from polymethylene-interrupted polyunsaturated fattyacids in animal cells Tamotsu Tanaka1, Jun-ichi Morishige1, Dai Iwawaki1, Terumi Fukuhara1, Naomi Hamamura1, Kaoru Hirano1, Takashi Os
Trang 1from polymethylene-interrupted polyunsaturated fatty
acids in animal cells
Tamotsu Tanaka1, Jun-ichi Morishige1, Dai Iwawaki1, Terumi Fukuhara1, Naomi Hamamura1, Kaoru Hirano1, Takashi Osumi2and Kiyoshi Satouchi1
1 Department of Applied Biological Science, Fukuyama University, Hiroshima, Japan
2 Graduate School of Life Sciences, University of Hyogo, Japan
Linoleic acid (18:2 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-polymethylene-interrupted-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: tamot@fubac.fukuyama-u.ac.jp
(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.
Trang 2Cupressaceae [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 C20PUFA 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
a Chain-elongated metabolite.
Trang 3pathway 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 C16fatty 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 (RF0.41) was located just below the methyl
linol-eate (RF0.45) 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 treatmpar-ent gave two species of
monoenoate which can be separated by AgTLC (RF
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
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.
Trang 4of 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 C20PMI-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
16:2( -7,10) 18:2( -9,12) 20:4( -5,8,11,14) A
0
2
4
20:4 ( -5,11,14,17) (µM )
18:3( -9,12,15)
20 :5( -5,8,11,14,17) B
Fig 3 Supplementation of C20PMI-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.
Trang 516:2 (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 C20polymethylene-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.
Trang 6cellular 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
[2-14C]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
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.
Trang 7sciadonic acid, because we and others conducted
experiments using [1-14C]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 C16, 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 C16rather than C20[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 C20PUFA
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 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
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
Trang 8were 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
10 mL DMEM containing 10% fetal bovine serum in a
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
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
the isolated methyl ester of 16:2 D-7,10 was partially
[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
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;
temperature of the injection port, ion source and interface
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
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
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
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