Comparative analyses of DHA-Phosphatidylcholine and recombination of DHA-Triglyceride with Egg-Phosphatidylcholine or Glycerylphosphorylcholine on DHA repletion in n-3 deficient mice
Wu et al. Lipids in Health and Disease
Comparative analyses of DHA- Phosphatidylcholine and recombination of DHA-Triglyceride with Egg- Phosphatidylcholine or Glycerylphosphorylcholine on DHA repletion in n-3 deficient mice
Fang Wu 2
Dan-dan Wang 2
Min Wen 0
Hong-xia Che 2
Chang-hu Xue 1 2
Teruyoshi Yanagita 3
Tian-tian Zhang 2
Yu-ming Wang 1 2
0 Institute of BioPharmaceutical Research, Liaocheng University , Liaocheng 252059 , China
1 Qingdao National Laboratory for Marine Science and Technology, Laboratory of Marine Drugs & Biological Products , Qingdao, Shandong Province 266237 , China
2 College of Food Science and Engineering, Ocean University of China , No. 5 Yushan Road, Qingdao 266003 , China
3 Laboratory of Nutrition Biochemistry, Department of Applied Biochemistry and Food Science, Saga University , Saga 840-8502 , Japan
Background: Docosahexaenoic acid (DHA) is important for optimal neurodevelopment and brain function during the childhood when the brain is still under development. Methods: The effects of DHA-Phosphatidylcholine (DHA-PC) and the recombination of DHA-Triglyceride with egg PC (DHA-TG + PC) or α-Glycerylphosphorylcholine (DHA-TG + α-GPC) were comparatively analyzed on DHA recovery and the DHA accumulation kinetics in tissues including cerebral cortex, erythrocyte, liver, and testis were evaluated in the weaning n-3 deficient mice. Results: The concentration of DHA in weaning n-3 deficient mice could be recovered rapidly by dietary DHA supplementation, in which DHA-PC exhibited the better efficacy than the recombination of DHA-Triglyceride with egg PC or α-GPC. Interestingly, DHA-TG + α-GPC exhibited the greater effect on DHA accumulation than DHA-TG + PC in cerebral cortex and erythrocyte (p < 0.05), which was similar to DHA-PC. Meanwhile, DHA-TG + PC showed a similar effect to DHA-PC on DHA repletion in testis, which was better than that of DHA-TG + α-GPC (p < 0.05). Conclusion: We concluded that different forms of DHA supplements could be applied targetedly based on the DHA recovery in different tissues, although the supplemental effects of the recombination of DHA-Triglyceride with egg PC or α-GPC were not completely equivalent to that of DHA-PC, which could provide some references to develop functional foods to support brain development and function.
DHA-Phosphatidylcholine; Tissue accretion kinetics; Repletion; N-3 Fatty acid deficiency; Weaning
Docosahexaenoic acid (DHA) is highly accumulated
in the brain and retina, which is critical for normal
nervous development and function [
Docosahexaenoic acid can be synthesized from its essential
fatty acid precursor, α-linolenic acid (ALA).
However, the capacity of brain for synthesizing the
longchain polyunsaturated fatty acids is very limited
especially in early life stage, thus DHA is mainly
supplied via the uteroplacental circulation during
pregnancy and the breast milk during nursing .
Many pregnant women cannot intake sufficient n-3
polyunsaturated fatty acids (PUFAs) during
pregnancy and lactation in modern western diets, which
is likely responsible for the DHA deficiency in
developing brains of infants and the increasing
incidence of neurological disorders [
]. There have
been many nutritional means such as the dietary
supplements of ALA and DHA to promote the
recovery of organ DHA during the timeframe of
gestation and/or lactation [
particular aspects of neurodevelopment such as synaptic
pruning and gliogenesis still continue in childhood,
suggesting that the nutrition on brain function is
critical during this period [
]. Previous studies
showed that direct DHA supplements could increase
dendritic spine density and neuritogenesis in the
hippocampus of mouse [
]. However, to the best of
our knowledge, very few studies have investigated
the DHA accumulation kinetics in the brain and
other tissues during the childhood.
DHA in natural fish oil is normally esterified to
triglyceride (TG) and phospholipid (PL). DHA enriched
phosphatidylcholine (DHA-PC) is one of the
important forms in DHA-PLs. Several studies have shown
that DHA-PC exhibited the higher bioavailability and
more effective accumulation in brain compared with
]. Moreover, dietary ingestion of
DHA-PC produced a more significant improvement
in cognitive performance and emotional well-being
than DHA-TG . This might be attributed to its
special molecular form including both DHA and
phosphatidylcholine (PC). However, the source of
DHA-PC is relatively limited compared to DHA-TG,
so we expected that whether recombination of
DHATG with normal PC (not containing any DHA) could
be a substitution of DHA-PC on DHA accumulation
in tissues. Interestingly, DHA-PC containing one
molar of DHA (sn-2) and one molar of PC could be
hydrolyzed into sn-2-lysoPC and free DHA by
pancreatic phospholipase A2 in intestine. In addition,
triglyceride enriched with high levels of DHA was
predominantly hydrolyzed into sn-2-monoacylglycerol
and free DHA, and ordinary PC was hydrolyzed to
sn-2-lysoPC in the intestinal lumen [
reesterification of absorbed fatty acids in erythrocyte
was governed by pure availability of compounds, we
expected that the recombination of DHA-TG and
ordinary PL (not containing any DHA) could have the
same effect as intaking DHA-PC on DHA
supplementation. Egg PC is an economical and widely applied
functional food supplement, which is rich in saturated
fatty acids and monounsaturated fatty acids.
αglycerylphosphorylcholine (α-GPC) is water soluble
for its special chemical form with one molecule of
choline. The different chemical structure of PC might
influence the DHA supplement.
Therefore, the present work was investigated to
comparatively analyze the effects of DHA- PC and
recombination of DHA-TG with egg PC or α-GPC
on DHA repletion by weaning n-3 deficient mice
model. The results of this study could provide a
meaningful reference to improve n-3 PUFAs
deficiency by dietary DHA supplement during childhood.
Preparation of DHA-TG and DHA-PC
DHA-PC was separated following the methods as
previously performed [
]. Briefly, total lipids were
extracted from squid roe (S. oualaniensis) and then
mixed with one-fifth volume of 0.15 M NaCl
solution. The mixture was placed into a separatory funnel
and kept for 24 h to completely clear the bottom
(chloroform) phase. The chloroform solution was
evaporated to dryness under vacuum. Then
phospholipids were separated from neutral lipids and
glycolipids by silica-gel column chromatography using
sequentially chloroform, acetone, and methanol as
eluents. The methanol eluent was collected and
DHA-PC was obtained after removal of organic
solvent under vacuum. The purity of DHA-PC was
confirmed according to the HPLC-ELSD analysis (purity
>90%). The fatty acid (FA) composition of the
DHATG, DHA-PC and egg PC was given in Table 1.
Dietary α-GPC (purity >99%), egg PC (purity >95%) and
DHA-TG were obtained from Tianjin Bodi Chemical
Co., Ltd. (Tianjin, China), Suzhou Fushilai
Pharmaceutical Co., Ltd. (Suzhou, China) and Weihai Boow
Foods Co., Ltd. (Weihai, China), respectively.
Animals and diets
All aspects of the animal experiment were carried out in
Food Science and Human Health Laboratory of Ocean
University of China (Qingdao, P. R. China) and conducted
according to guidelines provided by the Ethical Committee
of the University (Approval No.: SPXY2015012). The study
design was depicted in Fig. 1. Female ICR strain mice aged
7 weeks were purchased from Vital River (Beijing, China)
on the second day after conception and immediately
randomized into n-3 adequate or n-3 deficient groups, which
were fed with n-3 adequate or n-3 deficient diets during
pregnancy, respectively. On the first postnatal day,
pups were adjusted to 8 per dam and the dams were
continually fed with their assigned diets during
lactation. All pups were weaned on the 21st postnatal
day. The n-3 deficient pups were randomly assigned
to four groups as follows: n-3 deficient (n-3 Def )
group fed with n-3 deficient diet; DHA-PC group
fed with n-3 deficient diet including DHA-PC;
DHATG + PC group fed with n-3 deficient diet including
DHA-TG and egg PC; DHA-TG + α-GPC group fed
with n-3 deficient diet including DHA-TG and
αGPC. In addition, the n-3 adequate pups fed with
n3 adequate diet were served as the reference point.
The mice were maintained in individual cages under
a 12-h light/dark cycle at 23 °C with a 60 ± 10%
relative humidity and provided with food and water ad
libitum. The basal diets were prepared according to
the AIN-93G growth diet and the fatty acid
concentration of all diets were summarized in Table 2. The
DHA-supplemented diets comprised equal content of
DHA + EPA at a dose of 5% of total fatty acid. And the
content of PC was adjusted to equimolar ratio of
DHA to PC in different DHA-containing diets. The
diets were stored at −20 °C and fresh supplies were
given to the mice every day. Mice were sacrificed by
decapitation after 12-h fast either at weaning or after
2, 4, 7 and 14 days postweaning.
Erythrocytes were obtained from trunk blood by
centrifugation and washed with phosphate-buffered
saline. The cerebral cortex was separated from the
whole brain on ice and weighed. The liver and testis
from each pup were dissected out and weighed
immediately before snap frozen in liquid nitrogen and then
stored at −80 °C until further use. Body weights and
food intake were recorded every day throughout the
Fatty acid analysis
Total lipids were extracted from samples with a
mixture of pentadecanoic acid (15:0) as an internal
standard according to the Folch method [
samples with internal standard were extracted by
chloroform/methanol (2:1, v/v). The lipid extract was
evaporated to dryness under nitrogen flux for
analysis of fatty acid composition. The total lipids from
liver were separated by thin layer chromatography
with a mobile phase of petroleum ether: diethyl
ether: acetic acid (82:18:1, v/v/v) as previous study
]. The hepatic phospholipids and triglycerides
were scraped off the plate and the obtained lipids
were transmethylated to fatty acid methyl esters
(FAMEs) with HCl/methanol by shaking at 90 °C for
3 h. The derivatives were extracted by hexane for
fatty acids analysis using standard mixture containing
28 kinds of components to identify the retention
times. FAMEs were analyzed by an Agilent 6890 gas
chromatograph equipped with aflame-ionization
detector and an HPINNOW-AX capillary column
(30 m × 0.32 mm × 0.25 μm). The detector and
injector temperatures were kept at 250 °C and 240 °
C, respectively. The oven temperature was increased
from 170 °C to 240 °C at 3 °C/min and then held at
240 °C for 15 min. Nitrogen was used as the carrier
gas at the flow rate of 1.2 mL/min.
Data were expressed as the mean ± the standard
error of the mean (SEM). All the statistical tests
were performed with SPSS 18.0 and Figures were
made by Graphpad Prism 13.0. Student’s t test was
used to compare means between n-3 Def and n-3
Adq groups at 3 weeks of age. Differences between
all dietary groups after 3 weeks of age were analyzed
by one-way ANOVA. The difference was considered
statistically significant when p < 0.05.
Results and discussion
Time course of fatty acids alteration in cerebral cortex
Previous clinical and preclinical studies suggested
that the early postnatal period was a critical interval
when insufficient ingestion of n-3 PUFAs might be
very detrimental [
]. During this period, particular
aspects of neurodevelopment were continuing [
and the impact of lower n-3 PUFA level was
correlated with a greater number of further learning and
behavior problems [
]. Therefore, a rapid and
efficient recovery of DHA in developing brain was
important for optimal function, so we studied the DHA
accumulation kinetics in weaning n-3 deficient mice
supplemented with different forms of DHA.
There were no significant differences observed in
food intake, body and tissues weight among all
groups during this experiment (data not shown).
Interestingly, the initial DHA content in the brain of
weaning pups was 14.3% of total fatty acids in n-3
Adq group, but only 7.5% in n-3 Def group,
representing a decrease of 47.5% (Fig. 2a; Table 3). As
seen in Fig. 2a, the present results also showed a
rapid DHA recovery in cerebral cortex, especially the
n-3 deficient weaning mice was capable of restoring
DHA to the level of the n-3 adequate mice within
two weeks by DHA supplementation. All groups
including DHA-PC, DHA-TG + PC and DHA-TG
+ α-GPC exhibited a slight increase of DHA content
at 4 post-weaning days but a substantially great
amount relative to the n-3 Def group from 4 to
7 days, rising to 12.2, 11.9 and 12.7%, respectively.
After 2 weeks, the DHA levels in DHA-PC (16.5%)
and DHA-TG + α-GPC (16.1%) groups displayed
considerable recovery and nearly reached that of n-3
Adq group (16.2%), which were significantly higher
than the DHA-TG + PC group (14.7%). Moreover, the
DHA contents of n-3 Def and n-3 Adq groups were
only marginally increased within two weeks (n-3 Def
group: 7.5–8.1%; n-3 Adq group: 14.3–16.2%). A
continually rapid decline in brain DPA was observed
in the three DHA-supplemented groups over the
subsequent 2 weeks (Fig. 2b; Table 2). The DPA
levels of DHA-PC, DHA-TG + PC and DHA-TG +
αGPC groups significantly decreased by 61.3, 62.3 and
57.4% compared to n-3 Def group. The brain DTA
and AA patterns for the five dietary groups were
quite similar to that of DPA (Fig. 2c and d; Table 3).
Compared with n-3 Adq group, the content of DTA
and AA in n-3 Def group increased by 55.1 and
14.7% at the weaning day. The AA concentration in
the three DHA-containing (DHA-PC, DHA-TG + PC
and DHA-TG + α-GPC) mice exhibited a contiguous
decline to 9.32, 9.17 and 8.31%, respectively, after
DHA supplementation for 4 days. Kitson et al.
showed that the DHA content of brain in adult n-3
deficient rat could increase to the normal level after
4 weeks of DHA supplementation [
]. The time
courses for the DHA recovery of brain also indicated
that the mice required 8 weeks to reach the n-3
adequate level after dietary supplementation of ALA
in n-3 deficient mice at 7 weeks of old [
Interestingly, the data in present study showed a faster
DHA recovery (only 2 weeks required) in the n-3
deficient weaning mice, which was possible that the
activity of desaturase enzymes in the young animals
was much higher than mature animals [
The fatty acid composition analysis of brain
showed that there were no significant differences in
the total saturated fatty acids, monounsaturated
fatty acids and PUFAs among all dietary groups,
which was consistent with the previous results [
(Table 3). The ratios of n-6 / n-3 PUFAs of n-3
Adq group (1.05) was significantly lower than that
of n-3 Def group (2.88). The ratios of n-6 / n-3
PUFAs in DHA-containing groups exhibited a
considerable ecovery after dietary DHA
supplementation and reached the normal level by the end of
experiment (Table 3). The present results showed
that the DHA content of brain was substituted by
n-6 PUFAs were likely due to the competition
between the n-3 and n-6 families for elongation and
desaturation enzymes [
Time course of PUFAs alteration in hepatic phospholipids and triglycerides
The liver was the most rapidly recovery of all tissues
examined in this study. The initial DHA values in
hepatic PL and TG of n-3 deficient mice were
significantly decreased by 73.3 and 89.3%, respectively,
compared to n-3 Adq group (Fig. 3a and e). The
DHA levels in hepatic TG and PL had fully
recovered to the normal values when the n-3 Def mice
were supplied with the three DHA-containing diets
for two days. There were continually marked
increase of DHA levels in hepatic PL and TG after
DHA supplementation for 4 days. Interestingly,
DHA-TG + PC (21.5%) and DHA-TG + α-GPC (20.24%)
groups had higher DHA levels than that of DHA-PC
group in hepatic PL (17.3%) (Fig. 3a). For liver TG, the
DHA concentration of DHA-PC group (2.46%) was also
significantly lower than those of DHA-TG + PC (3.49%)
and DHA-TG + α-GPC (3.61%) groups (P < 0.05) (Fig. 3e).
Previous results found that the ester specific
differences were observed only in the livers of normal
10week-old rats, where the labeled 4C-DHA-PL
delivered a 2-fold and 1.5-fold higher accretion of
radioactivity compared with 14C-DHA-TG and
14C-DHATG + PL, respectively [
]. It was possible that the
efficiency of DHA repletion might be influenced by
developmental stage of tissue and the initial
nutritional status [
]. Compared with the normal adult
rats, the mice used in present study were weaning
n-3 deficient pups which still in developmental stage
and required a mass of n-3 PUFAs to satisfy normal
growth. Therefore, we hypothesized that the
decreasing DHA accumulation in liver might be beneficial
to the availability of DHA for other tissues [
Accompanying the loss of DHA, the initial DPA levels in
hepatic PL and TG of n-3 Def group were significantly
increased by 3.7 and 3.6 folds compared with n-3 Adq
group (Fig. 3c and g). After 4 days of DHA
supplementation, the DPA content in hepatic PL of
DHAsupplemented groups decreased rapidly to nearly 0.5%.
And the DPA content in hepetic TG of mice supplied with
DHA-PC, DHA-TG + PC and DHA-TG + α-GPC
decreased dramatically to 1.11, 0.78 and 0.87%, respectively.
In hepatic PL and TG, the AA and DTA levels in n-3 Adq
group were significantly lower than those in n-3 Def group.
In hepatic PL, the concentration of AA in
DHAPC, DHA-TG + PC and DHA-TG + α-GPC groups
significantly fell from 17 to 8.32%, 7.84%, 7.91%,
respectively after 4 days DHA supplementation
(Fig. 3c). The AA values in liver TG drastically
decreased from 3.26 to 0.45%, 0.61 and 0.42%,
respectively after DHA supplementation with DHA-PC,
DHA-TG + PC and DHA-TG + α-GPC for 4 days
(Fig. 3g). The hepatic DTA pattern for all
DHAsupplemented groups was quite similar to that of
AA (Fig. 3d and h).
Time course of PUFAs alteration in erythrocyte
The phospholipids have been observed to be cleaved
off into lysophospholipids and free fatty acids by
Only major Fatty acid methyl esters were presented; thus they do not add up to 100%. EPA (20:5n-3) were not detected (<0.01%, trace). Each parameter was
presented as the mean ± SEM (n = 6). * p < 0.05, ** p < 0.01, significant difference compared to the n-3 Adq group at 3 weeks of age determined by Student’s t-test.
Different letters indicate significant difference at p < 0.05 among all dietary groups after 3 weeks of ageSFA saturated fatty acid, MUFA monounsaturated fatty acid,
PUFA polyunsaturated fatty acid, N-6 n-6 polyunsaturated fatty acids, N-3 n-3 polyunsaturated fatty acids
DHA-TG + α-GPC
0.74 ± 0.05
phospholipase A2 mediated partial hydrolysis [
Previous reports showed that lyso-DHA-PC combined with
albumin was the main source of DHA for the
erythrocyte using fatty acid labeled with 13C [
erythrocytes, the DHA concentration was significantly
lower in n-3 Def group (1.5%) compared to n-3 Adq
group (3%), indicating a decrease of 50% at 3 weeks
of old (Fig. 4a). Then the DHA value in n-3 Def
group rose very slowly to 2.3% over the course of
experiment. When the n-3 Def group was supplied with
DHA-PC, DHA-TG + PC, DHA-TG + α-GPC during the
first week, the DHA content increased rapidly to 4.75, 4.2
and 5.63%, respectively, with a completely recovery
compared to n-3 Adq group (4.03%). Then the DHA-TG + PC
and DHA-TG + α-GPC groups exhibited a relative
increase of DHA level to 8.12 and 8.5% after 2 weeks of
supplementation. A faster ascent of DHA from 4.2 to 9.73%
was noticed in DHA-PC group. One possible explanation
of these results was that a portion of phospholipids might
be directly absorbed without phospholipase A2 partial
]. Ingestion of DHA-PC could increase
erythrocyte DHA more effectively compared with
]. Although the n-3 deficient mice were
supplemented with equimolar PC in the three DHA-containing
groups, the lyso-DHA-PC concentration in blood of
DHA-TG + PC and DHA-TG + α-GPC groups might be
less than that in DHA-PC group.
Moreover, DHA in the chemical form of
lysophosphatidylcholine was uptake to brain by the primary
transporter named MFSD2a across the blood-brain
], and lyso-DHA-PC represented the
major part of DHA supplementation in erythrocyte
. A previous study indicated that the DHA level
of erythrocyte could been taken as a marker of
DHA accumulation in brain during the circulating
life span [
]. As seen in this paper, we found a
similar time course of DHA reversal in erythrocyte
and brain, in which the DHA concentration in DHA-PC
group was significantly higher than the other
The erythrocyte AA content in n-3 Adq pups
(6.93%) was much lower than that of n-3 Def pups
(13%, Fig. 4c). The DHA-supplemented groups
exhibited a considerable decline of AA after DHA
supplementation for 1 week, which nearly reached
the level of n-3 Adq group (9%). Then the AA values
in DHA-PC and DHA-TG + α-GPC groups decreased
gradually to 7 and 8.1% while the DHA-TG + PC
group rose slightly to 10% after 2 weeks of repletion.
For n-3 Adq group, there was a gradual but
continuous increment to 9.48% for AA concentration over
the experiment. The initial DPA and DTA levels in
n-3 Def groups were significantly higher than that of
n-3 Adq group and recovered with a time course
similar to that of AA after DHA supplementation
(Fig. 4b and d). As previous studies, the n-6 PUFAs
content of erythrocyte in n-3 deficient mice would be
substitited by DHA after dietary DHA
Time course of PUFAs alteration in testis
Testis fatty acid profiles could be influenced by
dietary fat and sensitive to n-3 PUFAs [
]. In mice,
high levels of DHA, AA, DPA were observed in
membrane phospholipids of round spermatids and
mature mouse spermatozoa, which suggested an
important role for proper spermatogenesis [
this study, the testis DHA concentration in n-3 Def
group reduced by 66.2% (p < 0.05) compared to n-3
Adq group (8.22%) at the weaning day (Fig. 5a). The
DHA levels in dietary DHA-containing groups
showed an obvious and continuous increment from
0 to 7 days after weaning. Thereafter, the DHA
content in DHA-TG + α-GPC group exhibited a relative
decrease from 8.33 to 6.62% from 1 to 2 weeks of DHA
supplementation, and DHA-PC group remained the DHA
level about 9.25%. Interestingly, DHA-TG + PC group
exhibited a slight increase from 8.09 to 8.61% in the DHA
value during this period, which nearly reach to that of
DHA-PC group. The results might be attributed to the
rapid growth of mouse testes from 3 to 4 weeks of age,
when the testis weight in DHA-PC, DHA-TG + PC and
DHA-TG + α-GPC groups significantly rose by 76.3%,
60.4%, 60.2%, respectively, during this period (Fig. 5b).
Further analysis showed that the total amounts DHA in
testis of DHA-PC, DHA-TG + α-GPC and DHA-TG + PC
groups increased by 93.7, 65.6, and 43.8%, respectively,
compared with that of n-3 def group, in which the effect
of DHA-TG + PC was similar to that of DHA-PC. Similar
to brain and retina, mouse contain excessive amount of
PUFAs in testis, with particularly high concentration of
DHA, which played an important role for sperm
development and function [
]. Previous results showed dietary
DHA had the positive effects on male fertility [
supplement with DHA could significantly increase DHA
content of testes in delta-6 desaturase-null mice, and as a
result that the observed impairment in male reproduction
was restored [
]. It was meaningful to explore the time
course of testis DHA recovery when the weaning n-3
deficient mice were supplemented with different forms of
DHA. The results in this study showed that dietary
supplementation with DHA-PC was much more effective for
testis DHA accretion than DHA-TG + α-GPC during the
The initial DPA content of testis in n-3 Adq group
(7.98%) was much lower than n-3 Def group (12.33%)
(Fig. 5b). The DPA content in DHA-TG + PC and
DHA-TG + α-GPC groups displayed a faster decline
than that in DHA-PC group within 4 days of DHA
supplementation (p < 0.05). Thereafter the DPA levels in
DHA-PC and DHA-TG + PC groups decreased slightly to
8.46 and 6.49%, respectively, while DHA-TG + α-GPC
group exhibited a rapid decline to 4.26% by the end of
DHA supplementation. The time course patterns of AA
and DTA in testis were similar to that of DPA as seen in
Fig. 5c and d.
The present study investigated the recovery kinetics
of tissue DHA after supplementation of DHA-PC
and the recombination of DHA-TG with egg PC or
α-GPC in weaning n-3 deficient mice induced by
maternal dietary deprivation of ALA during
pregnancy and lactation. Results showed that dietary
DHA supplementation could rapidly recover the DHA
concentration of tissues in n-3 deficient mice during the
childhood, in which DHA-PC exhibited the optimal
efficacy on DHA repletion. Interestingly, the hepatic DHA
levels of DHA-TG + PC and DHA-TG + α-GPC groups
were significantly higher than that of DHA-PC group after
short-term DHA supplementation for 4 days. In addition,
DHA-TG + α-GPC exhibited the greater effect on DHA
accumulation than DHA-TG + PC in cerebral cortex and
erythrocyte, which was similar to DHA-PC. Conversely,
DHA-TG + PC was more effective on DHA repletion
compared with DHA-TG + α-GPC in testis. Therefore,
DHA-PC could not be completely substituted by the
recombination of DHA-TG and ordinary PC (not
containing any DHA) for DHA supplementation.
These findings could pave the way for dietary DHA
supplementation in n-3 deficiency conditions
especially during childhood, which could provide some
references to develop functional foods to support
brain development and function.
AA: Arachidonic acid (20:4n6); ALA: a-linolenic acid (18:3n3);
DHA: Docosahexaenoic acid (22:6n3); DPAn-6: Docosapentaenoic acid
(22:5n6); DTA: Docosatetraenoic acid (22:4n6); n-3 Adq: n-3 adequate; n-3
Def: n-3 deficient; PC: Phosphatidylcholine; PL: Phospholipid;
PUFAs: Polyunsaturated fatty acids; TG: Triglyceride; α-GPC:
This work was funded by the National Natural Science Foundation of China
(No. 31371757), State Key Program of National Natural Science of China (No.
31330060), National Natural Science Foundation of China-Shandong Joint Fund
for Marine Science Research Centers (U1606403) and Fundamental Research
Funds for the Central Universities (No. 201762028).
Availability of data and materials
All data generated or analyzed during the current study are available from
the corresponding author on reasonable request.
FW, YW and TZ conceived and designed the experiments; FW, DW, MW and
HC performed the experiments; FW, DW, MW, YW and TZ analyzed the data;
FW, DW, TY and CX contributed reagents/materials/analysis tools; FW wrote
the paper. All authors have read and approved the final manuscript.
Animal experiments described in our study were approved by the Ethical
Committee of Food Science and Human Health Laboratory of Ocean University
Consent for publication
All authors agree to publish this article in the journal of Lipids in Health
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Brand A , Crawford MA , Yavin E. Retailoring docosahexaenoic acidcontaining phospholipid species during impaired neurogenesis following omega-3 α-linolenic acid deprivation . J Neurochem . 2010 ; 114 : 1393 - 404 .
2. Jr DMJ , Ma K , Bell JM , Rapoport SI . Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids . J Neurochem . 2004 ; 91 : 1125 .
3. Neuringer M , Connor WE , Bendich A . Infant vision and retinal function in studies of dietary long-chain polyunsaturated fatty acids: methods, results, and implications . Am J Clin Nutr . 2000 ; 71 : 256S .
4. Denomme J , Stark KD , Holub BJ . Directly quantitated dietary (n-3) fatty acid intakes of pregnant Canadian women are lower than current dietary recommendations . J Nutr . 2005 ; 135 : 206 .
5. Mccann JC , Ames BN . Is docosahexaenoic acid, an n 3 long-chain polyunsaturated fatty acid, required for development of normal brain function? An overview of evidence from co . Am J Clin Nutr . 2005 ; 82 : 281 - 95 .
6. Moriguchi T , Loewke J , Garrison M , Catalan JN , Salem N Jr. Reversal of docosahexaenoic acid deficiency in the rat brain, retina, liver, and serum . J Lipid Res . 2001 ; 42 : 419 - 27 .
7. Schiefermeier M , Yavin E. n-3 Deficient and docosahexaenoic acidenriched diets during critical periods of the developing prenatal rat brain . J Lipid Res . 2002 ; 43 : 124 .
8. Hughes D , Bryan J. The assessment of cognitive performance in children: considerations for detecting nutritional influences . Nutr Rev . 2003 ; 61 : 413 .
9. He C , Qu X , Cui L , Wang J , Kang JX . Improved spatial learning performance of fat-1 mice is associated with enhanced neurogenesis and neuritogenesis by docosahexaenoic acid . Proc Natl Acad Sci U S A . 2009 ; 106 : 11370 - 5 .
10. Ramprasath VR , Eyal I , Zchut S , Jones PJ . Enhanced increase of omega-3 index in healthy individuals with response to 4-week n-3 fatty acid supplementation from krill oil versus fish oil . Lipids Health Dis . 2013 ; 12 : 178 .
11. Liu L , Bartke N , Van DH , Lawrence P , Qin X , Park HG , Kothapalli K , Windust A , Bindels J , Wang Z . Higher efficacy of dietary DHA provided as a phospholipid than as a triglyceride for brain DHA accretion in neonatal piglets . J Lipid Res . 2014 ; 55 : 531 .
12. Tsushima T , Ohkubo T , Onoyama K , Linder M , Takahashi K. Lysophosphatidylserine form DHA maybe the most effective as substrate for brain DHA accretion . Biocatal Agric Biotechnol . 2014 ; 3 : 303 - 9 .
13. Wen M , Ding L , Zhang L , Zhou M , Xu J , Wang J , Wang Y , Xue C . DHA-PC and DHA-PS improved Aβ1-40 induced cognitive deficiency uncoupled with an increase in brain DHA in rats . J Funct Foods . 2016 ; 22 : 417 - 30 .
14. Folch J , Lees M , Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues . J Biol Chem . 1957 ; 226 : 497 - 509 .
15. Ding N , Xue Y , Tang X , Sun ZM , Yanagita T , Xue CH , Wang YM . Short-term effects of different fish oil formulations on tissue absorption of docosahexaenoic acid in mice fed high- and low-fat diets . J Oleo Sci . 2013 ; 62 : 883 .
16. Skinner MK , Manikkam M , Guerrero-Bosagna C . Epigenetic transgenerational actions of environmental factors in disease etiology . Trends Endocrinol Metab . 2010 ; 21 : 214 - 22 .
17. Crews F , He J , Hodge C . Adolescent cortical development: a critical period of vulnerability for addiction . Pharmacol Biochem Behav . 2007 ; 86 : 189 - 99 .
18. Robertson RC , Oriach CS , Murphy K , Moloney GM , Cryan JF , Dinan TG , Paul RR , Stanton C . Omega-3 polyunsaturated fatty acids critically regulate behaviour and gut microbiota development in adolescence and adulthood . Brain Behav Immun . 2016 ; 59 : 21 .
19. Kitson AP , Metherel AH , Chen CT , Domenichiello AF , Trépanier MO , Berger A , Bazinet RP . Effect of dietary docosahexaenoic acid (DHA) in phospholipids or triglycerides on brain DHA uptake and accretion . J Nutr Biochem . 2016 ; 33 : 91 .
20. Moriguchi T , Harauma A , Salem N Jr. Plasticity of mouse brain docosahexaenoic acid: modulation by diet and age . Lipids . 2013 ; 48 : 343 - 55 .
21. Bourre JM , Piciotti M. Delta -6 desaturation of alpha-linolenic acid in brain and liver during development and aging in the mouse . Neurosci Lett . 1992 ; 141 : 65 - 8 .
22. Brenner RR , Peluffo RO , Nervi AM , Tomas MED . Competitive effect of α- and γ-linolenyl-CoA and arachidonyl-CoA in linoleyl-CoA desaturation to γ- linolenyl- CoA. Biochim Biophys Acta , Lipids Lipid Metab . 1969 ; 176 : 420 - 2 .
23. Graf BA , Duchateau GSMJE , Patterson AB , Mitchell ES , Bruggen PV , Koek JH , Melville S , Verkade HJ . Age dependent incorporation of 14 C-DHA into rat brain and body tissues after dosing various 14 C-DHA-esters . Prostaglandins Leukot Essent Fatty Acids . 2010 ; 83 : 89 - 96 .
24. Harauma A , Salem N Jr, Moriguchi T. Repletion of n-3 fatty acid deficient dams with α-linolenic acid: effects on fetal brain and liver fatty acid composition . Lipids . 2010 ; 45 : 659 .
25. Porter CJ , Trevaskis NL , Charman WN . Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs . Nat Rev Drug Discov . 2007 ; 6 : 231 .
26. Brossard N , Croset M , Normand S , Pousin J , Lecerf J , Laville M , Tayot JL , Lagarde M. Human plasma albumin transports [13C]docosahexaenoic acid in two lipid forms to blood cells . J Lipid Res . 1997 ; 38 : 1571 .
27. Bloom B , Kiyasu JY , Reinhardt WO , Chaikoff IL . Absorption of phospholipides; manner of transport from intestinal lumen to lacteals . Am J Phys . 1954 ; 177 : 84 - 6 .
28. Nguyen LN , Ma D , Shui G , Wong P , Cazenavegassiot A , Zhang X , Wenk MR , Goh EL , Silver DL . Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid . Nature . 2014 ; 509 : 503 - 6 .
29. Zhang TT , Li W , Meng G , Wang P , Liao W. Strategies for transporting nanoparticles across the blood-brain barrier . Biomater Sci . 2016 ; 4 : 219 .
30. Brossard N , Croset M , Lecerf J , Pachiaudi C , Normand S , Chirouze V , Macovschi O , Riou JP , Tayot JL , Lagarde M. Metabolic fate of an oral tracer dose of [13C]docosahexaenoic acid triglycerides in the rat . Am J Physiol . 1996 ; 270 : 846 - 54 .
31. Kuratko CN , Jr SN . Biomarkers of DHA status . Prostaglandins Leukot Essent Fatty Acids . 2009 ; 81 : 111 .
32. Levant B , Ozias MK , Carlson SE . Diet (n-3) polyunsaturated fatty acid content and parity affect liver and erythrocyte phospholipid fatty acid composition in female rats . J Nutr . 2007 ; 137 : 2425 - 30 .
33. Chanmugam PS , Boudreau MD , Hwang DH . Dietary (n-3) fatty acids alter fatty acid composition and prostaglandin synthesis in rat testis . J Nutr . 1991 ; 121 : 1173 .
34. Rejraji H , Sion B , Prensier G , Carreras M , Motta C , Frenoux JM , Vericel E , Grizard G , Vernet P , Drevet JR . Lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation . Biol Reprod . 2006 ; 74 : 1104 .
35. Martínezsoto JC , Landeras J , Gadea J . Spermatozoa and seminal plasma fatty acids as predictors of cryopreservation success . Andrology . 2013 ; 1 : 365 - 75 .
36. Wathes DC , Abayasekara DRE , Aitken RJ . Polyunsaturated fatty acids in male and female Reproduction1 . Biol Reprod . 2007 ; 77 : 190 - 201 .
37. Roquetarivera M , Stroud CK , Haschek WM , Akare SJ , Segre M , Brush RS , Agbaga MP , Anderson RE , Hess RA , Nakamura MT . Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice . J Lipid Res . 2010 ; 51 : 360 - 7 .