2576 mice do not develop neurobrillary tangles or
neuronal loss like human AD patients (Irizarry et al., 1997).
Although, this may be a drawback of the model, the absence of
these pathological features may also help to clarify the effects of
manipulations specically on Ab pathology, without the added
complication provided by, for example, the incorporation of tau
mutations that are not specic to AD.
6. Using the Tg2576 model to compare cognition
with biochemistry
In our experiments, we used Tg2576 mice generated and
genotyped as before (Lelos et al., 2011). Their wild type (WT)
littermates were used as controls. Four month old animals were
used and fed one of two dietsa control diet that contained
adequate PUFA levels and one that was enriched in DHA. The
importance of diet comparisons has been highlighted and dis-
cussed in the excellent review of Cunnane et al. (2009).
As noted above, the choice of diet is crucial for feeding
experiments and the only signicant difference should be the
content of DHA. Moreover, the total fat content should be similar.
Many feeding studies can be criticised because the control diet
may contain higher amounts of pro-inammatory (e.g. n-6
PUFAs) components and/or different amounts of total lipid
compared to the test diet. For these reasons we chose to supple-
ment a normal rodent chow (Special Diet Services, Essex, UK;
which had adequate PUFAs for good health) with a moderate
amount (5%) of additional lipid. One supplement diet was
enriched in DHA while the other contained a blend of fat (made
from lard, palm oil, olive oil, coconut oil in 3:3:3:1 by wt.), which
gave typical fatty acid percentages of the average UK diet. The
fatty acid composition of the two diets is shown in Table 3 and it
will be seen that the DHA in the test diet is mainly replaced with
palmitate and oleate in the control diet. The n-6/n-3 PUFA ratios
were 10.3 and 0.8 for the control and test diets, respectively. Thus,
they were equivalent to the lower end of the range for typical
Western diets and to someone taking DHA supplements,
respectively.
There were no signicant differences in body weight gain over
the treatment period between the WT or Tg groups whether on
control or DHA-diets. Final body weight for transgenics on the
DHA-diet was 29.970.5 g, transgenics on the control diet was
30.770.7 g, WT on DHA-diet was 32.170.8 g and WT on control
diet was 32.470.9 g. ANOVA was performed using repeated
measures 3-factor ANOVA with week of age as a repeated
Table 3
Diets used in the experiments.
Main fatty acids % Diet weight
Oil-blend DHA-enriched
12:0 0.0570.01 0.0470.01
14:0 0.087tr. 0.387tr.
16:0 1.6570.07 0.867tr.
16:1 0.037tr. 0.087tr.
18:0 0.3770.01 0.077tr.
18:1 n-9 2.8770.13 1.6670.05
18:2 n-6 2.0670.09 1.6070.02
18:3 n-3 0.2070.01 0.1770.01
22:6 n-3 n.d. 1.8670.04
Total n-3 PUFA 0.2070.01 2.4070.03
Total n-6 PUFA 2.0670.09 1.6070.02
n-6/n-3 ratio 10.3 0.8
Data show means7S.E.M. (n3). n.d.: none detected, tr.: trace (o0.005). Both
diets contained 8% fat.
A.M. Falinska et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 159166 162
measure and diet and genotype as between-subject factors. None
of the above factors produced signicant differences nor were
there any signicant interactions.
During the feeding period, four Tg mice died in the DHA-fed
group and three Tg mice on the control diet. Of the WT mice there
was only one deathon the control diet.
Although the mortality rate was higher in the Tg mice, it was
not statistically signicant by the Fisher exact test (p0.123).
Increased mortality rates for Tg2576 animals have, however, been
noted before. Inclusion of DHA in the diet caused an increase of
approximately 18% in the amount of this fatty acid in the cortex,
hippocampus and cerebellum compared to mice on the oil-blend
diet by the end of the experiment.
Behaviour testing used a forced-choice alternation (FCA) task
in a T-maze, which is a well-established behaviour test that
assesses spatial working memory. Most of the earlier work, which
established the memory requirements of FCA and its relevance to
hippocampal and pre-frontal cortex function, was conducted in
rats. The method was later successfully adapted for use in mice to
detect memory defects (Chapman et al., 1999).
Fig. 1 shows results for the experiment. Training was for 10
days and each mouse underwent 6 trials per day. The percentage
correct choice for each mouse was calculated daily. Repeated
measures 3-factor ANOVA showed a highly signicant effect of
genotype, indicating that Tg mice were signicantly impaired.
The time of training had a signicant effect (F8.91, po0.0001),
conrming an improvement during training (except for Tg on control
diet). There was no main effect of diet except when the interaction
between genotype and diet was considered. A signicant result
(F5.3, po0.025) was found indicating a protective effect of the
DHA-enriched diet for Tg mice. Post-hoc Tukeys pair-wise compar-
isons conrmed that Tg mice on DHA diets performed signicantly
better (po0.05) then Tg mice on control diets. It can be noted in
Fig. 1 that the Tg animals on DHA diets improved at the same rate as
WT mice but that Tg animals on control diets showed little, if any
improvement, during the 10 days of the experiment.
The percentage of mice reaching the learning criterion
(dened as 80% correct or better on at least 3 consecutive days
during training) was not signicantly affected by DHA adminis-
tration. A smaller percentage of transgenic mice reached the
learning criterion relative to non-transgenic littermates. Thus,
although transgenics fed DHA were clearly able to improve during
training (Fig. 1), DHA did not inuence the numbers attaining the
80% learning criterion.
Fig. 2 shows results obtained from Sandwich ELISA analysis of
Tg2576 mice of soluble or insoluble b-amyloid 1-40 and 1-42.
Non-transgenic mice are not included because they only show
traces, as previously reported by Kawarabayashi et al. (2004).
Further our ELISA method is specic for the human peptides, as
produced in transgenic animals only. As can be seen in Fig. 2 there
were no signicant differences caused by diet. As expected, the
amyloid 1-40 fragment was more abundant than the b-amyloid
1-42 peptide.
Fig. 1. Effect of diet or genotype on the learning ability of mice in the T-maze.
Transgenic mice were bred and genotyped using the procedures detailed in Lelos
et al. (2011). Feeding began at 4 months age and the mice were tested (Chapman
et al., 1999) at 9 months age. Testing consisted of six trials per day for 10 days.
Mice failing to complete more than 50% of the trials in the rst 3 days were
excluded from the study. Tg, Tg2576 mice; WT, wild-type litter mates; con, control
diets; DHA, DHA-enriched diet. Repeated measures ANOVA revealed a highly
signicant effect of genotype (f 68.6, po0.001) and a signicant effect of diet
with genotype (f 5.3, po0.025). The effect of time (days of training) was highly
signicant for all subjects (f 8.91, po0.0001). Post hoc analysis using Tukeys
pair-wise comparisons revealed that Tg2576 mice on either diet did less well than
their age-matched control littermates (po0.001). Transgenic mice on DHA-
enriched diets performed better than these on the control diet (p0.049).
Numbers of mice used were non-Tg control diet13, non-Tg DHA diet14, Tg
control diet12, Tg DHA diet16.
Fig. 2. b-Amyloid levels in transgenic Tg2576 mice aged 9 months. For details of experimentation, see (Lelos et al., 2011). Soluble (SDS) and insoluble (FA) levels of
b-amyloid 1-40 and 1-42 were analysed separately and values for individual mice are shown. The mice were fed from 4 months of age on diets as detailed in Table 3.
A.M. Falinska et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 159166 163
Because we had failed to see statistically signicant alterations
in behaviour or b-amyloid levels with diet (albeit that trends
were obvious, Fig. 1), we examined individual mice. Fig. 3 shows a
regression analysis for fatty acids (arachidonate (AA), DHA) in
brain extracts plotted against behaviour in the T-maze. There was
a positive correlation between the averaged learning score for the
last 4 days and the DHA/AA ratio in total fatty acids. Furthermore,
individual mice with higher AA levels showed a negative correla-
tion (p0.045). In addition, there was a negative correlation
between insoluble b-amyloid, 1-40 (the main accumulating
b-amyloid: see Fig. 2) levels and the %DHA in individual mouse
brains (p0.047).
The behavioural results show that dietary n-3 PUFAs can
ameliorate the decits in spatial working memory displayed by
Tg2576 mice. Nevertheless, the ability to learn to a high learning
criterion was not improved by the diet. However, the improve-
ment in acquisition indicates that DHA had benecial effects on
performance in Tg2576 mice.
When we examined DHA levels in the brains of Tg2576 mice
and compared it to behaviour we found a signicant positive
correlation with higher DHA/AA ratios and a negative correlation
with AA levels (Fig. 3). This agrees with the generally accepted
conclusion that DHA and a high DHA/AA ratio in brain is
important for healthy functions (Bazan, 2006; Harwood and
Caterson, 2006; Cunnane et al., 2009; Hashimoto et al., 2006).
Previous studies have shown that raised levels of dietary n-3
PUFAs can reduce amyloid burden in APP overexpressing mice
(Hashimoto et al., 2006; Lim et al., 2005) and indeed DHA may
further enhance the amyloid reducing properties of other dietary
compounds (Giunta et al., 2010). In keeping with these results, we
found lower insoluble b-amyloid (1-40) levels when mouse brains
had higher DHA levels (Fig. 4).
However, in contrast to previous studies, we did not observe a
general effect of diet on specic amyloid levels. This may well
reect the large differences in the age of the animals (present
study, 9.5 months: Lim et al. (2005; 22.5 months). One possible
mechanism for the behavioural improvement in DHA-treated
mice is via DHA-related improvements in synaptic function.
Synaptic transmission and plasticity is compromised in Tg2576
mice in an age-dependent manner (Jacobsen et al., 2006) and DHA
has been shown to improve hippocampal synaptic plasticity (Cao
et al., 2009; Su, 2010; Oster and Pillot, 2010).
7. Prospects for biotechnological solutions to the shortage
of n-3 PUFA
Given the very strong evidence that raising dietary levels of
n-3 PUFAs, especially EPA and DHA, is of benet in reducing
dementia (Cunnane et al., 2009) and knowing that the supply of
sh oil (as the main source) is limited, there is an urgent need to
look for alternative sources (Venegas-Caleron et al., 2010).
Farmed sh require a source of n-3 PUFA in their diets, even
for fresh-water species, which have some capacity to produce EPA
and DHA from dietary a-linolenate (ALA) (Sargent, 1997; Tocher,
2003). However, all available evidence shows that, even with
substantial ALA in the diet, the accumulated levels of DHA in sh
oils are never as high as for sh fed sh oils or growing wild
(Stubhaug, 2005).
An obvious source of EPA and/or DHA is to use the primary
sources of these fatty acids, namely microorganisms. Accordingly,
there have been considerable efforts made to nd suitable species
that can be grown economically (Cohen and Ratledge, 2005). For
certain specialist formulations (e.g. baby foods, nutraceuticals)
the cost of producing n-3 PUFA is sufciently low for microbio-
logical culture to be useful (Barclay et al., 2005).
However, microalgal (and similar) mass culture systems are
too expensive for bulk production of EPA or DHA, such as would
be needed in agriculture or to satisfy medically recommended n-3
PUFA levels for human populations. Many of the systems devel-
oped have also proven difcult to scale-up and have a signicant
environmental footprint (Venegas-Caleron et al., 2010).
In the long-term, therefore, the only really feasible option is to
genetically engineer plants to produce EPA or DHA (Venegas-
Caleron et al., 2010). However, there are two main problems. First,
the conversion of ALA to DHA, even by the simplest pathway,
involves three desaturases and two elongases (Fig. 5). Secondly, in
general the desaturases use complex lipids as substrates while the
elongases use acyl-CoAs. This means that, during the synthesis of
DHA, there is a contrast swapping of acyl chains from one substrate
class to the other. This is believed to be the major constraint for
efcient DHA function. To date only small amounts of DHA have
Fig. 3. Correlation between brain fatty acid composition and behaviour. Lipids
were extracted (Garbus et al., 1963) and aliquots used for quantitative and
qualitative measurement by GLC (Guschina et al., 2011). Mice were 9 months
old when tested and has been on their diets (Table 3) for 5 months. Regression
analysis of T-maze behaviour and mice brain fatty acid percentages revealed a
positive correlation with AA percentage (p0.045).
Fig. 4. Higher brain percentages of docosahexaenoic acid correlate with reduced
insoluble b-amyloid 140. Mice were fed their diets (Table 3) from 4 months of
age and analysed at age 9 months. The correlation graphs were prepared using the
linear trend line function in Excel and regression analysis gave a p value for
correlation of 0.047.
A.M. Falinska et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 159166 164
been produced in engineered plants (Venegas-Caleron et al., 2010),
the best being about 3% of total oil fatty acids (Kinney et al., 2004).
For EPA, with two less steps in its biosynthesis, the best yields
have approached 20% (Kinney et al., 2004). In a recent review of
transgenic plant oils as alternatives to sh oils, Venegas-Caleron
et al. (2010) have discussed a number of strategies for increasing
yields of EPA and DHA, as well as minimising the build-up of
undesirable intermediates. Of course, in some parts of the world,
such as Europe, there is a strong consumer lobby against the use
of transgenic crops. This may well prove a severe disincentive for
crop development for quite some time.
There are a few other biotechnical advances, which are being
developed or may be shortly. These include the use of krill as a
new source of n-3 PUFA (Watkins, 2007). This is already con-
tributing to the overall supply but cannot provide a long-term
solution because of the amount needed. Methods to enrich sh
waste, using enzymatic methods, have been developed (Sun et al.,
2002; Zuta et al., 2003) and could be scaled up to more efciently
use material that is often discarded. Finally, the polyketide
synthase pathway, which is used by Schizochytrium to produce
DHA (Barclay et al., 2005), could also be used in transgenic crops.
By these various means we should be able to better ensure
sustainable production of EPA and DHA. In turn this will provide
materials for a better and healthy diet.
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