Lipids
DOI 10.1007/s11745-014-3902-y
COMMUNICATION
Retroconversion of Docosapentaenoic Acid (n-6): an Alternative
Pathway for Biosynthesis of Arachidonic Acid in Daphnia magna
Ursula Strandberg • Sami J. Taipale
Martin J. Kainz • Michael T. Brett
•
Received: 27 November 2013 / Accepted: 20 March 2014
Ó AOCS 2014
Abstract The aim of this study was to assess metabolic
pathways for arachidonic acid (20:4n-6) biosynthesis in
Daphnia magna. Neonates of D. magna were maintained on
[13C] enriched Scenedesmus obliquus and supplemented
with liposomes that contained separate treatments of unlabeled docosapentaenoic acid (22:5n-6), 20:4n-6, linoleic
acid (18:2n-6) or oleic acid (18:1n-9). Daphnia in the
control treatment, without any supplementary fatty acids
(FA) containing only trace amounts of 20:4n-6 (*0.3 % of
all FA). As expected, the highest proportion of 20:4n-6
(*6.3 %) was detected in Daphnia that received liposomes
supplemented with this FA. Higher availability of 18:2n-6
in the diet increased the proportion of 18:2n-6 in Daphnia,
but the proportion of 20:4n-6 was not affected. Daphnia
supplemented with 22:5n-6 contained *3.5 % 20:4n-6 in
the lipids and FA specific stable isotope analyses validated
that the increase in the proportion of 20:4n-6 was due to
retroconversion of unlabeled 22:5n-6. These results suggest
that chain shortening of 22:5n-6 is a more efficient pathway
U. Strandberg (&)
Department of Biology, University of Eastern Finland,
PO Box 111, 80101 Joensuu, Finland
e-mail: [email protected]
S. J. Taipale
Department of Biological and Environmental Science,
University of Jyväskylä, PO Box 35 (YA), 40014 Jyväskylä,
Finland
to synthesize 20:4n-6 in D. magna than elongation and
desaturation of 18:2n-6. These results may at least partially
explain the discrepancies noticed between phytoplankton
FA composition and the expected FA composition in
freshwater cladocerans. Finally, retroconversion of dietary
22:5n-6 to 20:4n-6 indicates Daphnia efficiently retain long
chain n-6 FA in lake food webs, which might be important
for the nutritional ecology of fish.
Keywords Polyunsaturated fatty acid Retroconversion Docosapentaenoic acid (22:5n-6) Arachidonic acid
Abbreviations
ARA
Arachidonic acid (20:4n-6)
C18PUFA
Polyunsaturated fatty acid(s) with 18 carbons
C20PUFA
Polyunsaturated fatty acid(s) with 20 carbons
C22PUFA
Polyunsaturated fatty acid(s) with 22 carbons
DW
Dry weight
GC-IRMS Gas chromatography-isotope ratio mass
spectrometry
DPAn-6
Docosapentaenoic acid (22:5n-6)
EPA
Eicosapentaenoic acid (20:5n-3)
FA
Fatty acid(s)
FAME
Fatty acid methyl ester(s)
LNA
Linoleic acid (18:2n-6)
OLA
Oleic acid (18:1n-9)
PUFA
Polyunsaturated fatty acid(s)
M. J. Kainz
WasserCluster-Biological Station Lunz, Dr. Carl Kupelwieser
Prom. 5, 3293 Lunz am See, Austria
Introduction
M. T. Brett
Department of Civil and Environmental Engineering, University
of Washington, PO Box 352700, Seattle, WA 98195-2700, USA
Polyunsaturated fatty acids from the n-3 and n-6 families
(n-3 and n-6 PUFA) are required by most animals since
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Lipids
these fatty acids (FA) support important physiological
functions, but cannot be synthesized de novo. In crustacean
zooplankton, dietary PUFA, specifically eicosapentaenoic
acid (20:5n-3, EPA), have been suggested to support
growth and reproduction [1–3]. The role of arachidonic
acid (20:4n-6, ARA) in zooplankton is less studied [4, 5],
but in addition to being an important component of cell
membranes, 20:4n-6 is likely involved in the signal transduction, e.g., as a precursor for eicosanoids [6–8]. In
addition to direct dietary supply, zooplankton may regulate
the pool of the physiologically important C20 PUFA via
fatty acid modifications, e.g., by converting C18 PUFA to
C20 PUFA [9, 10]. The desaturation and chain elongation
of C18 PUFA to C20 PUFA seems to be taxon specific, but
is generally limited and not sufficient to support optimal
growth and reproduction [9, 10]. In addition to FA elongation and desaturation, an alternative metabolic pathway
is retroconversion of C22 PUFA to C20 PUFA. Studies on
the retroconversion of C22 PUFA in invertebrates are
scarce, but chain shortening of 22:6n-3 to 20:5n-3 has been
confirmed in marine Artemia nauplii by using radiolabeled
tracers [11]. Findings on increased proportions of C20
PUFA related to the availability of dietary C22 PUFA lead
to a similar hypothesis in daphnids, but retroconversion of
C22 PUFA to C20 PUFA has not been directly confirmed
with tracers [4, 5, 9].
The increased application of fatty acid biomarkers in
food web studies demands detailed knowledge on fatty acid
metabolism in consumers. Particularly interesting are the
fatty acid modifications in zooplankton that transfer energy
and essential molecules (such as n-3 and n-6 PUFA) from
producers to consumers at upper trophic levels, in particular fish [12]. The objective of this study was to use liposomes and stable isotopic tracers [13C] to study the
metabolic origin of 20:4n-6 in Daphnia magna. This study
used a reverse-labeling methodology to test whether the
retroconversion of n-6 docosapentaenoic acid (22:5n-6,
DPAn-6) is a more efficient pathway for 20:4n-6 synthesis
than elongation and desaturation of linoleic acid (18:2n-6,
LNA).
Materials and Methods
Daphnia magna neonates (\12 h old) were maintained on
[13C] labeled Scenedesmus obliquus for 6 days. S. obliquus
was chosen for the experimental diet because it is deficient
in 20:4n-6 and 22:5n-6 [13]. To achieve [13C] labeled S.
obliquus, 3 % of the NaHCO3 in the L16 growth medium
was replaced with NaH13CO3. Neonates were initially
reared in groups of *30 individuals per glass jar
(500 mL). On day 6, Daphnia were transferred to 40-mL
vials (1 Daphnia/vial) and maintained on [13C] labeled
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S. obliquus which was supplemented with liposomes. The
liposomes were enriched with one of the following unlabelled fatty acids: oleic acid (18:1n-9, OLA), 18:2n-6,
20:4n-6 or 22:5n-6. Liposomes were prepared from 1,2distearoyl-sn-glycero-3-phosphocholine
(pre-liposomes
formulation 5, Sigma) by adding water and heating the
formula above the phase transition temperature (64 °C).
For additional details on liposome preparation, see [1].
Fatty acids were added to the solution and mixed vigorously for 20 min. Encapsulated fatty acids were added to S.
obliquus cultures at a concentration of 30 lg of fatty acid
mg-1 of algae (DW), which corresponds to the approximate amount of 18:2n-6 in S. obliquus. The fresh algaeliposome mixtures were prepared daily (4 mg DWL-1 of
S. obliquus in the beginning of the experiment, which was
increased by 2 mg DWL-1 daily during a 5-day experimental period.
Daphnia were freeze-dried and pooled so that for each
treatment 3–5 samples were analyzed, with each sample
containing 3–4 animals. Total lipids were extracted with
chloroform/methanol/water (2:1:0.8, by volume). Extraction was facilitated with 10 min sonication. Total lipid
extracts were concentrated under a nitrogen stream. Lipids
were dissolved in toluene and transmethylated at 50 °C for
16 h, using an acidic catalyst (1 % H2SO4 in methanol).
The resulting fatty acid methyl esters (FAME) were analyzed at the University of Washington, with a HP6890 gas
chromatograph (GC), equipped with flame ionization
detection (FID), using a DB-23 capillary column (length
30 m, ID 0.25 mm, film thickness 0.25 lm, Agilent) with
helium as carrier gas (1 mL min-1). The temperature
program was as follows: initial oven temperature was set at
50 °C and maintained for 1 min, then the temperature was
increased at 10 °C min-1 to 100 °C, and subsequently
increased at 2 °C min-1 to 140 °C, thereafter the temperature was increased 1 °C min-1 to 180 °C and kept stable
for 5 min, then heated up at 2 °C min-1 to 200 °C, and
finally heated up at 10 °C min-1 to 240 °C. An external
fatty acid standard mix (37 FAME Mix, 47885-U; Supelco)
was used for peak qualification. Identification of DPAn-6
was verified with mass spectrometry by C.C. Parrish
(Ocean Sciences Centre, Memorial University, NL,
Canada).
Fatty acid specific stable isotopes were analyzed at the
University of Eastern Finland. FAME were injected into an
Agilent 6890 N GC with a DB-23 column (length 30 m, ID
0.25 mm, film thickness 0.15 lm, Agilent). The GC was
equipped with a Finnigan Delta Plus XP isotope ratio mass
spectrometer (IRMS) via the GC-III combustion interface
(Thermo-Finnigan). The temperature program was as follows: initial oven temperature 60 °C was maintained for
1.5 min, then the temperature was increased at
10 °C min-1 to 100 °C, followed by 2 °C min-1 to
Lipids
140 °C, and 1 °C min-1 to 180 °C, and finally 2 °C min-1
to 210 °C that was held for 6 min. The temperature in the
oxidation reactor was 940 °C and in the reduction reactor
630 °C. IRMS samples were analyzed as duplicates.
Hexadecanoic acid methyl ester (Indiana University, Arndt
Schimmelmann), with a d13C value of -30.74 % was used
as the internal standard for calibration and drift correction.
Fatty acid d13C values were corrected for the carbon added
during methyl esters derivatization and the d13C value of
fatty acids was manually calculated using individual
background values. The fatty acid d13C data were normalized to the total intensity of d13C within the sample (FA
d13C/total FA d13C) to correct for variability in the total
intensity between samples (which could be caused by
variable enrichment of algal cultures). Statistical analyses
were conducted with SPSS version 11.0.4.
Results and Discussion
Retroconversion of 22:5n-6 was a more efficient pathway
to synthesize 20:4n-6 than was the elongation and desaturation of 18:2n-6 in D. magna. Without any fatty acid
supplementation, the proportion of 20:4n-6 in Daphnia
maintained on S. obliquus averaged 0.3 % (SD 0.1) of total
fatty acids, whereas with 20:4n-6 and 22:5n-6 supplementation the proportion of 20:4n-6 in Daphnia was 6.2 %
(SD 1.5) and 3.5 % (SD 0.9) of all fatty acids, respectively
(Fig. 1). The fatty acid specific stable isotope analyses
Fig. 1 Mole percentage of 20:4n-6 in Daphnia magna maintained on
Scenedesmus that was supplemented with liposomes containing either
18:1n-9 (OLA), 18:2n-6 (LNA), 20:4n-6 (ARA) or 22:5n-6 (DPAn-6).
Daphnia in the control treatment were maintained on Scenedesmus
supplemented with liposomes without fatty acid additions. Columns
represent means (error bars ± SD), *P \ 0.001 compared to the
control treatment (n = 3–5), tested by ANOVA, followed a Tukey
HSD post hoc test
showed that the d13C values in 20:4n-6 were clearly
depleted in the DPAn-6 treatment, validating that this
20:4n-6 originated from the unlabelled 22:5n-6 delivered
by the liposomes (Fig. 2). The stable isotope data also
confirmed that the liposomes were ingested and assimilated
by the Daphnia; the d13C values were low for the saturated
fatty acid 18:0, which is the unlabelled fatty acid moiety of
the phospholipids in the liposomes (Fig. 2).
The enriched d13C values of the 20:4n-6 in the control
treatment indicates that small proportions of 20:4n-6
originated from the enriched algae. S. obliquus did not
contain 20:4n-6 or 22:5n-6, thus the most probable source
of 20:4n-6 in Daphnia was via chain elongation and
desaturation of 18:2n-6 [9]. Previously, negligible bioconversion of 18:3n-3 to the longer chain analog 20:5n-3
has been reported for various zooplankton taxa, including
Daphnia [9, 10]. In the current study, dietary 18:2n-6
supplementation did not influence the proportion of 20:4n6 in Daphnia (Fig. 1), but did increase the proportion of
18:2n-6 (Fig. 3). This suggests that elongation and desaturation of 18:2n-6 in Daphnia is inefficient and not regulated by substrate availability, but more likely by the
activity of elongases and desaturases, as previously suggested [10].
The proportion of 18:2n-6 in the DPAn-6 treatment did
not differ from that of the control treatment (Fig. 3). Also,
the 18:2n-6 in Daphnia was [13C] enriched in the DPAn-6
treatment indicating that this 18:2n-6 did not originate from
chain shortening of 22:5n-6 (Fig. 2). Thus the chain shortening of C22 PUFA in Daphnia does not seem to proceed
beyond C20 PUFA. The pathway for chain shortening of
PUFA is peroxisomal b-oxidation [14, 15]. Peroxisomes are
essential and ubiquitous intracellular organelles that participate in several metabolic processes, including b-oxidation. The b-oxidation in peroxisomes resembles that in
mitochondria, and one cycle shortens the chain length by
two carbons, and hydrogenates possible cis-D3 or cis-D4
double bonds [14, 15]. But contrary to mitochondria, peroxisomes do not have a major role in energy metabolism
[14, 15]. Instead the function of peroxisomal fatty acid boxidation seems to be associated with fatty acid modifications. The modified fatty acids may be transported to
mitochondria for complete catabolism, or incorporated to
lipids or processed further to, e.g., eicosanoids [14, 15].
Retroconversion of C22 PUFA to C20 PUFA may be
linked with a need to maintain a steady supply of C20
PUFA. The physiological requirement of 20:4n-6 in animals is generally attributed to its importance in signal
transduction, e.g., as precursors for eicosanoids as well as
its role in maintaining cell membrane structure and function [6, 16]. Eicosanoids are oxygenated fatty acid
metabolites that are involved in regulating reproduction
and immunity, and other physiological processes [7, 8].
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Lipids
0.25
Control
DPAn-6
13C
0.20
Normalized
Fig. 2 Normalized d13C values
(means and ranges, n = 2) for
different fatty acids in Daphnia
maintained on [13C] enriched
Scenedesmus and supplemented
with DPAn-6 via liposomes
(white bars). Daphnia in the
control treatment (grey bars)
were maintained on
Scenedesmus supplemented
with liposomes without fatty
acid additions
0.15
0.10
0.05
0.00
-0.05
16:3n-3
16:4n-3
18:0
18:2n-6
18:3n-3
18:4n-3
20:4n-6
20:5n-3
Fatty acid
Fig. 3 Mole percentage of 18:2n-6 in Daphnia magna maintained on
Scenedesmus that was supplemented with liposomes containing either
18:1n-9 (OLA), 18:2n-6 (LNA), 20:4n-6 (ARA) or 22:5n-6 (DPAn-6).
Daphnia in the control treatment were maintained on Scenedesmus
supplemented with liposomes without fatty acid additions. Columns
represent means (error bars ± SD), *P \ 0.001 compared to control
treatment (n = 3–5), tested by ANOVA, followed a Tukey HSD post
hoc test
Food quality as assessed by proportion of PUFA has been
directly linked with changes in gene expression related to
eicosanoid metabolism and reproduction in Daphnia [17].
In field surveys the content of n-3 and n-6 PUFA in seston
has been shown to correlate with zooplankton growth and
reproduction [18].
In aquatic environments, phytoplankton are the most
important source of n-3 and n-6 PUFA, but the fatty acid
profiles are taxon specific. The FA 18:2n-6 is abundant in
many phytoplankton classes, while most freshwater algae
contain only minor proportions of 20:4n-6 [13].
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Chlorophyceae and Bacillariophyceae do not contain any
22:5n-6, but this fatty acid accounts for up to 5–10 % of all
fatty acids in Chrysophyceae and Cryptophyceae [13],
which may provide, via retroconversion, an important
source of 20:4n-6 for cladocerans.
It is likely that C22 PUFA from the n-3 family undergo
similar retroconversion, although 20:5n-3 is much more
abundant in phytoplankton than is 20:4n-6 [13]. In addition
to meeting their own metabolic needs, modifications of
PUFA in zooplankton potentially alter the composition of
fatty acids that are available for consumers at the next
trophic level. The proportional enrichment of C20 PUFA at
the algae-zooplankton interface has been documented in
field surveys, suggesting potential ecosystem scale effects
of specific metabolic adaptations at taxon level [19].
Dietary C22 PUFA do not accumulate in cladocerans [20],
and our results indicate that instead of complete oxidation,
C22 PUFA are converted to C20 PUFA. This is ecologically
important if, as previously suggested [21], freshwater fish are
more efficient at converting C20 PUFA to C22 PUFA than
they are at converting C18 PUFA to C20 PUFA.
Acknowledgments We would like to thank Joseph L. Ravet for
helping with the preparation of the liposomes. In addition Tom Le and
Roxanne Russell are gratefully acknowledged for their assistance in
the laboratory and maintenance of algal cultures. This study was
financially supported by the Valle international exchange program, by
the National Science Foundation Grant 0642834 to MTB, and by the
Academy of Finland Grant (139786) to P. Kankaala.
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