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National Food Research Institute, Kannondai, Tsukuba, Ibaraki 305-8642, Japan
3To whom correspondence should be addressed. E-mail: hirasuzu{at}nfri.affrc.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • docosahexaenoic acid (DHA) • maze-learning ability • brain fatty acids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The effects of dietary PUFA on the visual system, where
DHA is a major component of photoreceptor membrane, have been
extensively investigated (Benolken et al. 1973
,
Birch et al. 1992
, Christensen et al. 1998
, Neuringer and Conner 1986
). Findings from
these studies have suggested that DHA is required for the normal
functioning of the visual system, particularly the photoreceptor, the
retina and the visual areas (Neuringer et al. 1994
).
Delion et al. (1994
and 1996
) suggested a relationship
between the modifications in lipid composition of cerebral membranes
that occur in both (n-3)-deficient rats and old subjects and the
observed alteration of the monoaminergic function and cognitive
deficits. Previous studies on the role of DHA in cognitive function
have been inconsistent. Willatts et al. (1998
) reported
higher means-end problem-solving scores in term infants fed a
formula containing DHA and AA. However, Wainwright et al. (1999
) did not find any relationship between brain fatty acid
composition and performance on a working-memory task in rats in the
Morris water maze.
In our previous work with mice, maze-learning ability was increased
after feeding with fish oil that contains large amounts of DHA
(Suzuki et al. 1998
). More recently, we demonstrated
that the intake of DHA improved the learning ability in both young and
old mice, but old mice had a poorer learning ability than young mice
(Lim and Suzuki 2000
). Furthermore, our results have
shown that old mice had a lower DHA level in brain phosphatidylcholine
(PC) than young mice (Lim et al. 2000
). We suggested
that these lower levels of DHA in brain PC in old mice were associated
with an inferior learning ability and perhaps due to an influence on
synaptic membrane fluidity. Based on our previous studies, we
questioned whether there is a relationship between the intake period of
DHA and maze behavior in mice. In this study, we attempted to determine
the period of intake required to improve this learning ability after
the incorporation of DHA into the brain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Crj:CD-1 mice aged 3 mo were used. All mice originated from the
same colonies and were obtained from Charles River Japan (Atsugi,
Kanagawa, Japan). DHA (DHA-95E, ethyl ester derivative of all
cis-4,7,10,13,16,19-DHA, 95% pure) was obtained from
Harima Chemicals (Tsukuba, Japan). Eighty mice were randomly divided
into two dietary groups: a group fed 5 g palm oil/100 g diet
(control group) and a group fed 2 g DHA ethyl ester/100 g diet
plus 3 g palm oil/100 g diet (DHA-EE group). The 40 mice in each
dietary group were divided into four groups of 10 according to the
feeding period: a group fed for 1 wk, a group fed for 2 wk, a group fed
for 1 mo and a group fed for 3 mo. Diets contained 5 g lipids/100
g diet and are presented in Table 1
. The main fatty acid composition of the lipids in each diet group is
presented in Table 2
. The palm oil and DHA-EE diets contained 5 and 3 g/100 g
triglycerides, respectively. There was no phospholipid in the palm oil
and DHA-EE diets. The palm oil contained no ethyl esters. The diets
were stored at -25°C, and fresh portions were fed to the mice every
2 d. All diets were handled so as to minimize oxidation of the
fatty acids. Mice consumed the diet and water ad libitum. All mice were
housed in a standard environment, in which temperature was maintained
at 24 ± 0.5°C, and the relative humidity was kept at 65 ± 5% with 12-h periods of light and dark. Body weights were measured
once a week. All mice were maintained according to the guidelines for
experimental animals of the National Food Research Institute, Japan.
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To determine maze-learning ability in mice, a video tracking and
motion analysis system (EMTEC; Tama, Tokyo, Japan) was used. The
analysis system, which measured the rapid real-time picture
acquisition, was previously described by Lim and Suzuki (2000
). A program to monitor the pattern of animal movement and
the time spent getting from the maze entrance to exit was adopted. This
program allowed direct recording of the X-Y coordinates
of mouse movement on a computer disk file. The conditioning of all mice
involved training mice to drink water and was carried out using a
simple maze of three partition walls before each maze-learning
ability test. Maze-learning ability was assessed at 1 and 2 wk and
1 and 3 mo after the start of the feeding experiment. The first maze
trials (trial 1) were conducted after 24 h of water deprivation so
that the thirsty mice sought water that was placed outside of the maze
exit. Each mouse was allowed to drink water after reaching the exit.
Trial 2 was performed under the same conditions on d 4 after the first
trial, and trial 3 was conducted on d 4 after the second trial. The
time required to reach the exit, the number of times that a mouse
strayed into blind alleys and the behavior of a mouse in the maze were
measured.
Preparations of plasma samples and brain homogenates.
After the last maze-learning examination for each diet period, all mice in the group were deprived of food for 24 h before being anesthetized with diethyl ether. Blood was then collected from the inferior vena cava, and the mice were killed by decapitation. The blood plasma was separated by centrifugation at 900 x g for 20 min at 4°C. The whole brain of each mouse was rapidly removed and homogenized in ice-cold 0.32 mol sucrose/L (9 mL/g tissue) using a Teflon-glass homogenizer. The blood plasma and brain homogenates were kept at -25°C until required for fatty acid analysis.
Fatty acid analysis.
Total lipid was extracted according to the method of Bligh and Dyer (1959
).
Samples of the plasma and brain were extracted in
chloroform/methanol/water (1:2:0.8, v/v/v). The methanol/water phase
was extracted again with chloroform. KCl (8.8 g/L) was added to the
combined extracts, and sufficient time was allowed for the lipid phase
to separate from the aqueous phase. Water left in the lipid phase was
removed by the addition of Na2SO4, and the
lipid phase was then dried under nitrogen. Methylation of fatty acids
was carried out according to the American Oil Chemists’ Society
official method Ce-1b-89 (American Oil Chemists’ Society 1998
). NaOH (0.5 mol/L) was added to the lipid fraction and
heated at 100°C for 5 min. After cooling, the lipid was heated with
boron trifluoride-methanol reagent (140 g/L) in a sealed vial at
100°C for 30 min. The fatty acid methyl esters (FAME) were extracted
with hexane, dried under a stream of nitrogen, redissolved in hexane
and stored at 4°C until analysis. The FAME were separated by gas
liquid chromatography with a flame ionization detector (Shimadzu,
Kyoto, Japan). It was fitted with a 30 m x 0.25 mm i.d. capillary
column (Supelcowax 10; Supelco, Bellefonte, PA). The column temperature
was programmed from 175°C to 225°C at a rate of 3°C/min, and the
carrier gas was helium. The injector temperature was 250°C, and the
detector temperature was 270°C. The chromatograms were recorded, and
the percentage composition of individual peaks was calculated with a
Chromatopac C-R6A (Shimadzu, Kyoto, Japan). The fatty acid esters were
identified by comparison of their retention times with authentic
standards.
Statistical analysis.
All results were expressed as means ± SD, and
statistical significance was determined by two-way (diet x period) analysis of variance using the SigmaStat statistical program
package (Jandel, Erkrath, Germany). When the F-test was
significant, comparisons among different intake periods of each dietary
group were made with Tukey’s test at 
= 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There was no difference in the final body weights (means ± SE g) of mice in the control (1 wk 38.6 ± 0.4, 2 wk 38.4 ± 0.3, 1 mo 42.6 ± 0.5, 3 mo 43.3 ± 0.4) and DHA-EE (1 wk 39.0 ± 0.4, 2 wk 40.8 ± 0.5, 1 mo 42.3 ± 0.4, 3 mo 44.4 ± 0.3) groups at any time. Food consumption was 4.0 ± 0.1 g/d for both control and DHA-EE groups.
Effect on maze-learning ability.
The time required to reach the maze exit was not significantly
different among control mice fed the diet for different times (data not
shown). In trial 2, mice fed the control diet for 3 mo strayed into
blind alleys in the maze significantly fewer times than did those fed
for 1 wk (P < 0.05) (Fig. 1
). However, during trials 1 and 3, there were no differences in the
numbers of times the control dietary groups strayed into blind alleys
of the maze after different intake periods.
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, P < 0.05). In trial 3, mice fed the DHA-EE diet
for 1 or 3 mo required significantly less time to find the exit than
did those fed the control diet for the same periods (P
< 0.05).
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). During this trial, mice fed the DHA-EE diet for 2 wk strayed into
blind alleys of the maze fewer times than did those fed this diet for 1
wk or 3 mo (P < 0.05). During trial 3, the DHA-EE
groups fed for 1 and 3 mo strayed into blind alleys of the maze fewer
times than their controls (P < 0.05) and straying
incidence was significantly lower in mice fed DHA-EE for 3 mo than
those fed this diet for 2 wk (P < 0.05).
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There was a striking difference in the mean percentage of 20:4(n-6),
20:5(n-3) and 22:6(n-3) in plasma lipids between the DHA-EE and
control dietary groups at all time points (P < 0.05)
(Table 3
). In the DHA-EE group, levels of 20:5(n-3) and 22:6(n-3) increased
with the time of intake, whereas the levels of 20:4(n-6) decreased. The
mice fed this diet for 3 mo had the highest levels of 22:6(n-3) and
20:5(n-3) and the lowest level of 20:4(n-6) compared with those fed the
control diet for all periods and DHA-EE for shorter periods
(P < 0.05). The control group at all time points had
the lowest level of 22:6(n-3).
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Diet and period of intake affected some fatty acids in brain lipids
(Table 4
). Levels of 20:4(n-6) in the brain of mice fed the DHA-EE diet
decreased with increasing time of intake, whereas levels of 20:4(n-6)
in the control group were unchanged. High levels of 22:6(n-3) were
found in the brain of mice fed the DHA-EE diet after 2 wk compared
with those fed for 1 wk, and this was maintained up to 3 mo. In mice
fed the control diet, no significant differences in the levels of
22:6(n-3) were observed after feeding for 1 wk, 2 wk and 1 mo, but
there was a significant decrease after 3 mo. There were significant
differences in 22:6(n-3) levels among the DHA-EE and control groups
at 2 wk, 1 mo and 3 mo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Suzuki et al. 1998
). Higher proportions of DHA are found in the synaptosomal
plasma membranes than in other plasma membranes (Breckenridge et al. 1972
), and it may be that DHA has a specific functional
role in the synaptic membrane. It has been proposed that the high
levels of DHA in membranes of the nervous system influence the
biophysical properties of the membranes due to the extent of
polyunsaturation and perhaps via other specific properties, such as the
shape of these molecules (Neuringer et al. 1988
). Our
previous study demonstrated that large amounts of DHA incorporated into
the brain are recovered in microsomal, synaptosomal and mitochondrial
fractions (Suzuki et al. 1997
). We have also reported
that synaptic membranes in mice fed a DHA-rich diet are more fluid
than those in control mice (Suzuki et al. 1998
). This
finding suggests that the improved learning ability that was observed
in mice fed DHA may be associated with greater synaptic membrane
fluidity. An increased learning ability of DHA-fed animals may be
explained by modifications of neurotransmitter pathways by changes in
DHA levels in brain membrane lipids. Recent work has shown that (n-3)
PUFA deficiency specifically alters monoaminergic function in the
frontal cortex of rats and that this disturbance can be linked to
behavioral impairment (Delion et al. 1994
and 1996
).
The administration of the alternative dietary fats significantly
affects the fatty acid composition of brain and plasma lipids. These
results are consistent with our previous work (Lim et al. 2000
) and suggest a relationship between modifications of fatty
acid composition in the brain, particularly an increase in DHA, and a
reciprocal decrease in AA levels, and the improved learning ability
after the intake of DHA. Plasma lipids reflect what has been absorbed
and metabolized from the dietary fatty acid pool and provide an
indication of the bioavailability of circulating PUFA. Our results
showed that the higher level of DHA in the plasma of the DHA-EE
dietary group observed at 1 wk compared with the control dietary group
continued to increase up to 3 mo. In the brain of DHA-EE fed mice, the
levels of DHA started to increase at 2 wk, and this was maintained up
to 3 mo. These findings indicated that it took a period of time after
the intake of dietary DHA-EE for DHA to be incorporated into the
brain. We found that there was little difference in the
maze-learning ability of the control dietary groups at different
time points despite the observation that levels of DHA had decreased in
the brain of mice fed this diet for 3 mo.
In summary, our results showed that an improvement in maze-learning ability due to the intake of DHA was evident at 1 mo after the start of the feeding trial and was maintained up to 3 mo, whereas the increased DHA levels in brain were apparent after feeding for just 2 wk. These results suggest that it may take some time after the incorporation of DHA into the brain for an improvement in learning ability to occur.
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FOOTNOTES |
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2 Current address: LMBB, NIAAA, National Institutes of Health, Bethesda, MD 20892-8115.
4 Abbreviations used: AA, arachidonic acid; DHA, docosahexaenoic acid; DHA-EE, docosahexaenoic acid ethyl ester; PC, phosphatidylcholine; PUFA, polyunsaturated fatty acids.
Manuscript received July 5, 2000. Initial review completed September 12, 2000. Revision accepted November 15, 2000.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1. American Oil Chemists’ Society Official Methods and Recommended Practices of the American Oil Chemists’ Society: AOCS Official Method Ce 1b-89 5th ed. 1998 American Oil Chemists’ Society Champaign, IL.
2.
Benolken R. M., Anderson R. E., Wheeler T. G. Membrane fatty acids associated with the electrical response in visual excitation. Science 1973;182:1253-1254
3.
Birch E. E., Birch D. G., Hoffman D. R., Uauy R. Dietary essential fatty acid supply and visual acuity development. Invest. Ophthal. Vis. Sci. 1992;33:3242-3253
4. Bligh E. G., Dyer W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959;37:911-917
5. Breckenridge W. C., Gombos G., Morgan I. G. The lipid composition of adult rat brain synaptosomal plasma membranes. Biochim. Biophys. Acta 1972;266:695-707[Medline]
6.
Christensen M. M., Lund S. P., Simonsen L., Hass U., Simonsen S. E., Hoy C. E. Dietary structured triacylglycerols containing docosahexanoic acid given from birth affect visual and auditory performance and tissue fatty acid profiles of rats. J. Nutr. 1998;128:1011-1017
7. Clandinin M. T., Field C. J., Hargreaves K., Morson L., Zsigmond E. Role of diet fat in subcellular structure and function. Can. J. Physiol. Pharmacol. 1985;63:546-556[Medline]
8.
Delion S., Chalon S., Guilloteau D., Besnard J. C., Durand G. -Linolenic acid dietary deficiency alters age-related changes of dopaminergic and serotoninergic neurotransmission in the rat frontal cortex. J. Neurochem. 1996;66:1582-1591[Medline]
9.
Delion S., Chalon S., Hérault J., Guilloteau D., Besnard J. C., Durand G. Chronic dietary -linolenic acid deficiency alters dopaminergic and serotoninergic neurotransmission in rats. J. Nutr. 1994;124:2466-2476
10. Kohashi M., Takahashi A., Iwai K. Effect of a histidine-excess diet on a tetrahydrofolylpolyglutamate pattern in rat liver. J. Nutr. Sci. Vitaminol. 1990;36:11-19
11.
Lim S. Y., Suzuki H. Intakes of dietary docosahexaenoic acid ethyl ester and egg phosphatidylcholine improve maze-learning ability in young and old mice. J. Nutr. 2000;130:1629-1632
12. Lim S. Y., Suzuki H., Suzuki T. Effect of long-term feeding of dietary docosahexaenoic acid and egg-phosphatidylcholine on the fatty acid composition of plasma, brain total lipids and phospholipids in adult and old mice. Nutr. Neurosci. 2000;3:267-276
13. Neuringer M., Anderson G. J., Connor W. E. The essentiality of n-3 fatty acids for the development and function of the retina and brain. Annu. Rev. Nutr. 1988;8:517-541[Medline]
14. Neuringer M., Connor W. E. n-3 fatty acids in the brain and retina: Evidence for their essentiality. Nutr. Rev. 1986;44:285-294[Medline]
15. Neuringer M., Reisbick S., Janowsky J. The role of n-3 fatty acids in visual and cognitive development: Current evidence and methods of assessment. J. Pediatr. 1994;125:S39-S47[Medline]
16. Suzuki H., Manabe S., Wada O., Crawford M. A. Rapid incorporation of docosahexaenoic acid from dietary sources into brain microsomal, synaptosomal and mitochondrial membranes in adult mice. Int. J. Vitam. Nutr. Res. 1997;67:272-278[Medline]
17. Suzuki H., Park S. J., Tamura M., Ando S. Effect of the long-term feeding of dietary lipids on the learning ability, fatty acid composition of brain stem phospholipids and synaptic membrane fluidity in adult mice: A comparison of sardine oil diet with palm oil diet. Mech. Age. Dev. 1998;101:119-128[Medline]
18.
Wainwright P. E., Xing H. C., Ward G. R., Huang Y. S., Bobik E., Auestad N., Montalto M. Water maze performance is unaffected in artificially reared rats fed diets supplemented with arachidonic acid and docosahexanoic acid. J. Nutr. 1999;129:1079-1089
19. Willatts P., Forsyth J. S., DiModugno M. K., Varma S., Colvin M. Influence of long-chain polyunsaturated fatty acids on infant cognitive function. Lipids 1998;33:973-980[Medline]
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