Báo cáo khoa học: Metabolic pathway that produces essential fatty acids from polymethylene-interrupted polyunsaturated fatty acids in animal cells pot

Metabolic pathway that produces essential fatty acids
from polymethylene-interrupted polyunsaturated fatty
acids in animal cells
Tamotsu Tanaka
1
, Jun-ichi Morishige
1
, Dai Iwawaki
1
, Terumi Fukuhara
1
, Naomi Hamamura
1
,
Kaoru Hirano
1
, Takashi Osumi
2
and Kiyoshi Satouchi
1
1 Department of Applied Biological Science, Fukuyama University, Hiroshima, Japan
2 Graduate School of Life Sciences, University of Hyogo, Japan
Linoleic acid (18:2 D-9,12) and a-linolenic acid (18:3 D-
9,12,15) are fatty acids that are essential to animals for
maintenance of growth, reproduction and development
of brain function. Because they lack the ability to
introduce double bonds at both D-12 and D-15 positions
of a fatty acid, animals have to acquire these poly-
unsaturated fatty acids (PUFAs) from their diet.
The alignment of the double bonds of these essential
fatty acids as well as important metabolites, such as

arachidonic acid (20:4 D-5,8,11,14) and eicosapentae-
noic acid (EPA, 20:5 D-5,8,11,14,17), is interrupted by
one methylene group. On the other hand, gymnosperm
plants have PUFAs with the alignment of their double
bonds interrupted by two or more methylenes [1–4].
PUFAs with this characteristic alignment of double
bonds are categorized as polymethylene-interrupted-
PUFAs (PMI-PUFAs). Pinolenic acid (18:3, D-5,9,12),
sciadonic acid (20:3 D-5,11,14) and juniperonic acid
(20:4 D-5,11,14,17) are typical PMI-PUFAs found
in conifer plants such as Pinaceae, Taxodiaceae and
Keywords
essential fatty acids; peroxisomal
b-oxidation; polymethylene-interrupted
polyunsaturated fatty acids; polyunsaturated
fatty acid remodeling
Correspondence
T. Tanaka, Department of Applied Biological
Science, Fukuyama University, Higashimura,
Fukuyama, Hiroshima, 729-0292, Japan
Fax: +81 84 9362459
Tel: +81 84 9362111
E-mail:
(Received 22 January 2007, revised 9 March
2007, accepted 23 March 2007)
doi:10.1111/j.1742-4658.2007.05807.x
Sciadonic acid (20:3 D -5,11,14) and juniperonic acid (20:4 D-5,11,14,17) are
polyunsaturated fatty acids (PUFAs) that lack the D-8 double bond of
arachidonic acid (20:4 D-5,8,11,14) and eicosapentaenoic acid (20:5
D-5,8,11,14,17), respectively. Here, we demonstrate that these conifer oil-

derived PUFAs are metabolized to essential fatty acids in animal cells.
When Swiss 3T3 cells were cultured with sciadonic acid, linoleic acid
(18:2 D-9,12) accumulated in the cells to an extent dependent on the con-
centration of sciadonic acid. At the same time, a small amount of
16:2 D-7,10 appeared in the cellular lipids. Both 16:2 D-7,10 and linoleic
acid accumulated in sciadonic acid-supplemented CHO cells, but not in
peroxisome-deficient CHO cells. We confirmed that 16:2 D-7,10 was effect-
ively elongated to linoleic acid in rat liver microsomes. These results indi-
cate that sciadonic acid was partially degraded to 16:2 D-7,10 by two cycles
of b-oxidation in peroxisomes, then elongated to linoleic acid in micro-
somes. Supplementation of Swiss 3T3 cells with juniperonic acid, an n)3
analogue of sciadonic acid, induced accumulation of a-linolenic acid (18:3
D-9,12,15) in cellular lipids, suggesting that juniperonic acid was meta-
bolized in a similar manner to sciadonic acid. This PUFA remodeling is
thought to be a process that converts unsuitable fatty acids into essential
fatty acids required by animals.
Abbreviations
AgTLC, argentation thin-layer chromatography; EPA, eicosapentaenoic acid; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine;
PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; PMI-PUFA, polymethylene-interrupted polyunsaturated fatty acid; PUFA,
polyunsaturated fatty acid.
2728 FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS
Cupressaceae [1–4]. Some of these plant seeds are used
as food, condiments or traditional Chinese medicines
[5], and their oils are considered to be an alternative
source of edible oil [6].
PMI-PUFAs have been shown to be constituents of
membrane phospholipids of animal cells [6–11]. We
have demonstrated that sciadonic acid and juniperonic
acid are metabolized in a similar manner to arachido-
nic acid and EPA, respectively, in the process of acyla-

tion to phospholipids in HepG2 cells [12,13]. We also
have demonstrated that membrane phosphatidylinosi-
tol (PtdIns) containing sciadonate can be converted
into PtdIns 4,5-bisphosphate and subsequently to
diacylglycerol in response to agonistic stimulation.
Despite the resulting diacylglycerol containing an
unusual PUFA residue for animals, it can effectively
activate protein kinase C [14]. The metabolism of
PMI-PUFAs in the processes of oxidation [15] and
chain elongation [16] in animal cells has also been
investigated. However, the metabolic fate of PMI-
PUFAs in animal cells is not fully understood.
In this study, we found evidence that sciadonic acid
and juniperonic acid are converted into linoleic acid
and a-linolenic acid, respectively, in animal cells. This
metabolic pathway can be regarded as a way to produce
essential fatty acids from gymnosperm PMI-PUFAs.
Results
Sciadonic acid-dependent formation of linoleic
acid and 16:2 D-7,10 in Swiss 3T3 cells
The fatty acid compositions of phosphatidylcholine
(PtdCho) and triaclglycerol of Swiss 3T3 cells cultured
with purified sciadonic acid are shown in Table 1.
Consistent with our previous results and other reports
[6–14], sciadonic acid is available for acyl residues of
glycerolipids of animal cells. In fact, 31.1% and 51.9%
of the fatty acid acylated in PtdCho and triacylgly-
cerol, respectively, was sciadonic acid under our
experimental conditions. The sciadonic acid-supple-
mented cells showed increased concentrations of

linoleic acid as shown in Table 1 and Fig. 1. Dihomo-
c-linolenic acid, a common C
20
PUFA in animals, has
double bonds at D-8,11,14, and therefore it is an
isomer of sciadonic acid. Supplementation of cells with
dihomo-c-linolenic acid did not increase the concen-
tration of linoleic acid, as shown in Fig. 1.
Another feature of the fatty acid composition of
lipids of sciadonic acid-supplemented cells was the
appearance of an unknown fatty acid. This fatty acid,
identified as 16:2 D-7,10 by the following experimental
results, is an important metabolite in the metabolic
Table 1. Fatty acid composition of PtdCho and triacylglycerol of Swiss 3T3 cells incubated with sciadonic acid (20:3 D-5,11,14). Swiss
3T3 cells were incubated with or without 50 l
M sciadonic acid for 24 h. The cellular lipids were extracted and separated into each lipid class
by TLC. The fatty acid composition was analyzed by GC after methanolysis. Values are weight percentages of total fatty acid, given as the
mean ± SD (three cell harvests). The peak number corresponds to the number at the top of the peak on GC shown in Fig. 2A. 16:2 (D-7,10),
detected as unknown fatty acid. SciA, Sciadonic acid; ND, not detected.
Peak
no. Fatty acid
PtdCho Triacylglycerol
Control SciA Control SciA
1 14:0 0.8 ± 0.1 2.2 ± 0.7 2.1 ± 0.3 1.8 ± 1.0
2 16:0 36.7 ± 1.0 35.8 ± 2.2 24.1 ± 0.2 8.4 ± 1.0
3 16:1 (D-9) 5.5 ± 0.3 2.9 ± 0.2 5.9 ± 1.1 2.1 ± 0.7
4 16:2 (D-7,10) ND 1.2 ± 0.2 ND 3.2 ± 0.7
5 18:0 8.7 ± 0.5 2.8 ± 0.8 14.0 ± 0.3 2.7 ± 1.7
6 18:1 (D-9) 33.0 ± 2.2 5.8 ± 0.5 36.5 ± 1.3 6.7 ± 2.3
7 18:1 (D-11) 7.2 ± 0.3 1.8 ± 0.2 7.4 ± 0.4 1.5 ± 0

8 18:2 (D-9,12) 1.0 ± 0.1 8.5 ± 2.9 0.6 ± 0.2 8.2 ± 1.7
9 18:3 (D-6,9,12) ND ND ND 0.7 ± 0.2
10 18:3 (D-9,12,15) ND ND ND 0.9 ± 0.7
20:3 (D-5,8,11) 2.3 ± 0.2 0.5 ± 0.2 2.9 ± 0.5 ND
11 20:3 (D-5,11,14) (SciA) ND 31.1 ± 3.4 ND 51.9 ± 5.5
12 20:4 (D-5,8,11,14) 1.9 ± 0.4 1.7 ± 0.4 0.9 ± 0.1 1.7 ± 1.1
13 20:5 (D-5,8,11,14,17) 0.7 ± 0.2 ND 0.3 ± 0.1 0.3 ± 0.3
14 22:3 (D-7,13,16) (SciA + C2)
a
ND 3.8 ± 1.0 ND 5.3 ± 0.2
15 22:4 (D-7,10,13,16) 0.7 ± 0 0.4 ± 0.1 0.6 ± 0.1 1.6 ± 0.4
16 22:5 (D-7,10,13,16,19) 1.0 ± 0.1 0.7 ± 0 2.6 ± 0.4 2.5 ± 0.6
17 22:6 (D-4,7,10,13,16,19) ND ND 2.6 ± 0.2 0.8 ± 0.1
a
Chain-elongated metabolite.
T. Tanaka et al. Polymethylene-interrupted fatty acid metabolism
FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS 2729
pathway described in this paper. This unknown fatty
acid was detected just after the methyl palmitoleate
(16:1 D-9) on GC (Fig. 2A, peak 4), suggesting that it
was an unsaturated C
16
fatty acid. We isolated it from
cellular lipids of sciadonic acid-supplemented Swiss
3T3 cells by argentation TLC (AgTLC). In our
AgTLC system, the methyl ester of the unknown fatty
acid (R
F
0.41) was located just below the methyl linol-
eate (R

F
0.45) on the TLC plate, suggesting that it was
dienoic acid. The isolated methyl ester of the unknown
fatty acid was partially hydrogenated to yield mono-
enoates that retain one of the double bonds of the par-
ent fatty acid. This treatment gave two species of
monoenoate which can be separated by AgTLC (R
F
0.36 and 0.31) with another solvent system, and they
were separately converted into dimethyl disulfide
adducts for GC-MS. The mass spectra and possible
structures of fragment ions are shown in Fig. 2B. The
set of three intense fragment ions were generated by
cleavage between the methylthio-substituted carbons.
They clearly showed that the two species of monoeno-
ates formed by the partial hydrogenation of the
unknown fatty acid were 16:1 D-7 and 16:1 D-10.
Thus, the unknown fatty acid detected in PtdCho and
the triacylglycerol fraction of sciadonic acid-supple-
mented cells (Table 1) was identified as 16:2 D-7,10. It
was detected only in sciadonic acid-supplemented cells.
In neither the control cells nor the cells cultured with
dihomo-c-linolenic acid was it present at a detectable
level.
The formation of 16:2 D-7,10 and accumulation of
linoleic acid in cellular lipids depended on the concen-
tration of sciadonic acid in the culture medium
(Fig. 3A). Despite the considerable increase in the level
0
5

10
15
18:2 -9,12 concentration (%)
PtdCho PtdEtn PtdSer PtdIns
TG
Lipid class
+20:3 -5,11,14
+20:3
-8,11,14
Control
Fig. 1. Level of linoleic acid (18:2 D-9,12) in fatty acids acylated in
PtdCho, phosphatidylethanolamine (PtdEtn), phosphatidylserine
(PtdSer), PtdIns and triacylglycerol of Swiss 3T3 cells. Swiss
3T3 cells were cultured without fatty acid (Control) or with 50 l
M
sciadonic acid (20:3 D-5,11,14) or dihomo-c-linolenic acid (20:3
D-8,11,14) for 24 h. Each lipid class was isolated from the cellular
lipids by TLC, and the fatty acid composition was determined by
GC. Values are weight percentages, given as the mean ± SD (three
cell harvests).
A
B
Fig. 2. Identification of 16:2 D-7,10 detected in Swiss 3T3 cells cul-
tured with sciadonic acid (20:3 D-5,11,14).Swiss 3T3 cells were
cultured with 50 l
M sciadonic acid. (A) Fatty acid methyl esters pre-
pared from the triacylglycerol fraction of the cells were analyzed by
GC. The number at the top of the peak on the chromatogram cor-
responds to the peak number in Table 1. Peak number 4 corres-
ponds to the methyl ester of 16:2 D-7,10. (B) The isolated methyl

ester of 16:2 D-7,10 was partially hydrogenated with hydrazine
monohydrate. The two resulting species of 16:1 were separately
isolated, and converted into dimethyl disulfide adducts for analysis
by GC-MS.
Polymethylene-interrupted fatty acid metabolism T. Tanaka et al.
2730 FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS
of linoleic acid in cellular lipids, formation of arachi-
donic acid was not apparent. Possibly, enrichment of
sciadonate in cellular lipids inhibited the metabolic
conversion of linoleate into arachidonate. This effect is
not intrinsic to sciadonic acid because PUFA enrich-
ment of cells is known to inhibit the desaturase activity
required for PUFA synthesis [17]. 16:2 D-7,10 was
detected in the cellular lipids when the cells were incu-
bated in >50 lm sciadonic acid. We confirmed that
neither purified sciadonic acid nor Torreya nucifera
seed oil, the source of the sciadonic acid in this study,
contains 16:2 (D-7,10) at a detectable level. From
these observations, it was deduced that 16:2 D-7,10
and linoleic acid were formed as a result of the meta-
bolism of sciadonate.
A possible metabolic route for the synthesis of
16:2 D-7,10 and linoleic acid from sciadonic acid is
shown in Fig. 4. In this pathway, 16:2 D-7,10 emerges
as a result of two cycles of b-oxidation of sciadonic
acid. The linoleic acid is formed by chain elongation
of 16:2 D-7,10. The resulting linoleic acid is converted
into a higher metabolite, such as arachidonic acid. A
significant proportion of the sciadonic acid incorpor-
ated into the cells seemed to be metabolized by this

pathway. When Swiss 3T3 cells were incubated with
50 lm sciadonic acid, 24 lg sciadonic acid per dish
was present in the cellular lipids (data not shown). At
that time, increments in the concentration of
16:2 D-7,10, linoleic acid and arachidonic acid in the
cellular lipids were calculated to be 0.7 , 3.5 and
0 lg ⁄ dish, respectively (Fig. 3A). The sum of these
metabolites of sciadonic acid was calculated to be
4.2 lg ⁄ dish. The ratio between sciadonic acid and its
metabolites was  6:1.
Juniperonic acid, an n)3 analogue of sciadonic acid,
is also a conifer-derived PMI-PUFA (Fig. 4). When
Swiss 3T3 cells were cultured with purified juniperonic
acid, the amounts of a-linoleate and EPA in the cellu-
lar lipids increased depending on the concentration of
juniperonic acid (Fig. 3B). When we analyzed the fatty
acid of each lipid class in the control cells, a-linolenic
acid was not detected in any lipid class. In contrast, it
emerged as a fatty acid residue of PtdCho and phos-
phatidylethanolamine (PtdEtn) at 2.9% and 1.0%,
respectively, in juniperonic acid-supplemented cells. In
control cells, the concentrations of EPA in fatty acids
of PtdCho, PtdEtn and PtdIns were 0.7%, 2.5% and
1.7%, respectively. On the other hand, the concentra-
tions of EPA in PtdCho, PtdEtn, and PtdIns in junipe-
ronic acid-supplemented cells were 2.5%, 4.5% and
4.5%, respectively. These results indicate that the
metabolic pathway works not only for sciadonic acid
but also for juniperonic acid, an n) 3 analogue of scia-
donic acid.

Involvement of peroxisomal b-oxidation in the
chain shortening of C
20
PMI-PUFAs
It is known that peroxisomal b-oxidation of long-chain
fatty acids in animals results in chain shortening with
only a few b-oxidation cycles [18]. Such metabolism of
long-chain fatty acids is called retroconversion and is
known to be an important process in the biosynthesis
of docosahexaenoic acid (22:6 D-4,7,10,13,16,19) from
24:6 D-6,9,12,15,18,21 [19–21]. We investigated the
possibility that partial degradation of sciadonic acid to
0
5
10
µ
g/dish
0 25 50 75 100
20:3 ( -5,11,14) (
µ
M)
16:2(
-7,10)
18:2(
-9,12)
20:4(
-5,8,11,14)
A
0
2

4
µ
g/dish
0 25 50 75 100
20:4 (
-5,11,14,17) (
µ
M)
18:3(
-9,12,15)
20 :5(
-5,8,11,14,17)
B
Fig. 3. Supplementation of C
20
PMI-PUFAs results in the accumula-
tion of common PUFAs in Swiss 3T3 cells. Swiss 3T3 cells were
incubated with an increasing concentration of sciadonic acid (20:3
D-5,11,14) (A) or juniperonic acid (20:4 D-5,11,14,17) (B) for 30 h.
After extraction, the cellular lipids were mixed with 15 lg 20:0 as
an internal standard and subjected to methanolysis for GC analysis.
The amounts of fatty acids were determined from the peak area
and expressed as lg ⁄ dish ( 2 · 10
6
cells). Experiments were con-
ducted in duplicate, and the mean values are given. Similar results
were obtained in another two independent experiments with differ-
ent cultures of Swiss 3T3 cells.
T. Tanaka et al. Polymethylene-interrupted fatty acid metabolism
FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS 2731

16:2 ( D-7,10) occurs in peroxisomes. The cells used for
this experiment were CHO-K1 and ZP102, the wild-
type and a peroxisome-deficient mutant of CHO cells,
respectively [22]. The loss of PEX5, which encodes the
peroxisome targeting signal-1 receptor, has been shown
to cause deficiency of peroxisomes in ZP102 [22].
When CHO-K1 and ZP102 cells were incubated with
sciadonic acid, sciadonic acid was incorporated into
the cellular lipids of both (Fig. 5A,C). Sciadonic acid-
dependent accumulation of linoleic acid and
16:2 D-7,10 was observed in the cellular lipids of
the wild-type cells (Fig. 5B). On the other hand, the
Fig. 4. Schematic representation of meta-
bolic pathway for synthesis of essential fatty
acids from C
20
polymethylene-interrupted
polyunsaturated fatty acids.
Fig. 5. Requirement of peroxisomes for for-
mation of 16:2 D-7,10 and linoleic acid (18:2
D-9,12) from sciadonic acid (20:3 D-5,11,14)
in CHO cells. CHO-K1 cells (A, B) and perox-
isome-deficient CHO cells (C, D), namely
ZP102 cells, were incubated with various
concentrations of sciadonic acid for 48 h.
The cellular lipids were mixed with 1 lg
20:0 as an internal standard and subjected
to methanolysis for GC analysis. The
amounts of fatty acids were determined
from the peak area, and expressed as

lg ⁄ dish ( 1 · 10
7
cells). Experiments were
conducted in triplicate, and values are given
as the mean ± SD.
Polymethylene-interrupted fatty acid metabolism T. Tanaka et al.
2732 FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS
cellular lipids of ZP102 cells did not accumulate
16:2 D-7,10 or linoleic acid, even though a high con-
centration of sciadonic acid was used (Fig. 5D). These
results clearly show that peroxisomes are required for
the formation of 16:2 D-7,10 from sciadonic acid, and
that 16:2 D-7,10 is essential for the biosynthesis of
linoleic acid. The absence of 16:2 D-7,10 in sciadonic
acid-supplemented ZP102 cells is an another guarantee
of the purity of the sciadonic acid used in this study.
In analogy with the results obtained with Swiss
3T3 cells, the peroxisomes of CHO-K1 cells meta-
bolized a significant proportion of sciadonic acid.
When the cells were cultured with 50 lm sciadonic acid
(Fig. 5A,B), 14.6 lg sciadonic acid per dish was pre-
sent in the cellular lipids. At that time, increments in
the concentration of 16:2 D-7,10, linoleic acid and
arachidonic acid in the cellular lipids were calculated
to be 0.3, 2.5 and 0 lg ⁄ dish, respectively. Thus, the
ratio between sciadonic acid and the sum of its meta-
bolites via peroxisomes was  5:1.
Conversion of 16:2 D-7,10 into linoleic acid
by fatty acid chain-elongation system of
microsomes

We isolated 16:2 D-7,10 from the cellular lipids of scia-
donic acid-supplemented Swiss 3T3 cells, and investi-
gated the efficacy of its conversion into linoleic acid by
the fatty acid chain-elongation system in microsomes
(Fig. 6). When 16:2 D-7,10 was incubated with
[2-
14
C]malonyl-CoA in the presence of microsomes, an
intensely radiolabeled fatty acid was formed. The labe-
led fatty acid migrated at a position corresponding to
linoleic acid on AgTLC, indicating that [2-
14
C]malo-
nyl-CoA was incorporated into 16:2 D-7,10 to form
linoleic acid. On the other hand, little linoleic acid
(18:2 D-9,12) was converted into 20:2 D-11,14 under
the same experimental conditions. Interestingly, the
efficacy of the conversion of 16:2 D-7,10 into linoleic
acid was similar to that of c-linolenic acid into
dihomo-c-linolenic acid, a common chain-elongation
process in PUFA synthesis in animal cells.
Discussion
Over the past 40 years, a number of studies on the
metabolism of PMI-PUFAs in animals have been
conducted. In 1965, Takagi [23] reported that feeding
sciadonate to rats that had been kept in an essential
fatty acid-deficient condition caused an increase in
the concentration of arachidonate in the lipids of the
liver. From this observation, he suggested the possi-
bility that desaturation of sciadonic acid at the D-8

position occurred in the rat liver. However, conclu-
sive evidence of the presence of desaturase activity
that directly converts sciadonic acid (20:3 D-5,11,14)
into arachidonic acid (20:4 D-5,8,11,14) has not yet
been reported. On the contrary, evidence of the inab-
ility of animals to synthesize arachidonic acid from
sciadonic acid has accumulated [24–28]. We also
showed that desaturation at the D-8 position of scia-
donic acid did not occur in rat liver microsomes [13].
In this report, we present evidence that animals can
synthesize arachidonic acid from sciadonic acid inde-
pendently of D-8 desaturase activity. The metabolic
pathway that synthesizes arachidonic acid from
sciadonic acid includes chain shortening and chain
elongation of the fatty acid (Fig. 4). Because the for-
mer process removes four carbon atoms from the
carboxyl terminus of sciadonic acid, arachidonic acid
formed from sciadonic acid does not contain the
original carboxy group. This is one of the reasons
why many investigators, including ourselves, did not
observe the formation of arachidonic acid from
0
0.1
0.2
0.3
0.4
0.5
Labeled fatty acid
(nmol / min / mg protein)
18:3

(
-6,9,12)
20:3
(
-8,11,14)
18:2
(
-9,12)
20:2
(
-11,14)
16:2
(
-7,10)
18:2
(
-9,12)
Fig. 6. Conversion of 16:2 D-7,10 into linoleic acid (18:2 D-9,12) by
the fatty acid chain-elongation system in rat liver microsomes.
c-linolenic acid (18:3 D-6,9,12), 16:2 D-7,10 or linoleic acid was incu-
bated with [2-
14
C]malonyl-CoA in the presence of rat liver micro-
somes. The fatty acids formed were analyzed by AgTLC as fatty
acid methyl esters. In the experiments with 16:2 D-7,10 and 18:2
D-9,12, the radioactivity located in the dienoate fraction was regar-
ded as the chain-elongated metabolite. In the experiments with
18:3 D -6,9,12, the radioactivity located in the trienoate plus tetra-
enoate fraction was regarded as the chain-elongated metabolite.
T. Tanaka et al. Polymethylene-interrupted fatty acid metabolism

FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS 2733
sciadonic acid, because we and others conducted
experiments using [1-
14
C]sciadonic acid.
The key metabolite in this process is 16:2 D-7,10.
This fatty acid was initially detected at a low level as
an unknown fatty acid in the cells cultured with
sciadonic acid. We determined it that it was a 16:2
fatty acid with double bonds at D-7 and D-10 by GC-
MS. It is known that peroxisomal b-oxidation does
not go to completion, but results in chain shortening
by only a few b-oxidation cycles [18]. Our data
obtained from experiments using CHO cells clearly
show that peroxisomal b-oxidation was involved in the
formation of 16:2 D-7,10 from sciadonic acid. It is not
known why peroxisomal b-oxidation of sciadonate
stops at C
16
, but it might be explained by the substrate
specificity of acyl-CoA thioesterase in peroxisomes.
This enzyme in purified peroxisomes has been shown
to have high activity towards acyl-CoAs with a chain
length of C
16
rather than C
20
[29].
The efficacy of the chain elongation of 16:2 D-7,10 to
linoleic acid was comparable to that producing di-

homo-c-linolenic acid (20:3 D-8,11,14) from c-linolenic
acid (18:3 D-6,9,12), the common process of C
20
PUFA
biosynthesis. These results are consistent with in vivo
results demonstrating the formation of linoleic acid
from 16:2 D-7,10 [30,31]. As not much 16:2 D-7,10
accumulated in cellular lipids compared with linoleic
acid, a large proportion of the 16:2 D-7,10, once expor-
ted from peroxisomes, must be quickly metabolized to
linoleic acid by the fatty acid chain-elongation system
in microsomes. The observed accumulation of linoleic
acid before 16:2 D-7,10 in cellular lipids may be
explained by this rapid conversion. Juniperonic acid
is an n )3 analogue of sciadonic acid. When we used
juniperonic acid as the supplement, the amounts of
both a-linolenic acid (18:3 D-9,12,17) and EPA (20:5 D-
5,8,11,14) increased in cellular lipids to an extent
dependent on the juniperonic acid supplementation.
This indicates that juniperonic acid is processed by a
metabolic pathway similar to that of sciadonic acid
(Fig. 4). It is not known why 16:3 D-7,10,13, an expec-
ted peroxisomal metabolite of juniperonic acid, was not
detected in cellular lipids. The efficiency of the conver-
sion of 16:3 D-7,10,13 into a-linolenic acid may be
greater than that of the corresponding n)6 analogue.
As described in the Results section, a significant pro-
portion of the sciadonate seemed to be processed by
peroxisomes in both Swiss 3T3 cells and CHO cells. It
should be noted that supplementation with dihomo-c-

linolenic acid, a common PUFA in animals, did not
affect the cellular concentration of linoleic acid
(Fig. 1). Dihomo-c-linolenic acid, an isomer of scia-
donic acid, might be an unsuitable substrate for this
metabolic pathway. It is known that arachidonic acid
is converted into linoleic acid by peroxisomal b-oxida-
tion, a process known as retroconversion [32]. It is
interesting to compare the efficiency of peroxisomal
metabolism of PMI-PUFAs with that of common
PUFAs to determine substrate preference. As men-
tioned above, feeding sciadonate to rats that had been
kept in an essential fatty acid-deficient condition
increased arachidonate in the liver. This increase was
comparable to that in a linoleate-fed group [23]. It is
therefore possible that peroxisomal b-oxidation of
sciadonate can supply the physiological requirement
for essential fatty acids in animals grown in essential
fatty acid-deficient conditions.
As the data presented here are limited to rodent
cells, it is not known whether this metabolic pathway
exists in human cells. However, the fact that peroxi-
somal retroconversion of long-chain fatty acids such as
24:6 is known to occur in humans cells [33] indicates
that the metabolic pathway identified here may also be
active in human cells.
Gymnosperms are widely distributed. Sciadonic acid
and juniperonic acid are known to be present in sev-
eral species of coniferous plants [1–4]. In the present
times, seeds of gymnosperms are not eaten on a com-
mercial scale, but some coniferous plant seeds are used

in traditional diets. For example, the roasted seeds of
Torreya nucifera, which contain sciadonic acid (10%
of total fatty acid), are eaten as a snack food in Japan.
The metabolism of PMI-PUFAs shown here is thought
to be the process by which unsuitable fatty acids are
converted into the essential fatty acids required by ani-
mals. It has been proposed that one of the roles of
peroxisomal b-oxidation is recycling of PUFAs [34].
The present observations add new insight into the role
of peroxisomal b-oxidation of fatty acids.
Experimental procedures
Materials
DMEM, penicillin, streptomycin and fetal bovine serum
were obtained from Gibco BRL and Life Technologies,
Inc. (Rockville, MD, USA). ATP, CoA, malonyl-CoA and
essentially fatty acid-free BSA were from Sigma Chemical
Co. (St Louis, MO, USA). [2-
14
C]Malonyl-CoA was from
Amersham (Arlington Heights, IL, USA). NADPH was
purchased from Oriental Yeast Co. (Tokyo, Japan). Lino-
leic acid, c-linolenic acid and dihomo-c-linolenic acid were
purchased from Serdary Research Laboratories (London,
ON, Canada). Sciadonic acid and juniperonic acid were
prepared from seeds of Torreya nucifera and Biota orient-
alis, respectively, as described previously [35]. The seeds
Polymethylene-interrupted fatty acid metabolism T. Tanaka et al.
2734 FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS
were milled in methanol, and lipids were extracted by the
method of Folch et al. [36]. The lipids were subjected to

methanolysis, and the methyl sciadonate and methyl juni-
peronate were purified from the fatty acid methyl esters by
AgTLC [35]. They were used after saponification. As
judged by GC analysis, the purities of sciadonic acid and
juniperonic acid were 99.0% and 100.0%, respectively. All
other reagents were of reagent grade.
Cell culture and analysis of cellular lipids
Swiss 3T3 cells were obtained from American Type Culture
Collection (Manassa, VA, USA). They were seeded in 100-
mm plastic dishes at 4 · 10
5
cells ⁄ dish and maintained in
10 mL DMEM containing 10% fetal bovine serum in a
humidified atmosphere of 10% CO
2
⁄ 90% air at 37 °C.
After they had grown to confluence, 50 lm sciadonic acid
was added to the cell cultures as a BSA complex [13]. After
24 h, the cells were harvested, and the lipids of the cells
were extracted by the method of Bligh & Dyer [37]. Each
phospholipid class and triacylglycerol was isolated by TLC
as described previously [13]. After preparation of fatty acid
methyl esters, the fatty acid compositions of the lipid clas-
ses were analyzed by GC (Shimadzu GC-14A; Shimadzu,
Kyoto, Japan) equipped with a capillary column coated
with CBP 20 (0.25 lm film, 30 m length; Shimadzu) [35].
The amount of each fatty acid in the cellular lipids was
determined by GC using arachidic acid (20:0) as the inter-
nal standard. CHO cells and a peroxisome-deficient mutant
of CHO cells, named ZP102 [22], were seeded in 100-mm

plastic dishes at 1 · 10
5
cells ⁄ dish. After 48 h, sciadonic
acid was added to the cell cultures as a BSA complex. Cel-
lular lipids were analysed as described above.
Structural analysis of 16:2 D-7,10 detected
in cellular lipids
The 16:2 D-7,10 detected in cellular lipids was analyzed by
GC-MS after derivatization. First, fatty acid methyl esters
were prepared by methanolysis of the triacylglycerol frac-
tion of the Swiss 3T3 cells, and the methyl ester of
16:2 D-7,10 was purified by AgTLC developed with petro-
leum ether ⁄ diethyl ether ⁄ acetic acid (80:15:2, v ⁄ v ⁄ v.). Then,
the isolated methyl ester of 16:2 D-7,10 was partially hydro-
genated with hydrazine monohydrate at 50 °C for 25 min
[35]. The two resulting isomers of the methyl ester of 16:1
that retained one of the double bonds in 16:2 D-7,10 were
separately isolated by AgTLC (R
F
0.36 and 0.31) developed
with petroleum ether ⁄ diethyl ether ⁄ acetic acid (90:10:2,
v ⁄ v ⁄ v), and converted into dimethyl disulfide adducts for
analysis by GC-MS as described in [35]. Analysis of the
dimethyl disulfide adducts of the methyl ester of 16:1
was carried out on a Shimadzu QP-2000 quadrupole mass
spectrometer equipped with an interface for capillary GC
(column coated with a 0.25 lm film of nonpolar CBJ1;
0.25 mm · 30 m; Shimadzu). The column temperature was
raised from 215 °C to 265 °C at a rate of 5 °CÆmin
)1

. The
temperature of the injection port, ion source and interface
was set at 250 °C. Helium was used as the carrier gas. The
ionization energy was 70 eV.
Fatty acid chain-elongation assay
The 16:2 D-7,10 used in this assay was prepared from the
cellular lipids from 160 (100-mm) dishes of sciadonic acid-
supplemented Swiss 3T3 cells grown as described above.
The purity of 16:2 D-7,10 was 92.0%. The remaining 8%
was linoleic acid. The fatty acid chain-elongation assay was
conducted as described previously [16]. In brief, each incu-
bation consisted of 0.5 mm nicotinamide, 1.5 mm glutathi-
one, 0.15 m KCl, 5 mm MgCl
2
, 0.25 m sucrose, 7.5 mm
ATP, 0.4 mm CoA, 1.5 mm NADPH, 0.1 mm [2-
14
C]malo-
nyl-CoA (0.05 lCi ⁄ 0.2 lmol), 50 mm potassium phosphate
buffer (pH 7.0), 3.0 mg microsomal protein prepared from
the liver of a Sprague-Dawley rat, and the desired fatty
acid in a total volume of 2.0 mL. Fatty acids were added
as a BSA complex, and incubation was conducted at 37 °C
for 30 min. After the incubation, lipids in the reaction mix-
ture were subjected to alkali hydrolysis, and the released
fatty acids were analyzed by AgTLC after conversion into
fatty acid methyl esters. When 16:2 D-7,10 and linoleic acid
were used as substrates, the radioactivity of the dienoate
fraction was determined. When c-linolenic acid was used,
the radioactivity of the trienoate plus tetraenoate fraction

was determined because of the rapid conversion of dihomo-
c-linolenic acid into arachidonic acid.
References
1 Smith CR Jr (1970) Occurrence of unusual fatty acids
in plants. In Progress in Chemistry of Fats and Other
Lipids (Holman RT, ed.), Vol. XI, Part 1, pp. 137–177.
Pergamon Press, London.
2 Takagi T & Itabashi Y (1982) cis-5-Olefinic unusual
fatty acids in seed lipids of gymnospermae and their
distribution in triacylglycerols. Lipids 17, 716–723.
3 Wolff RL (1998) Source of 5,11,14–20:3 (sciadonic)
acid, a structural analog of arachidonic acid. J Am Oil
Chem Soc 75, 1901–1902.
4 Wolff RL, Deluc LG & Marpeau AM (1996) Conifer
seeds: oil content and fatty acid composition. J Am Oil
Chem Soc 73, 765–771.
5 Nishitama N, Chu PJ & Saito H (1995) Beneficial
effects of biota, a traditional Chinese herbal medicine,
on learning impairment induced by basal forebrain-
lesion in mice. Biol Pharm Bull 18, 1513–1517.
6 Pasquier E, Ratnayake WMN & Wolff RL (2001)
Effects of D5 polyunsaturated fatty acids of maritime
pine (Pinus pinaster) seed oil on the fatty acid profile of
the developing brain of rats. Lipids 36, 567–574.
T. Tanaka et al. Polymethylene-interrupted fatty acid metabolism
FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS 2735
7 Sugano M, Ikeda I, Wakamatsu K & Oka T (1994)
Influence of Korean pine (Pinus koraiensis)-seed oil
containing cis-5,cis-9,cis-12 -octadecatrienoic acid on
polyunsaturated fatty acid metabolism, eicosanoid pro-

duction and blood pressure of rats. Br J Nutr 72, 775–
783.
8 Matsuo N, Osada K, Kodama T, Lim BO, Nakao A &
Sugano M (1996) Effects of c-linolenic acid and its posi-
tional isomer pinolenic acid on immune parameters of
Brown-Norway rats. Prostaglandins Leukot Essent Fatty
Acids 55, 223–229.
9 Ikeda I, Oka T, Koba K, Sugano M & Lie Ken Jie
MSF (1992) 5c,11c,14c-Eicosatrienoic acid and
5c,11c,14c,17c-eicosatetraenoic acid of Biota orientalis
seed oil affect lipid metabolism in the rat. Lipids 27,
500–504.
10 Berger A, Fenz R & German JB (1993) Incorporation
of dietary 5,11,14-icosatrienoate into various mouse
phospholipid classes and tissues. J Nutr Biochem 4,
409–419.
11 Berger A & German JB (1991) Extensive incorporation
of dietary D-5,11,14-eicosatrienoate into the phosphati-
dylinositol pool. Biochim Biophys Acta 1085, 371–376.
12 Tanaka T, Takimoto T, Morishige J, Kikuta Y, Sugiura
T & Satouchi K (1999) Non-methylene interrupted poly-
unsaturated fatty acids: effective substitute for arachido-
nate of phosphatidylinositol. Biochem Biophys Res
Commun 264, 683–688.
13 Tanaka T, Morishige J, Takimoto T, Takai Y &
Satouchi K (2001) Metabolic characterization of sciado-
nic acid (5c,11c,14c-eicosatrienoic acid) as an effective
substitute for arachidonate of phosphatidylinositol. Eur
J Biochem 268, 4928–4939.
14 Morishige J, Takai Y, Hirano K, Tanaka T & Satouchi

K (2005) Production and protein kinase C activation of
diacylglycerols containing polymethylene-interrupted
PUFA. Lipids 40, 155–162.
15 Ide T, Murata M & Sugano M (1995) Octadecatrienoic
acids as the substrates for the key enzymes in glyceroli-
pid biosynthesis and fatty acid oxidation in rat liver.
Lipids 30, 755–762.
16 Tanaka T, Hattori T, Kouchi M, Hirano K & Satouchi
K (1998) Methylene-interrupted double bond in poly-
unsaturated fatty acid is an essential structure for meta-
bolism by fatty acid chain elongation system of rat
liver. Biochim Biophys Acta 1393 , 299–306.
17 Ramanadham S, Zhang S, Ma Z, Wohltmann M,
Bohrer A, Hsu F-F & Turk J (2002) D6-, Stearoyl
CoA-, and D5-desaturase enzymes are expressed in
b
-cells and are altered by increases in exogenous PUFA
concentrations. Biochim Biophys Acta 1580, 40–56.
18 Kunau W-H, Dommes V & Schulz H (1995) b-oxida-
tion of fatty acids in mitochondoria, peroxisomes, and
bacteria: a century of continued progress. Prog Lipid
Res 34, 267–342.
19 Voss A, Reinhart M, Sankarappa S & Sprecher H
(1991) The metabolism of 7,10,13,16,19-docosapentae-
noic acid to 4,7,10,13,16,19-docosahexaenoic acid in rat
liver is independent of a 4-desaturase. J Biol Chem 266,
19995–20000.
20 Caruso D, Rise P, Galella G, Regazzoni C, Toia A,
Galli G & Galli C (1994) Formation of 22 and 24 car-
bon 6-desaturated fatty acids from exogenous deuter-

ated arachidonic acid is activated in THP-1 cells at high
substrate concentrations. FEBS Lett 343, 195–199.
21 Mohammed BS, Sankarappa S, Geiger M & Sprecher H
(1995) Reevaluation of the pathway for the metabolism
of 7,10,13,16-docosatetraenoic acid to 4,7,10,13,16-doco-
sapentaenoic acid in rat liver. Arch Biochem Biophys
317, 179–184.
22 Tsukamoto T, Bogaki A, Okumoto K, Tateishi K,
Fujiki Y, Shimozawa N, Suzuki Y, Kondo N &
Osumi T (1997) Isolation of a new peroxisome-defi-
cient CHO cell mutant defective in peroxisome target-
ing signal-1 receptor. Biochem Biophys Res Commun
230, 402–406.
23 Takagi T (1965) The dehydrogenation of all cis-5,11,
14-eicosatrienoic acid to arachidonic acid. Bull Chem
Soc Jpn 38, 2055–2057.
24 Dhopeshwarkar GA & Subramanian C (1976) Intracra-
nial conversion of linoleic acid to arachidonic acid: evi-
dence for lack of D 8 desaturase in the rat brain.
J Neurochem 26, 1175–1179.
25 Naval J, Martinez-Lorenzo MJ, Marzo I, Desportes P
& Pineiro A (1993) Alternative route for the biosynth-
esis of polyunsaturated fatty acids in K562 cells. Bio-
chem J 291, 841–845.
26 Dhopeshwarkar GA (1976) Biosynthesis of polyunsatu-
rated fatty acids in the developing brain. II. Metabolic
transformations of intracranially administrated [3-
14
C]
eicosapentanoic acid, evidence for lack of D8 desaturase.

Lipids 11, 689–692.
27 Sprecher H & Lee C-J (1975) The absence of an 8-desa-
turase in rat liver: a re-evaluation of optional pathways
for the metabolism of linoleic and linolenic acids. Bio-
chim Biophys Acta 388, 113–125.
28 Chen Q, Yin FQ & Sprecher H (2000) The questionable
role of a microsomal D8 acyl-CoA-dependent desaturase
in the biosynthesis of polyunsaturated fatty acids. Lipids
35, 871–879.
29 Hunt MC, Solaas K, Kase BF & Alexson SEH (2002)
Characterization of an acyl-CoA thioesterase that func-
tions as a major regulator of peroxisomal lipid meta-
bolism. J Biol Chem 277, 1128–1138.
30 Sprecher H (1968) The total synthesis and metabolism
of 4-decenoate, dodeca-3,6-dienoate, tetradeca-5,
8-dienoate and hexadeca-7,10-dienoate in the fat-
deficient rat. Lipids 3, 14–20.
31 Cunnane SC, Ryan MA, Craig KS, Brookes S,
Koletzko B, Demmelmair H, Singer J & Kyle DJ (1995)
Polymethylene-interrupted fatty acid metabolism T. Tanaka et al.
2736 FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS
Synthesis of linoleate and a-linolenate by chain elonga-
tion in the rat. Lipids 30, 781–783.
32 Hansen HS, Jensen B & von Wettstein-Knowles P
(1986) Apparent in vivo retroconversion of dietary ara-
chidonic to linoleic acid in essential fatty acid-deficient
rats. Biochim Biophys Acta 878, 284–287.
33 Su H-M, Moser AB, Moser HW & Watkins PA (2001)
Peroxisomal straight-chain acyl-CoA oxidase and d-
bifunctional protein are essential for retroconversion

step in docosahexaenoic acid synthesis. J Biol Chem
276, 38115–38120.
34 Sprecher H (2002) The role of anabolic and catabolic
reactions in the synthesis and recycling of polyunsatu-
rated fatty acids. Prostaglandins Leukot Essent Fatty
Acids 67, 79–83.
35 Tanaka T, Shibata K, Hino H, Murashita T, Kayama
M & Satouchi K (1997) Purification and gas
chromatographic-mass spectrometric characterization
of non-methylene interrupted fatty acid incorporated in
rat liver. J Chromatogr B 700, 1–8.
36 Folch J, Lees M & Sloane-Stanley GH (1957) A simple
method for the isolation and purification of total lipids
from animal tissues. J Biol Chem 226, 497–509.
37 Bligh EG & Dyer WJ (1959) A rapid method of total
lipid extraction and purification. Can J Biochem Physiol
37, 911–917.
T. Tanaka et al. Polymethylene-interrupted fatty acid metabolism
FEBS Journal 274 (2007) 2728–2737 ª 2007 The Authors Journal compilation ª 2007 FEBS 2737