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ORIGINAL CONTRIBUTION
A short-term n-3 DPA supplementation study in humans
Eliza Miller •Gunveen Kaur •Amy Larsen •Su Peng Loh •
Kaisa Linderborg •Harrison S. Weisinger •Giovanni M. Turchini •
David Cameron-Smith •Andrew J. Sinclair
Received: 4 March 2012 / Accepted: 30 May 2012
ÓSpringer-Verlag 2012
Abstract
Purpose Despite the detailed knowledge of the absorp-
tion and incorporation of eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA) into plasma lipids and red
blood cells (RBC) in humans, very little is known about
docosapentaenoic acid (DPA, 22:5 n-3). The aim of this
study was to investigate the uptake and incorporation of
pure DPA and EPA into human plasma and RBC lipids.
Methods Ten female participants received 8 g of pure
DPA or pure EPA in randomized crossover double-blinded
manner over a 7-day period. The placebo treatment was
olive oil. Blood samples were collected at days zero, four
and seven, following which the plasma and RBC were
separated and used for the analysis of fatty acids.
Results Supplementation with DPA significantly increased
the proportions of DPA in the plasma phospholipids (PL) (by
twofold) and triacylglycerol (TAG) fractions (by 2.3-fold,
day 4). DPA supplementation also significantly increased the
proportions of EPA in TAG (by 3.1-fold, day 4) and cho-
lesterol ester (CE) fractions (by 2.0-fold, day 7) and of DHA
in TAG fraction (by 3.1-fold, day 4). DPA proportions in
RBC PL did not change following supplementation. Sup-
plementation with EPA significantly increased the propor-
tion of EPA in the plasma CE and PL fractions, (both by 2.7-
fold, day 4 and day 7) and in the RBC PL (by 1.9-fold, day 4
and day 7). EPA supplementation did not alter the propor-
tions of DPA or DHA in any lipid fraction. These results
showed that within day 4 of supplementation, DPA and EPA
demonstrated different and specific incorporation patterns.
Conclusion The results of this short-term study suggest
that DPA may act as a reservoir of the major long-chain n-3
fatty acids (LC n-3 PUFA) in humans.
Keywords n-3 Polyunsaturated fatty acids (PUFA)
Docosapentaenoic acid (DPA) Eicosapentaenoic acid
(EPA) Docosahexaenoic acid (DHA) Fatty acid
metabolism
Introduction
A vast amount of information exists in relation to the
beneficial cardiovascular [CV] health actions of long-chain
Eliza Miller and Gunveen Kaur contributed equally to this work.
E. Miller A. Larsen
School of Exercise and Nutrition Sciences, Deakin University,
Burwood, VIC 3126, Australia
G. Kaur
Institute of Sport, Exercise and Active Living (ISEAL),
Victoria University, Melbourne, VIC 8001, Australia
S. P. Loh
Department of Nutrition and Dietetics,
Universiti Putra Malaysia, 43400 Selangor, Malaysia
K. Linderborg
Department of Biochemistry and Food Chemistry,
University of Turku, 20014 Turku, Finland
H. S. Weisinger A. J. Sinclair (&)
School of Medicine, Deakin University, Waurn Ponds,
VIC 3217, Australia
e-mail: andrew.sinclair@deakin.edu.au
G. M. Turchini
School of Life & Environmental Sciences,
Deakin University, Warrnambool, VIC 3280, Australia
D. Cameron-Smith
Liggins Institute, The University of Auckland,
Auckland 1142, New Zealand
123
Eur J Nutr
DOI 10.1007/s00394-012-0396-3
n-3 polyunsaturated fatty acids (LC n-3 PUFA), namely
EPA and DHA [1,2]. In contrast, very little is known about
the regularly consumed intermediary product DPA (22:5
n-3). DPA is found in most fish and marine foods and is
also present in lean red meat from ruminant animals [3,4].
On average, the intake of DPA in adult Australian popu-
lation is 71 mg/day, which represents approximately 29 %
of total LC n-3 PUFA intake [5].
The available literature, based on in vitro, ex vivo and
animal studies, suggests that n-3 DPA may exert benefi-
cial CV health effects [6–8]. DPA has been shown to be
the most potent inhibitor of platelet aggregation in rabbit
platelets, compared with either EPA or DHA. Platelet
aggregation is an early event in the development of
thrombosis or blood clot formation and is initiated by
thromboxane A
2
(TXA
2
)[9]. DPA inhibits cyclooxygen-
ase-1 which is required for the synthesis of TXA
2
thereby
inhibiting platelet aggregation. In human subjects, DPA
is equally effective as EPA and DHA in inhibiting
platelet aggregation (ex vivo) in female subjects; how-
ever, in male subjects, only EPA inhibited platelet
aggregation [10]. Furthermore, DPA exhibits additional
physiological actions, including the ability to suppress
the expression of lipogenic genes in cultured liver cells
and in mice receiving DPA supplementation [7,11].
Mechanistically, these actions may be due, in part, to the
ability of DPA to also induce the expression of peroxi-
some proliferator–activated receptor (PPARa), which
negatively regulates lipolysis in favour of increased fat
oxidation [12]. In addition, DPA is involved in the
reduction of the expression of inflammatory genes such as
tumour necrosis factor (TNF-a) in cell culture models
[13]. The beneficial role of DPA in CV health is also
supported by the studies investigating the metabolism of
DPA, which have shown that DPA is highly incorporated
in heart phospholipids (PL) compared with EPA [14,15].
In addition, there is evidence from in vitro [7] and in vivo
[14] studies that DPA can be metabolized into DHA and
retro-converted to EPA. Collectively, these studies suggest
that DPA may provide an additional source of beneficial
LC n-3 PUFA.
To date, there has only been one clinical study using a
supplement rich in DPA, namely seal oil. However, this
supplement also contained EPA and DHA in higher
proportions than DPA [16]. Therefore, the benefits
observed cannot be attributed purely to DPA rather than
the EPA or DHA present. The present study was con-
ducted to investigate the actions of a highly purified DPA
source, relative to pure EPA, on the incorporation into
plasma and RBC lipids, following a 7-day supplementa-
tion period. It was hypothesized that the pattern of
incorporation of DPA into human blood lipids would be
similar to EPA.
Materials and methods
Study population
Ten healthy lean females with a body mass index (BMI) of
20–25 kg/m
2
, aged between 21 and 30 years were recruited
for the study. Participants provided a written informed
consent and completed a medical questionnaire and PUFA
Food Frequency Questionnaire (FFQ). Participants were
excluded if they consumed more than 500 mg of LC n-3
PUFA per day (based on results of PUFA FFQ [17–19]),
were at high risk of any form of CVD (based upon family
history information obtained from medical questionnaire)
or were overweight as indicated by their BMI ([26 kg/m
2
).
Ethics approval was obtained from the Deakin University
Human Research Ethics Committee (EC2011-023).
Daily supplements
Purified EPA (99.8 %; w/w) and DPA (99.8 %; w/w), both
as free fatty acids, were sourced from Equateq Ltd, Bre-
asclete, Callanish, Scotland. The study investigators had
access to only 100 grams of pure EPA and DPA. To our
knowledge, this is the first ever study using pure DPA
because it has been very difficult to obtain the pure DPA,
given the prohibitive costs, technical expertise and time
required in its manufacture. Since DPA is in scarce supply,
we chose to feed a high dose for a short time to determine
into which lipids the DPA was incorporated, recognizing
that the dose was well above what might be ingested
through usual food sources of DPA.
The participants consumed the supplements for seven
consecutive days, after which time the study concluded.
The participants consumed 2 g of the supplement in
question on the first day of the study and 1 g daily for the
subsequent 6 days. The total DPA or EPA that was con-
sumed per supplementation period per subject was 8 g. The
initial dose was double that of the remaining doses in an
attempt to boost the DPA or EPA levels from the beginning
of the study.
Study design
Following screening, the participants received the olive oil
placebo treatment first, and they were then randomized to
receive the DPA or EPA supplements. The participants
consumed a standardized dinner meal (containing pasta
(dry 200 g), tomato stir through sauce (70 g) and a packet
pudding the night before the start of the study and were
given instructions to fast overnight for 10 h. On the first
study day, a fasted blood sample (day 0) was drawn, and
following this, the participants were asked to consume a
meal of 180 g of instant mashed potato (Continental,
Eur J Nutr
123
Deb
TM
, Unilever Australasia) that contained the 2 g of
DPA or EPA mixed with 18 ml of olive oil. The placebo
group consumed 20 ml of olive oil in the 180 g of mashed
potato. The participants consumed this ‘breakfast’ in
15 min.
For the next 6 days, participants were provided with
six, 2 g aliquots of a 1:1 mixture of DPA or EPA in olive
oil in 2 ml cryovials in a box (protecting the oils from
exposure to light). Participants were provided with
instructions to keep the supplements in the fridge and to
consume them each morning. During the placebo period,
the participants received six 2 g aliquots of olive oil. For
days 1–6, the participants were asked to pour the contents
of the cryovial into 200 ml of standard commercial
orange juice to aid in palatability. On the mornings of day
4 and 7 of their supplementation week, participants
attended the clinical facility to provide a fasting blood
sample.
During the three supplementation weeks, participants
were requested to refrain from consuming high LC n-3
PUFA products including fish, red meat and LC n-3
PUFA–fortified products (\2 marine and/or 2 red meat
meals/week and \2 LC n-3 PUFA–fortified products/
week), in order to prevent false increases in plasma-
circulating LC n-3 PUFA. The participants were asked
to give a recall of their diet 24 h before they came
into provide blood samples at days 0, 4 and 7. It was
found that the participants did not consume any fish
during the study period, and consumption of red meat
was \2 serves per week. Each supplementation period
lasted 7 days with a 2-week washout period prior to
crossover.
Plasma and red blood cell lipid analysis
Venous blood was collected into two 8-ml EDTA vacu-
tainers. Samples were immediately centrifuged for 15 min
at 5919gand 15 °C. Plasma at each time point was ali-
quoted and stored at -80 °C until further analysis. The
RBC remaining after the removal of the plasma were
washed twice with 0.9 % sodium chloride, centrifuged
each time and after the removal of the upper saline solu-
tion, the washed red cells were aliquoted into storage vials
and stored as the plasma.
Total plasma lipids were extracted from plasma as
described by Sinclair et al. [20]. In brief, 850 llof
plasma was extracted using 15 ml of dichloromethane:
methanol 2:1 (v/v) containing 0.01 mg butylated
hydroxytoluene (BHT) and reference internal lipid stan-
dards, specifically TAG-17:0, CE-17:0 (NuChek Prep,
Minnesota, USA) and phosphatidyl choline (PC)-17:0
(Avanti Polar Lipids, USA). The major neutral lipid
classes were separated by thin layer chromatography
(TLC), and the CE, TAG and PL fractions were scraped
from the TLC plates and transmethylated with 5 %
H
2
SO
4
in methanol prior to GC. The resulting fatty acid
methyl esters (FAME) were isolated and identified using
an Agilent Technologies GC 7890A (Agilent Technolo-
gies, Santa Clara, California, USA) equipped with an
Omegawax 250 capillary column (30 m 90.25 lm
internal diameter, 0.25 lm film thickness, Supelco,
Bellefonte, PA, USA), a flame ionization detector (FID)
and an Agilent Technologies 7,693 auto-sampler. Each of
the FAME peak was identified relative to known external
standards, a FAME mix of three PUFA, these being a
marine source, animal source and menhaden oil (Supleco,
Bellefonte, PA, USA). The resulting peaks were then
corrected by the theoretical relative FID response factors
and quantified relative to the internal standard used at the
lipid extraction stage [21].
Total RBC lipids were extracted from 200 llofRBC
with dichloromethane/methanol (1:1 v/v, containing a
known amount of PC-17:0 standard), similar to the Folch
method [22], with modifications by Armstrong et al.
[23]. The RBC lipids were separated by TLC, and the
PL fraction was scraped from the TLC plates and
transmethylated; the FAME were analysed as described
above.
Plasma TAG concentration
Plasma TAG concentrations were measured on a Roche
Cobas Integra 400 plus autoanalyser using a commercially
available enzymatic colorimetric method using a com-
mercially available kit (TRIGL) as per the manufacturer’s
instructions (Roche, Lavel, Quebec, Canada).
Statistical analysis
Data calculations and statistical analysis were performed
using the Minitab Statistical Software (Minitab Version 15;
Minitab Inc., USA). Data were analysed using two-way
ANOVA repeated measures, and pairwise comparisons
were made using Tukey’s test. A value of p\0.05 was
taken as significant.
Results
Subject characteristics
The 10 healthy female participants had a mean age of
25.5 ±3.3 years, with a BMI of 22.3 ±1.6 kg/m
2
; they
were non-diabetic, not taking CVD medication and did not
regularly consume fish oil capsules. All participants
Eur J Nutr
123
completed a PUFA FFQ [17–19] and were found to con-
sume 102 ±66 mg LC n-3 PUFA/day.
Acceptability of the supplements
Participants who experienced any adverse reaction were
requested to inform investigators immediately. Three cases
of mild diarrhoea were reported by participants during the
DPA and EPA supplementation periods, respectively, with
severity ranging from very mild to moderate. These events
were found to occur only during first 4 days of supple-
mentation and were most commonly reported to occur
within the first hour following supplement consumption.
Participants received daily reminders in person or by
e-mail to consume their supplement, and upon returning for
the final blood collection, participants were requested to
return the box containing all the vials; the supplements
were provided in a way to ascertain compliance with
consumption of the supplement. It was found that all
returned vials were empty.
Plasma and red blood cell fatty acid composition
Plasma PL
The average baseline levels of LC n-3 PUFA in plasma PL
were 0.8 % for EPA and DPA and 2.3 % for DHA (Fig. 1).
For those consuming the DPA supplement, there was a
significant increase in the proportion of DPA at day 4 (from
0.7 % to 1.4 %), compared with day 0 value (p=0.006).
Although, there was a trend for an increase in day 7 DPA
levels, this was not statistically significant (p=0.076). In
Overall Significance(ANOVA)
Combined supplementation
effect; p<0.01
Day effect; p<0.01
Breakfast*Day interaction; p<0.01
Overall Significance(ANOVA)
Combined supplementation
effect; p<0.01
Day effect; p<0.01
Breakfast*Day interaction; p=0.063
Overall Significance(ANOVA)
Combined supplementation
effect; p=0.393
Day effect; p=0.654
Breakfast*Day interaction; p=0.257
Fig. 1 Fatty acid composition of plasma phospholipids (PL) from
human participants supplemented with olive oil, EPA or DPA with a
dose of 2 g for the first day and 1 g for the subsequent 6 days. Results
are expressed as percentage mean ±SEM (n=10). Data were
analysed using two-way ANOVA repeated measures, and pairwise
comparisons were made using Tukey’s test. The superscripts with
capital alphabets represent a combined supplementation effect, and
different superscripts represent values that are significantly different
(p\0.05). The superscripts with small alphabets represent time
effect within each supplementation group, and different superscripts
represent values that are significantly different (p\0.05). The values
with no superscripts show no significant differences. OO olive oil,
EPA eicosapentaenoic acid, DPA docosapentaenoic acid, DHA
docosahexaenoic acid
Eur J Nutr
123
the EPA group, there was a significant increase in the
proportion of EPA (from 1 to 2.7 %) relative to baseline at
both days 4 and 7 (p\0.01). There were no significant
changes in the proportion of LC n-3 PUFA for participants
consuming the olive oil placebo.
Plasma TAG
The average baseline levels of LC n-3 PUFA in plasma
TAG were 0.3 % for EPA and DPA and 0.6 % for DHA
(Fig. 2). After DPA supplementation, there was a signifi-
cant rise in the proportion of DPA (from 0.5 to 1.2 %) at
day 4 (p=0.027), in the proportion of EPA (from 0.3 to
0.9 %) at day 4 (p=0.05), as well as in the proportion of
DHA (from 0.7 to 2.2 %) at day 4 (p=0.004). There were
no significant changes in the proportions of EPA, DPA or
DHA in plasma TAG for participants consuming the EPA
supplements or the olive oil placebo, compared with day 0.
Plasma CE
The average baseline levels of LC n-3 PUFA in plasma CE
were 0.9 % for EPA, 0.8 % for DPA and 0.7 % for DHA
(Fig. 3). As shown in Fig. 3, there were no significant
changes in the proportion of LC n-3 PUFA caused by the
olive oil supplementation. After DPA supplementation,
there was a significant rise in EPA proportions at day 7
(from 0.9 to 1.7 %, p=0.027) compared with day 0. After
supplementation with EPA, there was a significant rise in
the proportion of EPA at day 4 (from 0.9 to 2.4 %,
p\0.01) and day 7 (from 0.9 to 2 %, p\0.01), compared
with day 0.
Overall Significance(ANOVA)
Combined supplementation
effect;p=0.001
Day effect; p=0.079
Breakfast*Day interaction; p=0.192
Overall Significance(ANOVA)
Combined supplementation effect;
p<0.01
Day effect; p=0.04
Breakfast*Day interaction; p=0.116
Overall Significance(ANOVA)
Combined supplementation effect;
p<0.01
Day effect; p=0.07
Breakfast*Day interaction; p=0.023
Fig. 2 Fatty acid composition of plasma triacylglycerides (TAG)
from human participants supplemented with a dose of 2 g for the first
day and 1 g for the subsequent 6 days. Results are expressed as
percentage mean ±SEM (n=10). Data were analysed using two-
way ANOVA repeated measures, and pairwise comparisons were
made using Tukey’s test. The superscripts with capital alphabets
represent over all supplementation effect, and different superscripts
represent values that are significantly different (p\0.05). The
superscripts with small alphabets represent time effect within each
supplementation group, and different superscripts represent values
that are significantly different (p\0.05). The values with no
superscripts show no significant differences. OO olive oil, EPA
eicosapentaenoic acid, DPA docosapentaenoic acid, DHA docosa-
hexaenoic acid
Eur J Nutr
123
RBC PL
The average baseline levels of EPA, DPA and DHA in
RBC PL were 1.0, 2.2 and 6.7 %, respectively (Fig. 4).
After the DPA supplementation, there was no significant
change in the proportion of DPA in RBC PL. After the
EPA supplementation, there was a significant increase in
the proportion of EPA at both day 4 (from 1.1 to 2.0 %)
and 7 (from 1.1 to 1.9 %), compared with the baseline
value (p\0.01). There were no significant changes in the
proportions of DPA or DHA in any of the treatment groups.
Plasma TAG concentrations
There were no significant changes in the concentration of
TAG in plasma samples between the control and LC n-3
PUFA supplement periods, or with time in any of the three
groups (data not shown).
Discussion
The ingestion of LC n-3 PUFA–rich marine oils, either as
fish or in purified oil supplements, is a widely accepted
strategy for the reduction in plasma TAG levels [24–27].
This is supported by a considerable quantity of data on the
effect of LC n-3 PUFA in cell models [28,29], experi-
mental animals [30,31] and intervention clinical studies
[32,33]. DPA is one of the three major LC n-3 PUFA in
marine oils; yet, there is no available data on the plasma
lipid or RBC PL distribution of DPA following supple-
mentation with pure DPA in humans. Therefore, the aim of
Overall Significance(ANOVA)
Combined supplementation effect;
p<0.01
Day effect; p<0.01
Breakfast*Day interaction; p<0.01
Overall Significance(ANOVA)
Combined supplementation
effect;p=0.207
Day effect; p=0.348
Breakfast*Day interaction; p=0.388
Overall Significance(ANOVA)
Combined supplementation
effect;p=0.001
Day effect; p=0.558
Breakfast*Day interaction; p=0.825
Fig. 3 Fatty acid composition of plasma cholesterol ester (CE) from
human participants supplemented with olive oil, EPA or DPA with a
dose of 2 g for the first day and 1 g for the subsequent 6 days. Results
are expressed as percentage mean ±SEM (n=10). Data were
analysed using two-way ANOVA repeated measures, and pairwise
comparisons were made using Tukey’s test. The superscripts with
capital alphabets represent over all supplementation effect, and
different superscripts represent values that are significantly different
(p\0.05). The superscripts with small alphabets represent time
effect within each supplementation group, and different superscripts
represent values that are significantly different (p\0.05). The values
with no superscripts show no significant differences. OO olive oil,
EPA eicosapentaenoic acid, DPA docosapentaenoic acid, DHA
docosahexaenoic acid
Eur J Nutr
123
this study was to investigate the partitioning of pure DPA
into human plasma and red blood cell lipid fractions fol-
lowing a 7-day dietary supplementation period.
The most striking finding, contrary to expectations, was
that DPA and EPA partitioned into different lipid fractions
in both plasma and RBC phospholipids. In the steady state
(baseline values), DPA and EPA were both present in RBC
PL, plasma CE and plasma PL in higher proportions than in
the plasma TAG fraction. With DPA supplementation,
there was a significant increase in the proportions of DPA
in the plasma TAG and PL fraction, but not in RBC PL or
plasma CE fractions. Consistent with the steady state, EPA
supplementation significantly increased the proportion of
EPA in plasma CE and PL fractions and RBC PL, but not
in the plasma TAG fraction. The failure of DPA to be
incorporated into the plasma CE fraction and RBC PL
fraction was unexpected and reveals in the time frame of
this study a highly interesting difference between how DPA
and EPA are processed in the body. This differential pro-
cessing might occur perhaps at the level of incorporation of
the PUFA into chylomicron TAG and/or at the level of the
liver following the uptake of the PUFA from chylomicron
remnants, subsequent processing into VLDL lipids and
exchange between lipoproteins and red blood cell lipids.
The average baseline levels of LC n-3 PUFA in the
plasma PL in our study were 0.8 % for both EPA and DPA
and 2.3 % for DHA, which is consistent with previously
reported data for Australian subjects [34,35]. In our study,
supplementation with DPA led to a significant increase in
DPA levels (by twofold) in plasma PL which peaked by
Overall Significance(ANOVA)
Combined supplementation
effect;p<0.01
Day effect; p<0.01
Breakfast*Day interaction; p<0.01
Overall Significance(ANOVA)
Combined supplementation
effect;p=0.055
Day effect; p=0.328
Breakfast*Day interaction; p=0.600
Overall Significance(ANOVA)
Combined supplementation
effect;p=0.140
Day effect; p=0.538
Breakfast*Day interaction; p=0.838
Fig. 4 Fatty acid composition of red blood cell phospholipids (PL)
from human participants supplemented with olive oil, EPA or DPA
with a dose of 2 g for the first day and 1 g for the subsequent 6 days.
Results are expressed as percentage mean ±SEM (n=10). Data
were analysed using two-way ANOVA repeated measures, and
pairwise comparisons were made using Tukey’s test. The superscripts
with capital alphabets represent over all supplementation effect, and
different superscripts represent values that are significantly different
(p\0.05). The superscripts with small alphabets represent time
effect within each supplementation group, and different superscripts
represent values that are significantly different (p\0.05). The values
with no superscripts show no significant differences. OO olive oil,
EPA eicosapentaenoic acid, DPA docosapentaenoic acid, DHA
docosahexaenoic acid
Eur J Nutr
123
4 days. Seal oil supplementation (which contains a higher
proportion of DPA than other marine oils) led to a signif-
icant increase in DPA proportions in plasma PL [36]. No
changes were observed in EPA levels in plasma PL as a
result of DPA supplementation, which means any DPA that
was retro-converted to EPA, say in the liver, was not
incorporated into the plasma PL in the time frame of this
study. In our study, supplementation with EPA signifi-
cantly increased the EPA levels by approximately 2.7-fold
in plasma PL. Similar findings have also been reported by
Mori et al. [37] who fed 4 g/day of pure EPA to human
subjects for 6 weeks and showed increases in EPA levels in
plasma PL. In addition, Mori et al. showed an increase in
the DPA level. In our study, although DPA levels in the
EPA group were higher than baseline levels, this increase
did not achieve statistical significance. It should be noted
that the Mori et al.’s study was over an extended period (6
vs. 1 week in the present study). Studies utilizing fish oil
supplements have also shown increased levels of all three
LC n-3 PUFA in plasma PL after 6 weeks of supplemen-
tation [38].
The mean baseline values of LC n-3 PUFA in plasma
TAG in our study were 0.3 % for EPA and DPA and 0.6 %
for DHA, comparable to the previous reports [35]. In the
present study, DPA supplementation significantly increased
plasma TAG DPA, EPA and DHA levels at day 4. Previous
studies have reported that high doses of EPA lead to the
accumulation of EPA in the plasma TAG fraction [39]. The
increases in all 3 major n-3 PUFA species indicate that
DPA is both being retro-converted back to EPA and further
elongated onto DHA. Retro-conversion involves both per-
oxisomal acyl-CoA oxidase and b-oxidation [40,41].
Retro-conversion has been demonstrated in hepatocytes
[12], and may also be present in endothelial cells [42] and
fibroblasts [40,43]. Recently, evidence of retro-conversion
of DPA to EPA was found to be present in a wide variety of
rat tissues including liver, heart and skeletal muscle [14,
15]. The current study demonstrated that in healthy female
volunteers, 7 days of supplementation of DPA increased
plasma TAG EPA, demonstrating retro-conversion and
augmentation of this EPA pool. While we cannot be certain
of the reason for the increase in DHA levels in plasma
TAG fractions following DPA supplementation, this result
demonstrates DPA can act as a source of DHA in the body.
This increase in DHA levels is supported by animal studies
with pure DPA supplementation by Kaur et al. [15], Holub
et al. [14] and Gotoh et al. [11] who all reported increased
DHA levels in liver tissue.
The mean baseline values for LC n-3 PUFA in plasma
CE in our study were 0.9 % for EPA, 0.8 % for DPA and
0.7 % for DHA, consistent with previously published data
[44]. In the current study, DPA supplementation trended
towards an increased DPA and DHA levels in plasma CE;
however, statistical significance was not achieved. It is
possible that long-term supplementation might result in
increased DPA and DHA levels in plasma CE. There was
further evidence of DPA retro-conversion to EPA, since
there was a significant increase in EPA in the plasma CE
fraction (by twofold at day 8). This suggests that DPA can
act as a source of EPA in the body. Our data show that EPA
supplementation significantly increased plasma CE EPA
levels by approximately 2.7- and 2.3-fold at day 4 and day
8, but did not impact on DPA levels. A previous long term
study [44] has shown that an EPA-rich fish oil leads to
significant increases in plasma CE proportions of EPA
within 30 days of commencing the study (12-month study).
The present study showed that EPA can be incorporated
into plasma CE within 4 days of commencing the
supplement.
The mean baseline values of LC n-3 PUFA in RBC PL
in our study were 1.0 % for EPA, 2.2 % for DPA and
6.7 % for DHA, consistent with data published previously
[38,45]. In our study, there was no increase in DPA levels
in RBC PL in any group. A seal oil supplementation study
showed a considerable increase in EPA (0.8-fold) and
DHA (onefold) levels in erythrocytes and an only modest
increase in DPA (0.2-fold) [46]. Since seal oil also contains
EPA along with DPA, the modest increases in DPA levels
could be from the conversion of EPA into DPA, rather than
direct incorporation of DPA itself.
Pure EPA supplementation for 6 weeks has been
reported to increase EPA levels in RBC membrane [45].
Similarly, our data show that EPA supplementation for
1 week significantly increased RBC PL EPA levels at day
4. As RBC lifespan is approximately 120 days [44],
incorporation of EPA into RBC PL is unlikely to be
achieved through a process involving RBC turnover.
Incorporation of EPA into the RBC is more likely achieved
through exchange between plasma and RBC PL, as spec-
ulated previously [47–49].
It is recognized that the washout period between the
EPA and DPA treatments was relatively short (2 weeks)
and that this might have been insufficient time to allow for
turnover of the LC n-3 PUFA in the RBC. Based on the
literature, DHA appears to have the longest half life of all
RBC omega 3 fatty acids [38]; however, in the present
study, there were no changes (increases) in the DHA pro-
portions in the RBC in any of the 3 groups. Furthermore,
there were no changes in DPA or EPA proportions in the
DPA group. The only significant change in RBC omega 3
PUFA proportions was that EPA was incorporated into the
RBC lipids in the EPA group, and the EPA value at the
start of the next treatment (DPA treatment) was not sig-
nificantly different to the EPA value at the start of the EPA
treatment (p[0.05). Therefore, we believe that in this
study, the 2-week washout period was sufficient length of
Eur J Nutr
123
time to avoid carryover of raised LC n-3 PUFA from one
treatment group to the next.
The metabolism of DPA has not been studied previously
in humans. This short-term supplementation study in
healthy volunteers demonstrated that DPA, along with
EPA, were incorporated differentially into plasma and
RBC lipid fractions. Future studies should examine the
incorporation of DPA into chylomicrons, chylomicron
remnants and VLD lipoproteins to help explain the dif-
ferential metabolism of DPA and EPA. The most novel
finding was that in the context of this short-term study,
DPA showed the evidence of metabolism to both EPA and
DHA. This suggests that DPA could function as a reservoir
or buffer of the other LC n-3 PUFA.
Acknowledgments Research support from Meat & Livestock
Australia for financial support, Equateq Ltd (UK) for the generous
provision of the pure supplements and Deakin University Strategic
Research Centre for Molecular Medicine for financial support is
gratefully acknowledged. EM, AL, DCS and AJS planned and
designed the study; EM and AL recruited the participants and col-
lected samples and dietary data; GK, EM and GT conducted the
plasma analyses; GK, SPL and GT conducted the RBC analyses; GK
conducted the statistical analysis; GK, AJS and DCS wrote the
manuscript; GK, AJS, DCS, KL and HSW made significant contri-
butions to the discussion.
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