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Clinical Interventions in Aging 2013:8 1247–1257
Clinical Interventions in Aging Dovepress
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ORIGINAL RESEARCH
open access to scientific and medical research
Open Access Full Text Article
http://dx.doi.org/10.2147/CIA.S50349
Effects of krill oil containing n-3 polyunsaturated
fatty acids in phospholipid form on human
brain function: a randomized controlled trial
in healthy elderly volunteers
Chizuru Konagai1,2
Kenichi Yanagimoto3
Kohsuke Hayamizu3
Li Han3
Tomoko Tsuji3
Yoshihiko Koga2
1Department of Food and Nutrition,
Japan Women’s University, Bunkyo-ku,
Tokyo, Japan; 2Department of
Neuropsychiatry, Kyorin University
School of Medicine, Mitaka, Tokyo,
Japan; 3Human Life Science R&D
Center, Nippon Suisan Kaisha Ltd,
Chiyoda-ku, Tokyo, Japan
Correspondence: Chizuru Konagai
Department of Food and Nutrition, Japan
Women’s University, 2-8-1 Mejirodai,
Bunkyo-ku, Tokyo 112-8681, Japan
Tel +813 5981 3427
Fax +813 5981 3427
Email konagai@fc.jwu.ac.jp
Background: Krill oil, rich in n-3 (omega-3) polyunsaturated fatty acids (PUFAs) incorporated in
phosphatidylcholine, has been reported to have many effects on physiological function. However,
there are few studies using psychophysiological methods published that describe the effects of krill
oil on brain function. We investigated the influence of ingestion of krill oil on cognitive function
in elderly subjects by using near-infrared spectroscopy and electroencephalography.
Methods: A randomized, double-blind, parallel-group comparative study design was adopted.
Forty-five healthy elderly males aged 61–72 years were assigned to receive 12 weeks of treatment
with: medium-chain triglycerides as placebo; krill oil, which is rich in n-3 PUFAs incorporated
in phosphatidylcholine; or sardine oil, which is abundant in n-3 PUFAs incorporated in
triglycerides. Changes in oxyhemoglobin concentrations in the cerebral cortex during memory
and calculation tasks were measured. The P300 component of event-related potentials was also
measured during a working memory task.
Results: During the working memory task, changes in oxyhemoglobin concentrations in the
krill oil and sardine oil groups were significantly greater than those in the medium-chain trig-
lyceride group at week 12. The differential value for P300 latency in the krill oil group was
significantly lower than that in the medium-chain triglyceride group at week 12. With regard
to the calculation task, changes in oxyhemoglobin concentrations in the krill oil group were
significantly greater than those in the medium-chain triglyceride group at week 12.
Conclusion: This study provides evidence that n-3 PUFAs activate cognitive function in the
elderly. This is especially the case with krill oil, in which the majority of n-3 PUFAs are incor-
porated into phosphatidylcholine, causing it to be more effective than sardine oil, in which n-3
PUFAs are present as triglycerides.
Keywords: eicosapentaenoic acid, docosahexaenoic acid, phosphatidylcholine, event-related
potential, near-infrared spectroscopy, dorsolateral prefrontal cortex
Introduction
Researchers in a variety of areas have demonstrated that n-3 (omega-3) polyunsaturated
fatty acids (PUFAs), including eicosapentaenoic acid (EPA; 20:5n-3) and docosa-
hexaenoic acid (DHA; 22:6n-3), have considerable effects on physiological function.
Animal and human studies1–4 in addition to epidemiological surveys5–7 have also shown
that n-3 PUFAs effectively enhance and improve learning capability as well as memory
and cognitive function. Moreover, intake of EPA and DHA is reported to reduce the risk
of developing Alzheimer’s disease.8 However, most studies in humans have assessed
the effects of n-3 PUFAs based on the results of psychological tests and cognitive
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Konagai et al
tasks. There are very few studies employing physiological
methods that target elderly participants, although there are
some studies targeting young adults or children.9–13
Fish oil contains abundant amounts of n-3 PUFAs, most
of which is stored as triglycerides. Sardine oil is one of the
most popular fish oils, and is abundant in n-3 PUFAs incorpo-
rated in triglycerides. In some cases, n-3 PUFAs are stored as
phospholipids. Krill is the common name given to the order
Euphausiacea of shrimp-like plankton. Krill oil is rich in
n-3 PUFAs incorporated in phosphatidylcholine. Vakhapova
et al14 and Richter et al15 demonstrated that phosphatidylser-
ine containing n-3 PUFAs was effective for improvement of
memory in the elderly. However, the differences in effects on
cognitive function between these two forms of storage (ie,
phospholipid and triglyceride), have not been clarified.
Among the neuroimaging methods available, near- infrared
spectroscopy and event-related potentials are most suitable
for measuring changes in brain function longitudinally and
noninvasively. Moreover, the devices required are easy to
operate and the cost is low compared with other neurophysi-
ological methods. When brain activity is activated locally, the
supply of oxygen to that area increases accordingly. Oxygen
binds with hemoglobin and is transported in the form of
oxyhemoglobin. Therefore, by measuring blood oxyhemo-
globin concentrations using near-infrared spectroscopy, it is
possible to identify the level of activation of regional cerebral
function resulting from the execution of tasks. Near-infrared
spectroscopy emits near-infrared light from above the scalp.
Analyzing near-infrared light at several wavelengths detected
after it has been absorbed by oxyhemoglobin while passing
through the tissues of the cerebral cortex makes it possible to
measure the relative changes in oxyhemoglobin concentra-
tion as a marker of neural activity in the cerebral cortex.16
Event-related potentials, obtained by analyzing electroen-
cephalograms (EEGs), represent information processing in
the brain electrophysiologically, and are elicited especially
while executing cognitive tasks. Among the event-related
potential components, P300 is considered to reflect cognitive
processes, such as making decisions and controlling behav-
iors and activities.17 Because near-infrared spectroscopy has
a relatively high spatial resolution, it is useful for localizing
activated areas. The event-related potential, in contrast, has
the advantage of high temporal resolution.
In this study, two types of continuous performance
tasks were used during measurement of changes in oxy-
hemoglobin concentration and event-related potentials,
ie, the working memory task and the calculation task. The
amount of working memory mobilized to perform each task
is considerably different. The former needs mobilization of
much working memory. On the contrary, the latter demands
little working memory. Working memory is a function that
serves as the basis of cognitive activity.
Prior to conducting a study with a large sample size, we
performed a preliminary examination to clarify the effects of
long-term ingestion of krill oil and sardine oil on cognitive
function in elderly males using the two neurophysiological
methods described previously. Further, we investigated dif-
ferences in effects of n-3 PUFAs on cognitive function in
relation to the different chemical forms of storage. Only
male subjects were recruited to participate in this study,
because cerebral blood volume changes during cognitive
activation have been found to be gender-dependent.18 Dif-
ferences in event-related potentials have also been reported
to be gender-dependent.19
Materials and methods
Subjects
Healthy male subjects in their 60s and 70s who had retired
from employment in the Japanese business sector were
recruited for this study; all participants were paid volunteers.
Eligibility for participation was as follows: right-handedness,
not requiring prescription medication, no history of mental
disorders or cerebrovascular disease, absence of serious liver,
kidney, heart, respiratory, endocrine, or metabolic diseases,
and absence of food allergies to fish or crustaceans. The
participants were clinically confirmed by a psychiatrist to
have no cognitive impairment.
We provided written and oral explanations in advance
about the purpose of our study in accordance with the
Declaration of Helsinki. All the participants gave their written
informed consent to participate in the study protocol, which
was approved by the ethics committee of Kyorin University
School of Medicine.
The participants were measured for their height, body
weight, and blood pressure, and then underwent a blood test
(white blood cell count, red blood cell count, hemoglobin, and
hematocrit), a serum chemistry test (aspartate aminotrans-
ferase, alanine aminotransferase, gamma-glutamyl transpep-
tidase, creatinine, total cholesterol, high-density lipoprotein
cholesterol, low-density lipoprotein cholesterol, triglycer-
ides, blood glucose, glycosylated hemoglobin, and uric acid)
and urinalysis (occult blood, urinary protein, urinary glucose,
and urobilinogen). Forty-five men of mean age (±standard
deviation) 67.1 ± 3.4 years with no markedly abnormal find-
ings for these parameters were randomly divided into three
treatment groups (n = 15 per group). The participants were
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Effects of n-3 PUFAs in krill oil on brain function
instructed to avoid excessive consumption of alcohol and
food, to limit strenuous exercise, and to refrain from taking
supplements that contained PUFAs during the study.
Experimental supplement
Three types of experimental supplement were used: krill oil
as a typical oil rich in PUFAs incorporated in phosphatidyl-
choline, sardine oil as a typical fish oil, and medium-chain
triglycerides as placebo. Each supplement was prepared by
Nippon Suisan Kaisha Ltd. Table 1 shows the composition of
the main ingredients in the supplements. They were supplied
in soft gelatin capsules containing 0.25 g oil per capsule.
There was no difference in color, size, form, smell, or taste
between the three types of capsules. Subjects were asked to
take four capsules twice a day after breakfast and dinner for
12 weeks (eight capsules daily), from the day following the
initial measurements to the day of the final measurements.
The participants were instructed to complete a self-recorded
checklist to document their supplement intake during the
study period.
Experimental design
A randomized, double-blind, parallel-group, comparative
study design was adopted. A computer-generated random
allocation sequence was used for participant randomization.
This was performed by a person who was not involved with
other aspects of the study. Information about assignment was
not revealed to the researchers until the key was opened. The
participants were not informed about which treatment was
included in their capsules. Before the participants started
treatment, and at week 6 and week 12, we simultaneously
recorded changes in oxyhemoglobin concentration and an
EEG during performance of the working memory task, and
measured changes in oxyhemoglobin concentration dur-
ing the calculation task. Near-infrared spectroscopy and
EEG recordings were performed in an electrically shielded
chamber in the laboratory of the neuropsychiatry division
at Kyorin University. The following were also performed:
a survey of food intake frequency, measurements of body
weight and blood pressure, a blood test, a serum chemistry
test, measurements of plasma fatty acids (dihomo-gamma-
linolenic acid, arachidonic acid, EPA, and DHA), and
urinalysis. Subjects showing any abnormal physical symp-
toms or findings on blood or urine testing during treatment
were withdrawn from the study.
Food frequency method
The Food Frequency Questionnaire Based on Food Groups
(FFQg, version 3.0, Kenpakusha, Tokyo, Japan) was used
to evaluate intake of nutrients during the study period.
The FFQg was developed for use in Japan and is based
on 29 food groups and 10 types of cooking methods,
and is used for estimating energy and nutrient intake per
week for the previous month.20 From the FFQg, the mean
dietary energy intake was calculated according to the fifth
revised and enlarged edition of the Standard Tables of
Food Composition in Japan.21 The participants’ responses
were obtained by interview. We calculated the intake of
energy and of individual nutrients per day (protein, fats,
carbohydrates, sodium, potassium, calcium, magnesium,
Table 1 Composition of each supplement (per daily dose)
Nutrients MCTs KO SO
Components
Water (g) ,0.02 Trace 0
Total lipids (g) 1.98 1.98 1.98
Tocopherol (mg) 0 14 20
Lipid class
Phospholipids in total lipids (g) 0 0.90 0
Fatty acid composition of total lipids
8:0 (mg) 1,497 13 0
10:0 (mg) 287 7 0
12:0 (mg) 5 5 0
14:0 (mg) 0 155 84
14:1 (mg) 0 4 0
15:0 (mg) 0 7 3
16:0 (mg) 0 280 106
16:1 (mg) 0 87 148
16:2 (mg) 0 9 27
16:3 (mg) 0 3 44
16:4 (mg) 0 11 77
17:0 (mg) 0 35 5
17:1 (mg) 0 4 0
18:0 (mg) 0 19 9
18:1 (mg) 13 249 154
18:2n-6 (mg) 4 25 22
18:3n-3 (mg) 0 13 14
18:4n-3 (mg) 0 34 82
20:1 (mg) 0 11 3
20:2n-6 (mg) 0 0 2
20:3n-6 (mg) 0 0 3
20:4n-6 (mg) 0 3 20
20:4n-3 (mg) 0 4 17
20:5n-3 (EPA) (mg) 0 193 491
21:5n-3 (mg) 0 5 19
22:0 (mg) 0 0 3
22:1 (mg) 0 12 0
22:5n-6 (mg) 0 0 5
22:5n-3 (mg) 0 5 46
22:6n-3 (DHA) (mg) 0 92 251
Unidentiable (mg) 0 70 54
Note: Subjects took four capsules (0.25 g oil/capsule) twice a day after breakfast
and dinner.
Abbreviations: MCTs, medium-chain triglycerides; KO, krill oil; SO, sardine oil;
EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
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Konagai et al
phosphorus, iron, zinc, copper, manganese, dietary fiber,
retinol equivalent, vitamin D, alpha-tocopherol, vitamin K,
vitamin B1, vitamin B2, niacin, vitamin B6, vitamin B12,
folic acid, pantothenic acid, vitamin C, saturated fatty
acids, monounsaturated fatty acids, PUFAs, n-3 PUFAs,
n-6 PUFAs, and cholesterol).
Tasks
Working memory task
A continuous performance task using the 2-back paradigm
was carried out. Numerals from 1 to 9 randomly appeared
successively on a computer screen placed 150 cm in front
of the participant for 500 msec each, with an interstimulus
interval of 2,250 msec. The participants were instructed to
push a button with their right thumb when the numeral “3”
was displayed only if they recalled that the numeral 2-back
was even, and to do nothing when the numeral 2-back was
odd. Two hundred numerals were presented in total, with the
target “3” numeral being shown at a rate of 10% (20 trials).
For 14 seconds before starting to show the stimulus, the
numeral “0” was shown on the display monitor for the same
duration and interval as the stimulus, and the participants
were instructed to watch the numeral carefully. The task
lasted 450 seconds.
Calculation task
A calculation task was conducted using the Uchida-Kraepelin
test paper. The participants were asked to add two adja-
cent numerals and write down only the last numeral of the
two-digit number that was obtained. The participants were
instructed to make the calculation as quickly and accurately
as possible. The task lasted 300 seconds.
Measurement and data analysis
Changes in oxyhemoglobin concentration
Changes in oxyhemoglobin concentration while participants
performed the tasks were measured using near-infrared spec-
troscopy (Optical Topography System ETG-4000, Hitachi
Medical Co, Tokyo, Japan). We used a two-sided 3 × 3 probe
holder, and placed the emitter probes and detector probes at
sites where we could measure changes in oxyhemoglobin
concentration in each participant’s bilateral dorsolateral
prefrontal cortex. That is, the front row inner optodes were
positioned symmetrically 0.5 cm outside of Fp1 or Fp2 accord-
ing to the International EEG 10-20 system, and changes in
each participant’s oxyhemoglobin concentration were mea-
sured from a total of 24 channels (Figure 1). Mean changes
in oxyhemoglobin concentration were calculated against
the mean concentration during a 10-second period for the
prestimulus baseline.
Electroencephalography
While the participants performed the working memory task,
we simultaneously measured changes in oxyhemoglobin con-
centration and event-related potentials. Ag/AgCl electrodes
were located at Cz (vertex) and Pz (midline parietal site)
according to the International EEG 10-20 system. The EEG
was recorded and filtered from 0.1 Hz to 100 Hz, using linked
electrodes at the earlobes as the reference. The EEG was
recorded for 450 seconds while the participants performed
the working memory task. The EEG was averaged off-line
for a period of 1,000 msec, beginning 100 msec prior to
the stimulus onset. The baseline was corrected to the mean
value of the voltage during a period of 100 msec prestimulus.
From ten to 20 trials without artifacts due to eye blinking or
body movement were averaged to obtain the event-related
potential waveform. The peak latency and amplitude of the
P300 component of the event-related potential waveform at
Cz and Pz were measured. The differential values of P300
latency and amplitude at week 6 and week 12 compared with
those at week 0 were calculated for both sites. P300 is the
positive potential that appears approximately 300 msec after
a stimulus is presented.
Statistical analysis
Statistical Package for the Social Sciences version 16.0
software (SPSS Inc., Chicago, IL, USA) was used for sta-
tistical processing. Two-way repeated-measures analysis
of variance with the Bonferroni post hoc test was carried
out on the following near-infrared spectroscopic and other
data: change in oxyhemoglobin concentration, body weight,
body mass index, blood pressure, blood test values, serum
1
23
4
56
78
9
10 11
12
14
17 13
16
19 15
22 18
21
24
23
20
Fp2Fp1
Figure 1 Channel positions for near-infrared spectroscopy. Changes in oxyhemoglobin
concentration were measured at 24 sites in the bilateral frontal areas of the brain.
Filled (black) circles and open (white) circles show the emitter and detector probes,
respectively. The numbers show the location of the measured channels.
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Effects of n-3 PUFAs in krill oil on brain function
chemistry test values, urinalysis, nutritional intake based on
food frequency method, and plasma fatty acid concentration.
For P300 latency and amplitude, the Kruskal–Wallis test was
used to analyze the differential values measured at week 6
and week 12 compared with those at week 0. The Mann–
Whitney U-test with Bonferroni correction was then used
to determine any differences between the treatments. The
statistical significance level was set at P , 0.05.
Results
Participants
A flow chart for study participation is shown in Figure 2.
As mentioned earlier, 45 males who met the inclusion cri-
teria agreed to participate in this study. They were randomly
assigned to three treatment groups. Age, height, body weight,
body mass index, smoking status, and alcohol consumption
were compared by one-way analysis of variance or the chi-
square test. No significant differences were found in any of the
items except for height (F[2,42] = 4.379, P = 0.019 by one-way
analysis of variance) between the three groups (Table 2).
Three of the 45 participants dropped out during the study
period. Data for 15 participants in the medium-chain triglyc-
eride group, 13 in the krill oil group, and 14 in the sardine
oil group were used for analysis. The reasons for dropping
out were epigastric pain (two participants) and dermatitis
(one participant). These cases were judged not to be directly
related to ingestion of supplements.
Supplement intake rate
According to the self-recording checklist, mean supplement
intake for the experimental period (excluding dropout
participants) was 98.2% ±2.4% in the medium-chain trig-
lyceride group (n = 15), 98.7% ±1.6% in the krill oil group
(n = 13), and 98.5% ±1.7% in the sardine oil group (n = 14).
No significant difference in intake was found between the
three groups (F[2,39] = 0.181, P = 0.835 by one-way analysis
of variance).
Food frequency questionnaire,
vital signs, and blood and urine tests
No significant differences between the treatments or inges-
tion periods were found with regard to any of the nutrients
measured by the food frequency method, such as amounts
of major components ingested or dietary fatty acids and
cholesterol. No harmful events due to the treatments were
observed in any of the treatment groups during the study.
None of the groups showed any significant variations in body
weight, body mass index, or blood pressure during the study
period. Moreover, no changes attributable to ingestion of the
supplements were observed in blood tests, serum chemistry
tests, or urinalysis.
Plasma fatty acids
With regard to the concentration of PUFAs in plasma
(Table 3), significant interactions were found between
the treatments and ingestion periods in terms of dihomo-
gamma-linolenic acid concentration (F[4,78] = 3.516,
P = 0.011 by two-way repeated-measures analysis of vari-
ance) and EPA concentration (F[4,78] = 5.141, P = 0.001
by two-way repeated-measures analysis of variance).
Further analysis using Bonferroni post hoc testing showed
that the EPA concentration in the sardine oil group was
higher than that in the medium-chain triglyceride group at
week 12 (P = 0.045).
Assessed for eligibility (n = 652)
Excluded (n = 600)
Not meeting inclusion criteria
(n = 193)
Declined to participate (n = 394)
Other reasons (n = 13)
Analyzed (n = 15) Analyzed (n = 14)
Randomized (n = 45)
Analyzed (n = 13)
MCTs (placebo) 2g/d
(n = 15)
SO 2g/d
(n = 15)
KO 2g/d
(n = 15)
Excluded
Epigastric pain
(n = 2)
Excluded
Dermatitis
(n = 1)
Medical examination (n = 52)
Excluded (n = 7)
Medical reasons (n = 7)
EnrollmentAllocation Analysis follow-up
Figure 2 Flow chart of participation throughout the study.
Abbreviations: MCTs, medium-chain triglycerides; KO, krill oil; SO, sardine oil.
Table 2 Baseline characteristics of the participants
MCT
(n =15)
KO
(n =15)
SO
(n =15)
P-value
Age (years) 67.3 ±3.4 67.1 ±3.7 67.0 ±3.3 0.962
Height (cm) 167.0 ±3.9 163.5 ±5.8 168.6 ±4.5 0.019
Body weight (kg) 66.2 ±10.7 64.9 ±8.3 63.0 ±5.9 0.604
BMI (kg/m2)23.7 ±3.5 22.8 ±2.6 23.6 ±2.2 0.666
Current smoker 1 (7) 2 (13) 4 (27) 0.306
Alcohol consumer 11 (73) 13 (87) 12 (80) 0.659
Notes: Mean ± standard deviation or n (%). P-values derived by one-way analysis of
variance or chi-square test.
Abbreviations: MCT, medium-chain triglyceride; KO, krill oil; SO, sardine oil; BMI,
body mass index.
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Working memory task
Changes in oxyhemoglobin concentration
Figure 3 shows a comparison of changes in oxyhemoglobin
concentration at 225.0 seconds after the start of executing the
task. With regard to changes in oxyhemoglobin concentration
in response to performance of the working memory task, the
results of two-way repeated-measures analysis of variance
showed a significant interaction in channel 10 (F[4,78] = 4.331,
P = 0.003 by two-way repeated-measures analysis of variance).
Compared with the medium-chain triglyceride group, the
sardine oil group (P = 0.043) and the krill oil group (P = 0.004)
showed significantly greater changes in oxyhemoglobin con-
centrations at week 12 (Figure 4).
Event-related potentials
Figure 5 shows the changes in P300 latency and amplitude
at Cz and Pz. There were slight differences in the differential
values for P300 latency between the treatments at week 6
Table 3 Polyunsaturated fatty acids in plasma
Week 0 Week 6 Week 12 P-value
MCT KO SO MCT KO SO MCT KO SO
DGLA
(μg/mL)
25.3 ±2.5 21.1 ±2.6 27.3 ±2.1 25.4 ±2.1 19.5 ±1.8 23.4 ±1.8 25.8 ±2.4 19.9 ±2.1 20.0 ±2.0 0.011
AA
(μg/mL)
156 ±8 130 ±5 143 ±8 158 ±8 136 ±6 152 ±7 159 ±10 128 ±7 141 ±70.700
EPA
(μg/mL)
110 ±10 122 ±20 99 ±10 113 ±12 155 ±21 155 ±14 108 ±12a125 ±18a,b 157 ±12b0.001
DHA
(μg/mL)
192 ±14 191 ±19 191 ±16 196 ±14 196 ±17 205 ±15 177 ±13 179 ±19 187 ±15 0.944
Notes: Mean ± standard error. MCT, n = 15; KO, n = 13; SO, n = 14. P-values show signicant probability of interaction between treatments and ingestion periods derived
by two-way repeated-measures analysis of variance. Numerical values within the same period given different superscript letters (a and b) show signicant differences between
treatments (P , 0.05 by Bonferroni post hoc test).
Abbreviations: DGLA, dihomo-gamma-linolenic acid (20:3n-6); AA, arachidonic acid (20:4n-6); EPA, eicosapentaenoic acid (20:5n-3); DHA, docosahexaenoic acid (22:6n-3);
MCT, medium-chain triglyceride; KO, krill oil; SO, sardine oil.
MCT (n = 15)
KO (n = 13)
SO (n = 14)
Week 0 Week 6 Week 12
−0.55
0.55
[mM × mm]
Figure 3 Topographic maps of changes in oxyhemoglobin concentration at 225.0 seconds during working memory task.
Note: MCT, n = 15; KO, n = 13; SO, n = 14.
Abbreviations: MCT, medium-chain triglyceride; KO, krill oil; SO, sardine oil.
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Effects of n-3 PUFAs in krill oil on brain function
(Cz, χ2 = 4.981, df = 2, P = 0.083; Pz, χ2 = 5.130, df = 2,
P = 0.077, Kruskal–Wallis test). At week 12 of the inges-
tion period, there were differences between the groups (Cz,
χ2 = 6.118, df = 2, P = 0.047; Pz, χ2 = 5.754, df = 2, P = 0.056,
Kruskal–Wallis test), and a significant difference was
observed between the krill oil and medium-chain triglyceride
groups (Cz, P = 0.027 and Pz, P = 0.030, Mann–Whitney
U-test with Bonferroni correction). Both sites showed no
significant difference between treatments for differential
values in P300 amplitude at either week 6 or week 12.
Calculation task
Figure 6 shows the mean changes in oxyhemoglobin
concentration at 150.0 seconds after starting the task. For
changes in oxyhemoglobin concentration in response to
execution of the calculation task, the results of two-way
repeated-measures analysis of variance showed a slight
interaction between the treatments and ingestion periods at
Week 0Week 6Week 12
oxy-Hb [mM × mm]
MCT KO SO
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
**
*
Figure 4 Comparison of changes in oxyhemoglobin (oxy-Hb) concentrations at
channel 10 during working memory task.
Notes: Values are expressed as the group mean ± standard error. MCT, n = 15;
KO, n = 13; SO, n = 14. Values were signicantly different from the MCT group.
*P , 0.05, **P , 0.01 by Bonferroni post hoc test.
Abbreviations: MCT, medium-chain triglyceride; KO, krill oil; SO, sardine oil.
−2.0
−1.0
0.0
1.0
2.0
0612
Δ amplitude (microV)
Week
−40
−30
−20
−10
0
10
20
30
0612
Week
*
−40
−30
−20
−10
0
10
20
30
0612
Δ latency (msec)
Δ latency (msec)
Week
*
AB
D
−2.0
−1.0
0.0
1.0
2.0
0612
Δ amplitude (microV)
Week
C
MCT
SO
KO
Figure 5 Comparison of differential values in P300 latencies and amplitudes during the working memory task. (A) Latency at Cz, (B) latency at Pz, (C) amplitude at Cz, and
(D) amplitude at Pz.
Notes: Values are expressed as the group mean ± standard error. MCT, n = 15; KO, n = 13; SO, n = 14. Values were signicantly different from the MCT group: *P , 0.05
by Mann–Whitney U-test with Bonferroni correction. No signicant difference in P300 amplitude was observed between the treatments at week 6 or week 12.
Abbreviations: MCT, medium-chain triglyceride; KO, krill oil; SO, sardine oil.
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Konagai et al
channel 15 (F[4,78] = 2.236, P = 0.073 by two-way repeated-
measures analysis of variance). The krill oil group showed
significantly greater changes in oxyhemoglobin concentration
compared with the medium-chain triglyceride group at week
12 (P = 0.006, Figure 7).
Discussion
The results of the working memory tasks show that the
groups taking krill oil or sardine oil for 12 weeks had greater
changes in oxyhemoglobin concentration in channel 10 than
the group taking medium-chain triglycerides. With regard to
the effect of n-3 PUFAs, Jackson et al have already demon-
strated in their pilot study using near-infrared spectroscopy
that supplementation with DHA-rich fish oil, in comparison
with placebo, resulted in significantly increased oxyhemo-
globin concentrations in the prefrontal areas in healthy young
adults.13 As the function of the cerebral cortex declines with
age, a smaller increase is observed in oxyhemoglobin con-
centrations resulting from execution of cognitive tasks.18,22
Near-infrared spectroscopy probes including channel 10 were
placed to measure the function of the dorsolateral prefrontal
cortex in the present study. The dorsolateral prefrontal cortex
performs a variety of functions. Among these, mobilization
of working memory is one of the important functions of the
dorsolateral prefrontal cortex.23–25 Aging reduces regional
cerebral blood flow, which causes deterioration of cerebral
function.26 Aging has also been shown to reduce the level
of dorsolateral prefrontal cortex activity during execution of
working memory tasks.27 The results of this study suggest that
long-term ingestion of krill oil and sardine oil promotes work-
ing memory function by activating the dorsolateral prefrontal
MCT (n = 15)
KO (n = 13)
SO (n = 14)
Week 0 Week 6 Week 12
−1.00
1.00
[mM × mm]
Figure 6 Topographic maps of changes in oxyhemoglobin concentration at 150.0 seconds during the calculation task.
Note: MCT, n = 15; KO, n = 13; SO, n = 14.
Abbreviations: MCT, medium-chain triglyceride; KO, krill oil; SO, sardine oil.
0.00
0.20
0.40
0.60
0.80
1.00
Week 0 Week 6 Week 12
oxy-Hb [mM × mm]
MCT KO SO
**
Figure 7 Comparison of changes in oxyhemoglobin (oxy-Hb) concentration at
channel 15 during the calculation task.
Notes: Values are expressed as the group mean ± standard error. MCT, n = 15;
KO, n = 13; SO, n = 14. Values were signicantly different from the MCT group:
**P , 0.01 by Bonferroni post hoc tests.
Abbreviations: MCT, medium-chain triglyceride; KO, krill oil; SO, sardine oil.
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Effects of n-3 PUFAs in krill oil on brain function
cortex in elderly people, and thus prevents deterioration in
cognitive activity.
Very few studies have been published using the event-
related potential to identify the effects of EPA and DHA.
Fontani et al compared event-related potential measurements
while subjects executed the Go/No-Go paradigm before
and after taking fish oil for 35 days, and reported improve-
ment in attentional functions after ingestion of n-3 PUFAs.9
Regarding P300, which was measured simultaneously with
near-infrared spectroscopy recording, we observed no differ-
ences in amplitude between our treatment groups, although
the group that took krill oil for 12 weeks showed a signifi-
cant decrease in latency compared with the medium-chain
triglyceride group. P300 latency is thought to reflect the rate
of information processing, while amplitude is thought to
reflect the amount of mobilization of processing resources.28
Continuous ingestion of krill oil was thus shown to expedite
the information processing rate. P300 latency is known to be
prolonged with aging,29,30 ie, ingestion of krill oil is likely to
ameliorate the reduction of cerebral function associated with
aging, or to maintain such function.
During the calculation tasks, the krill oil group showed
significantly greater changes in oxyhemoglobin concentra-
tions in the left frontal area (channel 15) as compared with
changes seen in the medium-chain triglyceride group. The left
hemisphere of the brain is generally regarded to be dominant
with respect to performing calculations. Our results suggest
that intake of krill oil enhances the function of the cerebral
hemisphere relating to calculation. A considerable decrease
in oxyhemoglobin concentration changes at week 12 was
observed for both tasks in the medium-chain triglyceride
group. Although the reason for these results is unclear, there
is a possibility that the practice effect was emphasized only
in the medium-chain triglyceride condition.
Physiological measurement by near-infrared spectroscopy
and EEG analyses demonstrated that krill oil and sardine
oil have the effect of activating the function of the cerebral
cortex. No difference was found between the three groups in
terms of food-derived fatty acid intake volume. Therefore,
the effects obtained in the krill oil and sardine oil groups are
considered to result from ingestion of the supplements.
Both krill oil and sardine oil were shown to promote activ-
ity of the dorsolateral prefrontal cortex in terms of the working
memory task. Given that these supplements contain EPA and
DHA, which are not contained in medium-chain t riglycerides,
these fatty acids were assumed to have influenced the activity
of the dorsolateral prefrontal cortex. However, sardine oil,
which contains the largest amounts of these fatty acids, was
not shown to possess any activation effects regarding the
calculation task, and only krill oil, containing lesser amounts
of these fatty acids, was shown to have an effect. The fatty
acids contained in fish oil are usually stored as triglycerides.
Krill oil, in contrast, has a far higher percentage of phospho-
lipids, in particular phosphatidylcholine, than other foods.31–34
Most of the n-3 PUFAs contained in krill oil are thought to
be incorporated in phosphatidylcholine. In this study, krill
oil had a lower EPA and DHA content than sardine oil.
Plasma EPA concentrations in the sardine oil group were
significantly higher than in the medium-chain triglyceride
group at week 12. On the other hand, there was no significant
difference in plasma EPA concentration between the krill oil
and medium-chain triglyceride groups. Nevertheless, krill oil
demonstrated effects that were equivalent to or better than
those of sardine oil. This led us to assume that the difference
in their chemical form of incorporation was the reason for the
different effects between the two oils rather than any differ-
ence in their fatty acid content.
Vaisman et al reported that ingestion of phospholipid-con-
jugated EPA and DHA increased the scores for a continuous
performance task compared with scores obtained when the
fatty acids in the ingested oil were stored as triglycerides. This
finding indicates that n-3 PUFAs incorporated in phosphati-
dylcholine act on brain function more efficiently than those
incorporated in triglycerides.35 Brossard et al36 and Lemaitre-
Delaunay et al37 reported that lysophosphatidylcholine, which
is derived from phosphatidylcholine by phospholipase A2, is
the preferred physiological carrier of DHA. Thies et al found
that PUFAs bound to lysophospholipids at the sn-2 position
are preferentially incorporated into the brain.38 PUFAs are
most often bound to the sn-2 position in phospholipids.39
The n-3 PUFAs bound to phosphatidylcholine in krill oil
may thus be taken up by the brain tissues more readily than
the n-3 PUFAs of sardine oil, most likely bringing about the
observed difference in their effects. However, the relation-
ship between brain function and fatty acid composition in
brain tissue has not been clarified, so further studies need to
be carried out.
The major limitations of the present study are its small
sample size and inclusion of male subjects only. However, its
results suggest that n-3 PUFAs activate cognitive function of
the brain in the elderly. The differences in the effects obtained
by krill oil and sardine oil seem to be related to differences
with regard to incorporation of fatty acids into lipids. It is
concluded that fatty acids present as phosphatidylcholine
have the potential to bring about more beneficial effects on
cognitive function. Further studies using large sample sizes
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Konagai et al
and including female subjects are needed to confirm the
results of this study.
Acknowledgment
We thank the volunteers who participated in this study.
Disclosure
This study was funded by Nippon Suisan Kaisha Ltd. KY, KH,
LH, and TT are employees of Nippon Suisan Kaisha Ltd. All
authors declare that there are no other conflicts of interest in
this work.
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