Sciknow Publications Ltd.
American Journal of Nutrition and Food Science
©Attribution 3.0 Unported (CC BY 3.0)
AJNFS 2015, 2(2):21-25
DOI: 10.12966/ajnfs.04.01.2015
Effect of Dietary Docosahexaenoic Acid Levels on Respiratory
Functions in Heart Mitochondria in Rats
Norihiro Yamada1,*, Jun Shimizu2
1
Food and Nutrition study major, Department of Life Science, Tsu City College, 157 Ishinden-Nakano, Tsu City, Mie 514-0112, Japan.
Department of Clinical Dietetics and Human Nutrition , Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyaki-dai, Sakado-shi,
Saitama 350-0295, Japan.
2
*Corresponding author (Email: [email protected])
Abstract –Docosahexaenoic acid (C22:6 n-3, DHA) has been attracting attention due to its beneficial effects on several
lifestyle-related diseases. However, an excessive intake of DHA has been suggested to decrease the percentage of linoleic acid
(C18:2 n-6, LA) in cardiolipin (CL) in heart mitochondria, which may reduce oxygen consumption by and cytochrome-c activity
in these mitochondria. Therefore, we examined the effects of dietary DHA levels on the respiratory functions of heart
mitochondria in rats. Animals were fed a diet that had 22.6 % (energy percent, en %) of its total energy from lipids for two weeks.
Dietary 1ipids were adjusted to a polyunsaturated fatty acid / saturated fatty acid ratio of 1.0 and contained different levels of
DHA (en % : 0.0, 1.8, 3.7, 6.5, and 8.6). The proportion of LA in the CL fraction of heart mitochondria decreased in response to
dietary DHA levels. On the other hand, the O2 consumption rate and cytochrome-c oxidase activity in heart mitochondria were
unaffected by the supplementation with DHA. In the present study, dietary DHA levels in rats were 3-fold higher than the
maximum levels practically obtained in humans. These results suggested that the respiratory functions of heart mitochondria
were not adversely affected, even when DHA was consumed at the maximum practically ingestible amount for humans.
Keywords –Docosahexaenoic acid, Linoleic acid, Respiratory function, State 3 respiration, Cytochrome-c oxidase
1. Introduction
Docosahexaenoic acid (C22:6 n-3, DHA) has been attracting
attention due to its anti-atherogenic, anti-thrombotic, and
cancer-preventing effects (Holub, 2009, Phang, et al. 2013,
Kato, et al. 2007). On the other hand, the excessive dietary
intake of DHA has been reported to increase the amount of
lipid peroxides (Saito, et al. 1996, Yamada, et al. 1997).
Furthermore, the excessive intake of DHA has been suggested
to decrease the percentage of linoleic acid (C18:2 n-6, LA) in
cardiolipin (CL) in heart mitochondria. Cortie and Else
reported that DHA was incorporated into CL in heart
mitochondria at the expense of LA (Cortie & Else, 2012).
Decreases in the percentage of LA in the CL fraction of the
heart have been associated with reductions in cytochrome
(cyt) c oxidase activity and respiratory rates in the heart
mitochondria of rats fed diets containing 40 percent of its
energy (en %) from sardine oil (Yamaoka, et al. 1988). On the
other hand, previous studies demonstrated that dietary DHA
did not affect the respiratory functions of isolated cardiac
mitochondria in rats at intake levels practically applicable to
humans (Kobayashi, et al. 1996, Khairallah, et al. 2012).
Among several kinds of phosphatides, CL has been shown
to play an important role in the respiratory functions of heart
mitochondria. CL primarily exists in the inner leaflet of the
inner mitochondrial membrane (Schlame & Haldar, 1993) and
has been referred to as the signature phosphor lipid of
mitochondria (Claypool & Koehler, 2012). CL has four acyl
chains (Schlame & Haldar, 1993), and the fatty acid
composition of CL in heart mitochondria is tightly regulated
by its high level of tetra-LA (4×LA) molecular species, which
have high affinity for cytochrome-c (Cortie & Else, 2012).
Although previous studies have examined the effects of
dietary fish oil or DHA on heart mitochondrial respiratory
functions, the findings obtained were inconsistent. The
excessive intake of DHA has recently been attracting interest
because it is often used as a dietary supplement.
Pharmacological levels of DHA are 2.5 en %, and Dietary
Reference Intakes for Japanese (2010) established that the
dietary goal for total fat intake was approximately 20~30 en %
for males and females aged over 1 year old (Khairallah, et al.
2012, Ministry of Health, Labour and Welfare of Japan;
2009).
The aim of the present study was to examine the effects of
different levels of dietary DHA (0~8.6 en %) under the
condition of 22.6 en % total lipids on changes in the fatty acid
composition of the CL fraction of and respiratory functions in
rat heart mitochondria.
22
American Journal of Nutrition and Food Science (2015) 21-25
2. Materials and Methods
The mitochondrial pellet obtained was washed twice with
buffer A.
2.1. Experimental animals, diet, and rearing method
Five-week-old male Sprague-Dawley rats (Clea Japan Inc.,
Tokyo) were reared in individual stainless steel apartment
cages in a room (23±1℃, humidity 50±10%) with a 12-hour
light/12-hour dark cycle (lights on from 8:00 to 20:00). They
were fed a basal diet for 1 week and were given the test diets
for 2 weeks. In order to prevent the oxidation of dietary lipids,
the diets were given at 17:00 and the remains were removed at
9:00 the next morning. Animals had free access to water.
Animal care and experiments were approved by the Animal
Committee of Tezukayama University.
The basal diet was composed of 20% casein, 0.3%
DL-methionine, 60% sucrose, 3.5% mineral mixture
(AIN-76TM), 1.0% vitamin mixture (AIN-76TM), 0.2% choline
bitartrate, 5% cellulose powder, and 10% lipids (lard; Hayashi
Chemicals Co., Ltd., Tokyo, Japan). Test lipids were prepared
by mixing coconut oil (Hayashi Chemicals Co., Ltd., Tokyo),
LA (Extra linoleic-90; LA content of 90%, Nippon Oil & Fat
Co., Ltd., Tokyo), and DHA (DHA-95E; DHA content of
95%, Harima Chemicals Inc., Tokyo). Table 1 shows the
composition of the major fatty acids in the test lipids, the en %
of DHA, and the P/S and n-3/n-6 ratios.
Table 1. Fatty acid compositions of experimental Lipids (%)
DHA level (energy%)
Fatty acids
0.0 1.8 3.7 6.5
2.3. Isolation of CL and analysis of fatty acid composition
The lipid fraction of the heart was extracted by the method of
Folch et al. (1957). The heart mitochondrial CL fraction was
separated by two-dimensional Silica Gel thin-layer chromato
graphy. Total phospholipid fractions were separated by
Kiesel-gel (Merck, Darmstadt) 60G using a solvent system
petroleum ether: diethylether: acetic acid (80:20:1), and the
CL fraction was separated by Kiesel-gel 60H using a solvent
system of chloroform: methanol: water (65:25:4), respectivel
y. Lipid fractions were visualized by spraying with
Rhodamine 6G (Kanto Chemical Co., Inc., Tokyo). After
scraping the CL spot on the plate, the lipids were extracted
with achloro form: methanol (1:1) mixture. After hydrolysis
in 1/2 N NaOH methanol at 120℃ for 60 minutes, the CL
fraction mixture was subjected to methylation according to
the method of Metcalfe and Schmitz (Metcalfe & Schmitz,
1961), and the fatty acid composition was analyzed by gas
chromatography (GC) using a 12A gas chromatograph
(Shimadzu Co., Ltd., Kyoto). The conditions used for GC
were as follows; column 0.25 mm × 40 m, a stainless steel
capillary column coated with Rascot Silar-5CP (Chromatotec
Ltd., Tokyo); column temperature, 200℃; inlet temperature,
250℃, and carrier gas, N2. Fatty acids were identified by
comparing retention times with that of each standard fatty
acid compound (Sigma Chemical Co., Ltd., St. Louis).
8.6
C8:02.82.4
2.5 2.2 2.4
C10:0 2.82.7
2.7
2.6 2.9
C12:0 25.024.0 24.2 24.8 25.7
C14:0 9.39.6
9.7 10.3 10.4
C16:0 5.15.3
5.3
5.7 5.8
C18:0 1.71.8
1.6
1.8 1.8
C18:1
7.67.1 6.3 5.6
4.6
C18:2 n-6 LA 46.838.7 30.516.9 6.7
C20:5 n-3 EPA- 0.4 0.8 1.3
1.7
C22:6 n-3 DHA- 8.0 16.5 28.8 38.1
LA level (energy%)10.5 8.7 6.8 3.8
P/S ratios1.031.031.04 0.99 0.95
n-3/n-6*
0.000.220.571.785.91
1.5
*n-3/n-6 = (EPA+DHA)/LA
2.2. Preparation of heart mitochondria
After being reared on the test diets for two weeks, rats were
anesthetized with sodium pentobarbital (50mg/kg body
weight) and the heart was excised immediately. The heart was
washed in cooled buffer A (220mM mannitol, 70mM sucrose,
10mMTris, 0.1mM EDTA, and 0.2% BSA) at pH 7.2.
Approximately 1.0 g of the heart was minced and gently
homogenized in 2 ml of buffer a containing 2 mg protease
(Nagase, Nagase Chemtex Corp., Osaka) at 4 ℃ . The
homogenate was left to stand for 5 min at 4 ℃ with the
addition of 8 ml of buffer A. After centrifugation of the
homogenate at 500 × g for 10 min at 4℃, the supernatant
obtained was again centrifuged at 5,000 ×g for 10 min at 4℃.
2.4. Measurement of respiratory control
Respiratory control and ADP/O ratios were measured
according to the method reported by Hagiwara (Hagiwara,
1961). The composition of the reaction solution was 5mM
potassium phosphate (pH 7.4), 187.5mM mannitol, 62.5mM
sucrose, 0.2mM EDTA・2K, 10mM KCl, 5mM MgCl2, and
10mMTris-HCl buffer (pH 7.4). Ten millimolar succinate was
used as the substrate. After equilibration of the medium at
25℃, 100μl of the mitochondrial suspension was added and
the ratio of O2 consumption was measured using a
Clarke-type electrode (Rank Brothers, Ltd., Cambridge).
State 3 respiration was induced by the addition of 234 nmol
ADP. Total protein concentrations were measured in heart
mitochondria by Lowry’s method (Lowry, et al. 1951).
2.5. Enzyme assays
The activity of cyt-c-oxidase (EC1.9.3.1) was measured in
heart mitochondria by the method of Orii and Okunuki (1965).
Cytochrome-c type III from the horse heart was purchased
from Sigma Chemical Co., Ltd. (St. Louis). Oxidation by
reduced cyt-c-oxidase was determined from decreases in
absorbance at 550 nm. F1-F0-ATPase (EC 3.6.7.3) activity
was measured by the method of Kagawa (1974). ATP was
purchased from Wako Pure Chemical Ind. Co., Ltd. (Osaka).
American Journal of Nutrition and Food Science (2015) 21-25
2.6. Statistical analysis
Data obtained from experiments were expressed as the mean
± SEM. Significant intergroup differences in the means were
performed by a one-way ANOVA and the Steel-Dwass test
for multiple comparisons using the statistical software
package R (Version 2.8.1, http://www.r-project.org/).
Significance was confirmed at the level ofp<0.05.
3. Results
3.1. Fatty acid composition of phospholipids in the heart
CL fraction
Data are shown in Table 2. The proportions of C16:0, C18:0,
and C18:1 in the CL fraction of the heart increased slightly
when the diet contained DHA at or greater than 6.5 en %. The
proportion of LA (C18:2 n-6) was significantly lower in the
group fed the diet with 3.7 en % DHA than in the 0 en % DHA
group, and decreased further as DHA levels in the
23
dietincreased. No significant differences were observed in the
proportion of arachidonic acid (C20:4 n-6) up to 6.5 en %
DHA, but was significantly higher in the 8.6 en % DHA group
than in the other groups. The proportions of eicosapentaenoic
acid (C20:5 n-3, EPA) n-3 and DHA increased as the DHA
level was elevated in the diet. The proportion of DHA was
significantly higher in the DHA groups with 6.5 or greater
en % DHA than in the 0 en % DHA group. The P/S ratio
decreased as the DHA level in the diet increased. The n-3/n-6
ratio in the CL fraction was significantly higher in the group
with 8.6 en % DHA than in the 4 other groups.
3.2. Respiration and oxidative phosphorylation in isolated
intact heart mitochondria
No significant intergroup difference was observed in State 3
respiration by heart mitochondria with the DHA
supplementation (Figure), or in State 4 respiration, respiratory
control ratio (RCR), or the ADP/O ratio (Table 3)
Table 2. Fatty acid composition of cardiolipin fraction from hearts of rats fed experimental diets for 14 days 1.
DHA level(energy%)
Fatty acids
0.0
1.8
3.7
6.5
8.6
C16:0 3.67±0.58a5.29±0.48ab5.53±0.97ab7.42±1.03b7.97±1.46b
C18:0 2.99±0.51a3.69±0.34a4.38±0.70a5.98±0.96a11.16±2.60b
C18:1 5.23±0.33a6.16±0.56a5.13±0.26a6.59±0.81a9.02±0.81b
C18:2 n-6 78.33±2.05a71.63±1.69ab64.48±4.21bc55.90±3.65c41.12±6.05d
C20:4 n-6 1.21±0.08a0.94±0.06a0.85±0.08a1.26±0.10a2.59±0.48b
C20:5 n-3 0.07±0.02a0.13±0.02a0.16±0.04a0.39±0.07b0.57±0.09c
C22:6 n-3 0.71±0.08a1.63±0.23ab2.12±0.22ab4.61±0.20b12.11±2.23c
P/S ratios9.57±1.18a6.67±0.70b4.21±0.74bc3.12±0.57c3.04±0.82c
n-3/n-6 ratios0.02±0.00a0.03±0.00a0.04±0.00a0.10±0.00a0.36±0.10b
1
Means±SEM of 9 rats. Means in the same row not sharing a common superscript letters are significantly different (p<0.05).
Table 3. State 4 respiration, RC, ADP/O, and F1F0-ATPase activity in isolated heart mitochondria of rats fed experimental
Diets for 14 days1
DHA level(energy%)
Items
0.0 1.8
3.7
6.5
8.6
State 4233.7±2.3 31.5±1.2 30.1±1.5 32.7±2.2 31.5±2.7
n atom O2/min•mg protein
RCR35.61±0.174.87±0.415.23±0.314.96±0.335.31±0.40
ADP/O
2.78±0.11 3.29±0.15 3.14±0.11 3.05±0.13 2.71±0.10
F1F0-ATPase
777±18778±23801±41767±13786±22
Pi4nmol/min/mg protein
1
Means±SEM of 9 rats. Means in the same row not sharing a common superscript letters are significantlydifferent(p<0.05).
24
American Journal of Nutrition and Food Science (2015) 21-25
2
The substrate used was 5mM succinate.
RCR, respiratory control ratio.
4
Pi, phosphoric acid
3
3.3. Cyt-c-oxidase and F1-F0-ATPase activities in heart
mitochondria
Cyt-c-oxidase activity was slightly lower in the group with
6.5 en % DHA than in the 0 en % DHA group (p<0.05), and
no significant difference was observed among the other
groups (Figure). F1-F0-ATPase activity was unaffected by the
DHA supplementation (Table 3).
Figure. State 32 Respiration and Cytochrome
cOxidase-Specific Activities in Heart Mitochondria of Rats
Fed Experimental Diets for 14 days1.
1
Means±SEM of 9 rats. Means in the bar not sharing
acommon superscript letters are significantly different
(p<0.05).
2
The substrate used was 5mM succinate.
4. Discussion
In the present study, we investigated the effects of dietary
DHA from a low (0 en %) to a high level (8.6 en %) on
respiratory functions in the heart mitochondria of
Sprague-Dawley rats. Our results demonstrated that the
dietary administration of high levels of DHA (8.6 en %) did
not affect respiratory functions in heart mitochondria.
Previous findings on the effects of dietary DHA on
respiratory functions in heart mitochondria have been
inconsistent, and this has been attributed to differences in the
fatty acid composition of the source of dietary lipids, lipid
administration levels, and durations of the intake of
experimental diets. Yamaoka et al. previously reported that
feeding rats a 20% corn oil (CO, containing 60.9% LA) or
sardine oil (SO, containing 19.7 % EPA and 8.3 % DHA) diet
for 30 days led to State 3 respiration and cyt-c-oxidase
activity being 27.6% and 51.9% lower, respectively, in the SO
group than in the CO group (Yamaoka, et al. 1988).
Kobayashi et al. fed rats a diet containing 17.5～31.5 en %
total fat lipids, such as safflower, soybean, and fish oil (FO),
and found that heart mitochondrial cyt-c-oxidase activity was
not affected by FO (Kobayashi, et al. 1996). Khairallah et al.
also reported that supplementation with DHA had no effects
on respiratory functions in cardiac mitochondria when rats
were fed a diet with 14 en % total fat and 5.75 en % DHA for
10 weeks (Khairallah, et al. 2012).
In the present study, a decrease was observed in LA in the
heart CL fraction in conjunction with elevations in dietary
DHA levels; the percentage of LA was 45% lower in the 8.6
en % DHA group than in the 0 en % DHA group. On the other
hand, State 3 respiration, State 4 respiration, RCR, the ADP/O
ratio, and cyt-c-oxidase and F1-F0-ATPase activities in heart
mitochondria were not affected by dietary DHA
supplementation. Khairallah et al. proposed that high levels of
tetra-LA CL were not essential for normal mitochondrial
functions if they were replaced with very-long chain n-3 or
n-6 polyunsaturated fatty acids (Khairallah, et al. 2012). Our
results were supported by these findings. F1-F0-ATPase
activity was not affected by the DHA supplementation.
F1F0-ATPase has been shown to exhibit high binding affinity
for CL (Eble, et al. 1990, Haines 2009). Yamaoka et al.
previously reported that F1-F0-ATPase activity was
significantly higher in the SO group than in the CO group
(Yamaoka, et al. 1988).
Although the P/S ratio of dietary lipids was fixed at 1.0,
the ratio of CL decreased inversely with dietary DHA levels,
and was mainly attributed to DHA not being preferentially
incorporated into CL than LA. The fatty acid composition
results showed that LA was preferentially incorporated into
CL. In the 8.6 en % DHA group, the proportion of LA in the
diet was only 6.7%, whereas that in CL was 41.1 %.
Otsuka et al. examined ten-year trends in fish, shellfish,
EPA, and DHA intakes according to birth cohorts in
community-dwelling middle-aged and elderly men and
woman between 1997 and 2010 in Japan (Otsuka, et al. 2013),
and reported that the habitual maximum intake of EPA +
DHA per day was 4.097g/day, which corresponded to an
intake of 36.9 kcal/day. Dietary Reference Intakes for
Japanese (2010) established dietary reference intakes for
energy of between 1450 and 3000 kcal/day in individuals over
18 years of age (Ministry of Health, Labour and Welfare of
Japan; 2010). If the total energy intake each day is 1450 kcal,
the above maximum intake of total EPA + DHA is
approximately 2.5 en %. Khairallah et al. reported
pharmacological levels of DHA equivalent to 2.5 en %
(Khairallah, et al. 2012). Therefore, we considered the
realistic maximum intake of total EPA + DHA in humans to
be approximately 2.5 en %. Dietary Reference Intakes for
Japanese (2010) set the dietary goal for total fat intake to
approximately 20~30 en % in adults, while that in the United
States is 20~35 en % (Ministry of Health, Labour and Welfare
of Japan; 2010); therefore, dietary total fat supply needs to be
between 20 and 35 en %. Yamaoka et al. (1988) used a diet
with an en% total fat of 40.1%, but this lipid content was high
American Journal of Nutrition and Food Science (2015) 21-25
for humans. Kobayashi et al. and Khairallah et al. used diets
with fat levels that were approximately applicable to humans
(Kobayashi, et al. 1996, Khairallah, et al. 2012). In the present
study, we supplied 22.6 en % total fat containing several
levels of DHA (0~8.6 en %). Therefore, our experimental
conditions were applicable to human nutrition.
The results of the present study suggested that an
excessive supply of DHA did not affect respiratory functions
in heart mitochondria because LA was preferentially
incorporated into CL over DHA in the presence of a certain
level of LA in the diet.
5. Conclusion
We herein demonstrated that supplementation with more than
3-fold the realistic maximum intake of DHA by humans
(8.6en%) did not reduce heart mitochondrial respiratory
functions. These results suggested that DHA supplementation
is safe for heart mitochondrial respiratory functions under
conditions applicable to humans. Furthermore, since we
employed highly purified DHA, our results are valuable from
the viewpoint of observing the main effects of DHA.
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