Báo cáo khoa học: Mechanisms of accumulation of arachidonate in phosphatidylinositol in yellowtail A comparative study of acylation systems of phospholipids in rat and the fish species Seriola quinqueradiata pot

Mechanisms of accumulation of arachidonate in phosphatidylinositol
in yellowtail
A comparative study of acylation systems of phospholipids in rat and the fish
species
Seriola quinqueradiata
Tamotsu Tanaka, Dai Iwawaki, Masahiro Sakamoto, Yoshimichi Takai, Jun-ichi Morishige,
Kaoru Murakami and Kiyoshi Satouchi
Department of Applied Biological Science, Fukuyama University, Japan
It is known that phosphatidylinositol (PtdIns) contains
abundant arachidonate and is composed mainly of 1-stea-
royl-2-arachidonoyl species in mammals. We investigated if
this characteristic of PtdIns applies to the PtdIns from
yellowtail (Seriola quinqueradiata), a marine fish. In common
with phosphatidylcholine (PtdCho), phosphatidylethanol-
amine (PtdEtn) and phosphatidylserine (PtdSer) from brain,
heart, liver, spleen, kidney and ovary, the predominant
polyunsaturated fatty acid was docosahexaenoic acid, and
levels of arachidonic acid were less than 4.5% (PtdCho),
7.5% (PtdEtn) and 3.0% (PtdSer) in these tissues. In striking
contrast, arachidonic acid made up 17.6%, 31.8%, 27.8%,
26.1%, 25.4% and 33.5% of the fatty acid composition of
PtdIns from brain, heart, liver, spleen, kidney and ovary,
respectively. The most abundant molecular species of PtdIns
in all these tissues was 1-stearoyl-2-arachidonoyl. Assay of
acyltransferase in liver microsomes of yellowtail showed that
arachidonic acid was incorporated into PtdIns more effect-
ively than docosahexaenoic acid and that the latter inhibited
incorporation of arachidonic acid into PtdCho without
inhibiting the utilization of arachidonic acid for PtdIns. This
effect of docosahexaenoic acid was not observed in similar
experiments using rat liver microsomes and is thought to

contribute to the exclusive utilization of arachidonic acid for
acylation to PtdIns in yellowtail. Inositolphospholipids and
their hydrolysates are known to act as signaling molecules
in cells. The conserved hydrophobic structure of PtdIns
(the 1-stearoyl-2-arachidonoyl moiety) may have physio-
logical significance not only in mammals but also in fish.
Keywords: acyltransferase; arachidonic acid; fish; phospha-
tidylinositol; yellowtail.
Biological membranes are composed of several phospho-
lipid classes, and glycerophospholipid classes are further
separated into molecular species based on the combination
of acyl (alkyl, alkenyl) residues at positions sn-1 and sn-2.
One well-known characteristic of phosphatidylinositol
(PtdIns) is an abundance of arachidonate. This has been
demonstrated in several mammalian tissues [1–12] and
confirmed in this study in most tissues of the rat. At the
molecular level, PtdIns has been reported to be composed
mainly of 1-stearoyl-2-arachidonoyl species in guinea pig
brain [6], bovine brain [7], rat liver [8], human platelets
[9,10], human endothelial cells [11] and rabbit macrophages
[12]. We confirm here that this molecular conservation of
PtdIns is a feature distinct from other phospholipids in most
tissues of the rat. The molecular conservation of PtdIns is
thought to have physiological importance for (a) eicosanoid
precursor storage, (b) donation of potent activators of
protein kinase C (PKC), such as 1-stearoyl-2-arachidonoyl-
glycerol [13], and (c) donation of arachidonate-containing
biologically active molecules, such as 2-arachidonoylglycerol
[14]. However, the exact physiological meaning of the
conservation of PtdIns molecular species has not been fully

resolved.
Several enzymatic systems are involved in the accumu-
lation of arachidonate in PtdIns. CoA)1-acyl-2-lyso-PtdIns
acyltransferase activity, operating in the remodeling path-
ways of phospholipid biosynthesis, is known to utilize
arachidonoyl-CoA as substrate [15,16]. Both diacylglycerol
kinase [17–20] and CDP-sn-1,2-diacylglycerol synthase [21],
enzymes involved in the PtdIns cycle, have been reported to
contribute to the enrichment of arachidonate in PtdIns.
With respect to the biosynthesis of PtdIns, we and others
[22,23] have demonstrated that sciadonic acid (20:3,
D-5c,11c,14c), an n)6 series trienoic acid that lacks the D8
double bond of arachidonic acid, is metabolized in a similar
manner to arachidonic acid in the biosynthesis of PtdIns
[24,25]. We have also presented evidence suggesting that the
nonarachidonic acid and utilizable polyunsaturated fatty
Correspondence to T. Tanaka, Department of Applied Biological
Science, Fukuyama University, Fukuyama, 729-0292, Japan.
Fax: + 81 84 936 2459, Tel.: + 81 84 936 2111, Ext. 4056,
E-mail:
Abbreviations: PKC, protein kinase C; PtdCho, phosphatidylcholine;
PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol;
PtdSer, phosphatidylserine; PUFA, polyunsaturated fatty acid.
Enzymes: acylCoA:lysophospholipid acyltransferase (EC 2.3.1.23);
CDP-diacylglycerol synthase (CTP-phosphatidate:cytidylyltrans-
ferase; EC 2.7.7.41); diacylglycerol kinase (EC 2.7.1.107); phospho-
lipase A
2
(EC 3.1.1.4); phospholipase C (EC 3.1.4.3); protein kinase C
(EC 2.7.1.37).

(Received 26 September 2002, revised 13 December 2002,
accepted 10 February 2003)
Eur. J. Biochem. 270, 1466–1473 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03512.x
acid (PUFA) for PtdIns is a potential tool with which to
clarify the significance of the arachidonic acid residue of
bioactive lipids of PtdIns origin [25].
In general, lipids from terrestrial mammals are rich in n)6
series PUFAs, such as linoleic acid and arachidonic acid. In
contrast, the predominant PUFAs in lipids from marine fish
are n)3 series fatty acids such as docosahexaenoic acid and
eicosapentaenoic acid. Does the molecular conservation in
which PtdIns is composed mainly of arachidonate-contain-
ing molecular species apply to marine fish? Studies have
shown that 1-stearoyl-2-arachidonoyl-PtdIns is the pre-
dominant molecular species even in codfish roe [26] and
salmon sperm [27], salmon liver [28] and rainbow trout
retina [29]. If molecular conservation of PtdIns is found in
marine fish, what mechanisms operate to accumulate
arachidonic acid into PtdIns in an environment in which
arachidonic acid is limited?
In this study, we investigated the composition of PtdIns
from brain, heart, liver, spleen, kidney and ovary in Seriola
quinqueradiata, the marine fish known as yellowtail. The
results show that PtdIns is rich in arachidonic acid despite
the predominance of n)3 series PUFAs in these tissues. We
also investigated mechanisms for the accumulation of
arachidonic acid into PtdIns through experiments with
microsomes from the liver of yellowtail, and found that the
one-sided accumulation of arachidonic acid into PtdIns is
attained in the presence of large amounts of docosahexa-

enoic acid and that several acyltransferase activities are
involved in the process in yellowtail.
Materials and methods
Materials
Yellowtails (S. quinqueradiata) were obtained from a
local market. Standard fatty acids were purchased from
Serdary Research Laboratories (London, ON, Canada).
[1-
14
C]Arachidonic acid (55 mCiÆmmol
)1
)and[1-1
4
C]doco-
sahexaenoic acid (55 mCiÆmmol
)1
) were from NEM Life
Sciences Products, Inc. (Boston, MA, USA). Essentially
fatty acid-free BSA, ATP, CoA, 1-palmitoyl-2-lyso-phos-
phatidylcholine (lysoPtdCho), phospholipase C (from
Bacillus cereus) and phospholipase A
2
(from Crotalus
adamanteus venom) were from Sigma Chemical Co.
(St Louis, MO, USA). By treatment with phospholipase
A
2
[30], 1-acyl-2-lyso-PtdIns (lysoPtdIns) was prepared
from bovine liver PtdIns (Sigma). The resulting lysoPtdIns
was purified by TLC using chloroform/acetone/methanol/

acetic acid/water (50 : 20 : 10 : 13 : 5, v/v/v/v/v). Lyso-
PtdIns was extracted from the silica gel by the method of
Bligh & Dyer [31] under slightly acidic (HCl) conditions. All
other reagents were of reagent grade.
Fatty acid composition of phospholipids and molecular
species composition of PtdIns
Brain, heart, liver, spleen, kidney and ovary of yellowtails
were isolated, and total lipids were extracted by the method
of Bligh & Dyer [31]. After separation of the phospholipid
fraction by silicic acid column chromatography, PtdCho,
phosphatidylethanolamine (PtdEtn) and the mixture of
phosphatidylserine (PtdSer), PtdIns and sphingomyelin
were separated by TLC with the solvent system chloro-
form/methanol/28% ammonia (65 : 35 : 5, v/v/v). The
mixed fraction of PtdIns, PtdSer and sphingomyelin was
further separated by TLC with chloroform/acetone/meth-
anol/acetic acid/water (50 : 20 : 10 : 13 : 5, v/v/v/v/v) to
obtain PtdIns and PtdSer. Detection was with 0.01%
primuline (in acetone/water, 4 : 1, v/v) under UV light. The
fatty acid composition of each phospholipid was analyzed
by GC after transmethylesterification. A portion of the
PtdIns was subjected to phospholipase C treatment, and the
resulting diacylglycerol was converted into dinitrobenzoyl
derivatives as described by Kito et al. [32]. The diacyl-
glyceroldinitrobenzoyl derivative was analyzed by HPLC
with a 0.45 · 25 cm Inertsil ODS-2 column (GL Science
Inc., Tokyo, Japan) using acetonitrile/propan-2-ol (80 : 20,
v/v) as eluent. The major peaks were assigned by the direct
analysis with GC after transmethylesterification. Lipids of
male Sprague–Dawley rats (250–300 g) were analysed by

the same method as those of yellowtail.
Acyltransferase assay
The isolated liver of yellowtail was homogenized in 50 m
M
potassium phosphate buffer (pH 7.0) containing 1.5 m
M
glutathione, 0.15
M
KCl, 1 m
M
EDTA and 0.25
M
sucrose
(homogenizing buffer) with a Potter–Elvehjem glass/Teflon
homogenizer. The microsome fraction was prepared by
sequential centrifugation [25]. Microsomes from the liver of
male Sprague–Dawley rats (250–300 g) were prepared by
the same method. The final microsomal pellet was suspen-
ded in the homogenizing buffer (omitting EDTA), and the
protein content was determined by the method of Lowry
et al. [33]. Acyltransferase was assayed as described previ-
ously [25]. Each incubation contained 32 nmol lysoPtdCho
(1-acyl) or lysoPtdIns (1-acyl), 0.5 m
M
nicotinamide,
1.5 m
M
glutathione, 0.15
M
KCl, 5 m

M
MgCl
2
, 0.25
M
sucrose, 3.0 m
M
ATP, 0.1 m
M
CoA, 50 m
M
potassium
phosphate buffer (pH 7.0), 0.1 mg protein of the micro-
somal fraction, and radiolabeled fatty acid (0.05 lCi per
25 nmol) in a total volume of 1.0 mL. After incubation at
37 °C for 10 min, lipids were extracted, the resulting
PtdCho and PtdIns were isolated by 2D TLC as described
previously [25], and radioactivities were determined. The
inhibitory effect of unlabeled docosahexaenoic acid on the
incorporation of labeled arachidonic acid into lysoPtdCho
and lysoPtdIns was determined by experiments with 0.1 mg
liver microsomal protein, 10 nmol labeled arachidonic acid,
and the indicated amount of unlabeled docosahexaenoic
acid in the presence of 6.4 nmol lysoPtdIns and 6.4 nmol
lysoPtdCho.
Results
Fatty acid composition of phospholipids from tissues
of yellowtail and rat
The fatty acid compositions of PtdCho, PtdEtn, PtdSer and
PtdIns of brain, heart, spleen, kidney and ovary of yellowtail

were investigated (Tables 1–4). In all the tissues, the most
abundant PUFA in the PtdCho fraction was docosahexa-
enoic acid. The proportion of eicosapentaenoic acid in
PtdCho was relatively high compared with that in PtdEtn
Ó FEBS 2003 Arachidonate in phosphatidylinositol of yellowtail (Eur. J. Biochem. 270) 1467
and PtdSer, except in brain where oleic acid was abundant.
In both the PtdEtn and PtdSer fractions, docosahexaenoic
acid was the predominant PUFA in all the tissues. The
presence of dimethylacetals in PtdEtn suggested the exist-
ence of a substantial amount of an alkenylacyl subclass in
these tissues. In common with PtdCho, PtdEtn and PtdSer,
levels of arachidonic acid were very low compared with
those of docosahexaenoic acid in these tissues. In striking
contrast, larger amounts of arachidonic acid existed in
PtdIns from all tissues investigated (Fig. 1). The propor-
tion of it in PtdIns was highest in ovary (33.5%) and
lowest in brain (17.6%). In all tissues, the proportion of
Table 3. Fatty acid composition of PtdSer from various tissues of yellowtail. Values are weight percentages, given as the mean ± SD. Tissues were
obtained from three different yellowtails.
Fatty acid Brain Heart Liver Spleen Kidney Ovary
14:0 0.7 ± 0.3 0.9 ± 1.0 0.5 ± 0.2 0.8 ± 0.2 0.4 ± 0 0.4 ± 0.7
16:0 2.7 ± 1.6 7.2 ± 2.0 16.7 ± 3.2 6.3 ± 0.9 11.1 ± 2.0 8.5 ± 2.4
16:1 1.8 ± 1.0 1.3 ± 1.3 0.4 ± 0.2 1.6 ± 0.7 0.5 ± 0.3 0.4 ± 0.2
18:0 26.4 ± 2.4 31.3 ± 4.9 25.7 ± 3.4 32.0 ± 1.3 28.8 ± 1.1 31.7 ± 3.0
18:1(n)9) 19.6 ± 2.2 4.2 ± 0.7 3.3 ± 1.1 4.4 ± 1.3 4.2 ± 1.5 7.6 ± 3.1
18:1(n)7) – 2.5 ± 0.5 1.6 ± 0.2 3.2 ± 0.3 2.4 ± 0.4 2.8 ± 0.6
18:2(n)6) 2.1 ± 2.3 0.7 ± 0.4 – 2.1 ± 2.2 0.6 ± 0.3 0.7 ± 0.2
20:4(n)6) 0.5 ± 0.2 0.9 ± 0.4 1.4 ± 1.0 1.1 ± 0.3 2.0 ± 0.6 3.0 ± 2.0
20:5(n)3) 0.9 ± 0.1 1.5 ± 0.6 1.0 ± 0.2 1.5 ± 0.7 2.6 ± 2.0 1.5 ± 1.0
22:5(n)3) 4.3 ± 1.5 4.1 ± 0.8 2.1 ± 0.5 2.2 ± 0.7 2.8 ± 0.6 2.8 ± 1.2

22:6(n)3) 36.1 ± 1.4 33.7 ± 5.6 41.0 ± 0.9 36.4 ± 2.6 34.3 ± 2.4 26.0 ± 5.9
Table 1. Fatty acid composition of PtdCho from various tissues of yellowtail. Values are weight percentages, given as the mean ± SD. Tissues were
obtained from three different yellowtails.
Fatty acid Brain Heart Liver Spleen Kidney Ovary
14:0 0.7 ± 0.3 0.8 ± 0.3 1.4 ± 0.2 1.8 ± 0.8 2.0 ± 1.2 1.2 ± 0.9
16:0 21.4 ± 1.4 33.0 ± 1.6 32.4 ± 3.3 35.0 ± 1.8 32.9 ± 3.5 34.8 ± 1.0
16:1 5.5 ± 1.4 1.3 ± 0.5 1.8 ± 0.7 3.8 ± 0.6 3.7 ± 0.7 2.5 ± 1.0
18:0 8.6 ± 0.3 3.9 ± 0.5 3.9 ± 0.8 3.6 ± 0.3 3.7 ± 0.5 3.8 ± 1.0
18:1(n)9) 29.2 ± 1.2 7.2 ± 1.3 7.4 ± 0.6 13.1 ± 1.8 12.2 ± 1.9 12.5 ± 2.2
18:1(n)7) 0.9 ± 0.8 2.6 ± 0.3 2.1 ± 0.3 4.0 ± 0.3 3.3 ± 0.6 2.5 ± 1.3
18:2(n)6) 1.4 ± 1.2 1.0 ± 0.4 1.3 ± 0.1 2.6 ± 1.0 2.4 ± 0.5 2.1 ± 0.7
20:4(n)6) 1.2 ± 0.4 4.5 ± 0.3 2.1 ± 0.2 3.7 ± 0.8 3.6 ± 0.9 3.6 ± 0.8
20:5(n)3) 1.8 ± 0.2 10.6 ± 1.5 8.4 ± 0.5 10.7 ± 1.1 11.1 ± 2.6 11.1 ± 1.2
22:5(n)3) 6.5 ± 0.4 1.7 ± 0.4 2.1 ± 0.6 1.7 ± 0.7 1.6 ± 0.4 1.6 ± 0.2
22:6(n)3) 15.3 ± 0.2 28.3 ± 3.1 31.5 ± 3.4 14.3 ± 0.4 19.7 ± 3.1 19.6 ± 5.5
Table 2. Fatty acid composition of PtdEtn from various tissues of yellowtail. Values are weight percentages, given as the mean ± SD. Tissues were
obtained from three different yellowtails. DMA, Dimethylacetal.
Fatty acid Brain Heart Liver Spleen Kidney Ovary
14:0 – – 0.4 ± 0.2 0.4 ± 0.1 – 0.8 ± 0.2
16:0DMA 1.2 ± 0.2 6.0 ± 0.8 0.5 ± 0.1 7.9 ± 2.0 6.3 ± 0.7 8.3 ± 1.7
16:0 4.6 ± 0.4 6.5 ± 1.3 22.2 ± 3.2 8.0 ± 1.0 12.5 ± 4.3 11.8 ± 1.8
16:1 1.0 ± 0.1 0.4 ± 0.3 0.8 ± 0.4 0.5 ± 0.1 0.7 ± 0.4 1.0 ± 0.4
18:0DMA 19.3 ± 2.0 1.5 ± 0.9 – 4.1 ± 0.4 3.5 ± 2.1 4.9 ± 1.5
18:1DMA 4.0 ± 1.1 1.0 ± 0.4 – 5.1 ± 0.5 3.4 ± 0.8 3.2 ± 0.8
18:0 10.2 ± 0.6 21.0 ± 0.3 13.6 ± 2.0 8.8 ± 0.3 9.0 ± 1.2 7.4 ± 1.2
18:1(n)9) 24.3 ± 2.3 3.3 ± 1.3 5.2 ± 0.9 3.5 ± 0.6 3.9 ± 0.7 4.7 ± 1.0
18:1(n)7) – 3.0 ± 0.6 3.1 ± 0.2 3.2 ± 0.4 3.0 ± 0.4 2.6 ± 0.3
18:2(n)6) – 0.9 ± 0 0.7 ± 0.4 0.7 ± 0.3 0.8 ± 0.3 0.8 ± 0.2
20:4(n)6) 2.9 ± 0.7 2.6 ± 0.3 1.6 ± 0.2 4.4 ± 1.3 5.5 ± 0.5 7.5 ± 1.4
20:5(n)3) 3.6 ± 0.6 3.8 ± 0.6 3.6 ± 0.3 6.1 ± 0.3 8.7 ± 3.0 6.7 ± 2.1

22:5(n)3) 1.3 ± 0.2 4.0 ± 0.3 1.8 ± 0.7 2.1 ± 0.8 1.7 ± 0.6 1.5 ± 0.7
22:6(n)3) 19.2 ± 4.9 41.5 ± 5.0 39.1 ± 4.5 37.7 ± 1.9 32.9 ± 3.7 27.4 ± 4.3
1468 T. Tanaka et al.(Eur. J. Biochem. 270) Ó FEBS 2003
docosahexaenoic acid in PtdIns was lower than in other
phospholipids in corresponding tissues.
Fatty acid compositions of PtdCho, PtdEtn, PtdSer and
PtdIns from brain, heart, lung, liver, pancreas, kidney and
testis of rat were investigated. Only the proportions of
arachidonic acid in each phospholipid are presented in
Fig. 1. The proportions of arachidonic acid in PtdCho,
PtdEtn and PtdSer from these rat tissues varied from
4.7% to 21.4%, from 10.6% to 39.1% and from 4.8% to
32.6%, respectively. In contrast, 38.5%, 33.6%, 38.7%,
32.9%, 36.2% and 34.7% of total fatty acids in the
PtdIns of rat brain, heart, lung, liver, pancreas, kidney
and testis, respectively, were arachidonic acid. These
results confirm that PtdIns is rich in arachidonic acid in
rat tissues. Furthermore, it is evident that this characteri-
stic of PtdIns also applies to yellowtail, a fish species
living in seawater.
Molecular species composition of PtdIns from tissues
of yellowtail and rat
The molecular species compositions of PtdIns from various
tissues of yellowtail were analyzed by HPLC as diacyl-
glyceroldinitrobenzoyl derivatives. To assign major peaks of
these derivatives, we collected the eluate corresponding to
each molecular species peak, and directly analysed the fatty
acids of each fraction by GC. Under our analytical
conditions, one pair of molecular species, 18:1/22:6 and
16:0/20:5, could not be resolved. Therefore, the amounts of

these molecular species are shown as mixed components. As
expected from the fatty acid analyses, the most abundant
molecular species in all the tissues was 1-stearoyl-2-arachi-
donoyl-PtdIns (Table 5). Although the proportion of this
molecular species was relatively low in brain, about half of
the total molecular species of PtdIns were 1-stearoyl-2-
arachidonoyl species in liver, heart, spleen and ovary
(Table 5). The next most abundant molecular species was
18:0/20:5 in all tissues. We also analyzed the molecular
species composition of PtdIns obtained from tissues of rat:
65.6 ± 4.2%, 63.0 ± 7.8%, 54.4 ± 5.9%, 65.3 ± 4.8%,
65.7 ± 2.2%, 60.2 ± 2.7% and 53.5 ± 7.8% of the total
molecular species of PtdIns from rat brain, heart, lung, liver,
pancreas, kidney and testis, respectively, were 1-stearoyl-
2-arachidonoyl species. The molecular conservation
observed in mammalian tissues that PtdIns is composed
mainly of 1-stearoyl-2-arachidonoyl species also applies to
tissues of yellowtail.
Accumulation of arachidonic acid in PtdIns
in the presence of the large amounts
of docosahexaenoic acid in yellowtail
Lipids from yellowtail have a preponderance of docosa-
hexaenoic acid over arachidonic acid. In fact, docosahexa-
enoic acid and arachidonic acid made up 28.4% and 3.5%,
respectively, of the fatty acid composition of the total lipid
fraction of yellowtail liver (data not shown). Despite such a
one-sided PUFA composition, arachidonic acid is exclu-
sively accumulated in PtdIns. Therefore, there must be a
mechanism that selects arachidonic acid from the large
amounts of docosahexaenoic acid for acylation to lyso-

PtdIns in fish cells. To investigate this, we assessed the
efficacy of the acylation of [
14
C]arachidonic acid or
[
14
C]docosahexaenoic acid into sn-2 of lysoPtdIns (1-acyl)
or lysoPtdCho (1-acyl) in fish liver microsomes. In prelimi-
nary experiments, the optimum temperature for acylation of
arachidonic acid to lysophospholipids was found to be
37 °C, so the assay was conducted at this temperature.
When lysoPtdIns was used as an acyl acceptor, arachidonic
acid was incorporated into sn-2 of PtdIns more effectively
than docosahexaenoic acid (Fig. 2A). The saturation levels
of acylation for arachidonic acid and docosahexaenoic acid
were  70 and 7 nmol per 10 min per mg protein, respect-
ively. When lysoPtdCho was used as an acyl acceptor, the
acyltransferase activity of the fish liver microsomes acylated
docosahexaenoic acid more effectively than arachidonic
acid (Fig. 2B). At a fatty acid concentration of 50 l
M
,
the amounts of docosahexaenoic acid and arachidonic
acid incorporated into PtdCho were 129 and 94 nmol per
10 min per mg protein, respectively. The same experiments
were conducted with rat liver microsomes: docosahexaenoic
acid was found to be a poor acyl donor not only for
lysoPtdIns but also for lysoPtdCho compared with arachi-
donic acid (Fig. 2C,D). At a fatty acid concentration of
50 l

M
, the level of acylation of docosahexaenoic acid to
lysoPtdIns was 19.4 nmol per 10 min per mg protein, which
was about one-fifth of that obtained with the same con-
centration of arachidonic acid (90.4 nmol per 10 min per mg
Table 4. Fatty acid composition of PtdIns from various tissues of yellowtail. Values are weight percentages, given as the mean ± SD. Tissues were
obtained from three different yellowtails.
Fatty acid Brain Heart Liver Spleen Kidney Ovary
14:0 0.8 ± 0.5 0.4 ± 0.1 0.3 ± 0 0.4 ± 0 0.3 ± 0.2 0.5 ± 0.4
16:0 11.1 ± 2.4 4.6 ± 0.6 5.3 ± 0.9 6.2 ± 0.9 5.7 ± 0.7 6.1 ± 1.0
16:1 0.7 ± 0.1 0.6 ± 0.2 0.3 ± 0.1 0.5 ± 0 0.3 ± 0 0.6 ± 0
18:0 29.7 ± 1.3 33.7 ± 1.6 37.7 ± 3.2 34.0 ± 0.7 36.4 ± 3.2 32.8 ± 1.0
18:1(n)9) 8.0 ± 1.0 6.6 ± 0.6 7.3 ± 0.8 7.7 ± 0.7 6.0 ± 2.4 7.0 ± 0.9
18:1(n)7) 1.8 ± 0.1 2.0 ± 0.1 1.4 ± 0.2 2.1 ± 0.3 1.6 ± 0.5 1.9 ± 0.5
18:2(n)6) 0.5 ± 0.2 0.6 ± 0.2 0.3 ± 0.1 0.7 ± 0.2 0.4 ± 0.2 0.4 ± 0.1
20:4(n)6) 17.6 ± 1.7 31.8 ± 1.1 27.8 ± 6.5 26.1 ± 4.2 25.4 ± 3.5 33.5 ± 3.3
20:5(n)3) 11.5 ± 1.4 9.1 ± 1.6 12.5 ± 0.6 8.8 ± 0.4 13.0 ± 1.3 2.0 ± 0.1
22:5(n)3) 1.4 ± 0.4 0.7 ± 0.2 0.6 ± 0.3 1.7 ± 0.9 1.2 ± 0.3 0.9 ± 0.4
22:6(n)3) 13.1 ± 1.3 3.1 ± 0.8 3.5 ± 2.1 7.4 ± 1.3 7.0 ± 1.7 10.0 ± 1.1
Ó FEBS 2003 Arachidonate in phosphatidylinositol of yellowtail (Eur. J. Biochem. 270) 1469
protein) in rat liver microsomes. When experiments were
conducted with lysoPtdCho and 50 l
M
fatty acid, the
amount of docosahexaenoic acid incorporated into PtdCho
(28.4 nmol per 10 min per mg protein) was about one-
seventh of that of arachidonic acid (209.5 nmol per 10 min
per mg protein) in rat liver microsomes.
In an extended study, the inhibitory effect of docosa-
hexaenoic acid on the incorporation of [

14
C]arachidonic
acid into lysophospholipid was investigated. This experi-
ment was conducted in the presence of both lysoPtdIns and
lysoPtdCho to elucidate the distribution of [
14
C]arachidonic
acid incorporated into these phospholipids. We also modi-
fied the experimental conditions so that the addition of an
equimolar quantity of unlabeled arachidonic acid achieved
50% inhibition of the acylation of [
14
C]arachidonic acid to
lysophospholipids. In experiments using microsomes from
yellowtail liver (Fig. 3A), the distribution of [
14
C]arachi-
donic acid between PtdCho and PtdIns was approximately
1 : 1 in the absence of docosahexaenoic acid. In contrast,
the addition of docosahexaenoic acid at equimolar, two and
fourtimesmolarexcessover[
14
C]arachidonic acid modified
the distribution of [
14
C]arachidonic acid between PtdCho
and PtdIns from 1 : 1 to 1 : 3, 1 : 4 and 1 : 5, respectively.
These results obtained in the presence of large amounts of
docosahexaenoic acid are in good agreement with the
distribution patterns of arachidonic acid between PtdCho

and PtdIns observed in the fatty acid analysis of tissues of
yellowtail. Furthermore, they indicate that the one-sided
incorporation of arachidonic acid into lysoPtdIns can be
accomplished in the presence of large amounts of docosa-
hexaenoic acid. A similar experiment was conducted with
rat liver microsomes (Fig. 3B). Unlike the results obtained
with liver microsomes from yellowtail, the ratios of distri-
bution of [
14
C]arachidonic acid between PtdCho and PtdIns
were not much changed by the addition of docosahexaenoic
acid at equimolar, two and four times molar excess over
[
14
C]arachidonic acid, remaining about 1 : 0.6–0.7. This
ratio was similar to that obtained in the absence of
docosahexaenoic acid (1 : 0.6). Because of the difference
in the phospholipid acylation systems of rat and yellowtail
in the preference for docosahexaenoic acid over arachidonic
acid for acylation to lysoPtdCho (Fig. 2), the docosahexa-
enoic acid preference of the enzymatic activity of yellowtail
contributes to the one-sided distribution of arachidonic acid
between PtdCho and PtdIns in yellowtail.
Discussion
Unlike terrestrial animals, lipids from marine fish have a
preponderance of n)3 series PUFAs over n)6series
PUFAs. Despite this PUFA composition, phospholipid
acylation systems operating in yellowtail utilize arachidonic
acid exclusively for acylation to lysoPtdIns. In this study, we
have clarified several mechanisms concerning this point.

The key enzymatic activity for construction of the final
molecular species of PtdIns is considered to be acylCoA–
lysoPtdIns acyltransferase activity operating in the remode-
ling pathway of phospholipid biosynthesis. This enzymatic
activity in liver microsomes of yellowtail strictly recognized
arachidonic acid and hardly utilized docosahexaenoic acid
at all. This one-sided efficacy was remarkable compared
with that observed in rat liver microsomes. This strict
recognition must contribute to the accumulation of arachi-
donic acid in PtdIns in yellowtail.
Docosahexaenoic acid is predominantly acylated to
PtdCho in tissues of yellowtail like other marine fish species
[26–29,34]. Consistent with these observations, lysoPtdCho
acyltransferase activity in liver microsomes of the yellowtail
preferred docosahexaenoic acid. This enzymatic activity
also utilized arachidonic acid with significant efficacy.
Therefore, in the absence of docosahexaenoic acid, arachi-
donic acid was acylated into both lysoPtdCho and lyso-
PtdIns at similar levels (Fig. 3A). The result indicates that,
in yellowtail, there is no selectivity for incorporation of
Fig. 1. Arachidonic acid contents of PtdCho, PtdEtn, PtdSer and
PtdIns obtained from several tissues of yellowtail and rat.
1470 T. Tanaka et al.(Eur. J. Biochem. 270) Ó FEBS 2003
arachidonic acid itself, whether into PtdCho or PtdIns.
However, in the presence of a large amount of docosahexa-
enoic acid, docosahexaenoic acid effectively inhibits the
incorporation of arachidonic acid into PtdCho without
inhibiting the utilization of arachidonic acid for PtdIns
(Fig. 3A). A possible explanation of this phenomenon is
that docosahexaenoic acid competes with arachidonic acid

effectively only in the case of incorporation into PtdCho.
This docosahexaenoic acid effect would explain the relat-
ively low content of arachidonic acid in PtdCho, and may
contribute to the exclusive utilization of arachidonic acid for
acylation to PtdIns in living fish cells. In the experiment with
rat liver microsomes, a large amount of docosahexaenoic
acid did not affect the distribution of arachidonic acid
incorporated into PtdCho and PtdIns (Fig. 3B). This
observation is in good agreement with the results showing
that docosahexaenoic acid is a poor acyl donor compared
with arachidonic acid, not only for lysoPtdIns but also for
lysoPtdCho (Fig. 2C,D). The preference for arachidonic
acid over docosahexaenoic acid for acylation to these
lysophospholipids has been reported in microsomes of
porcine platelets, porcine liver and rat liver [35]. The
arachidonic acid-specific acyltransferase and acylCoA syn-
thase present in mammals could be involved in these
processes [36]. Besides the acylation systems in the remode-
ling of phospholipids, both diacylglycerol kinase [17–20]
and CDP-sn-1,2-diacylglycerol synthase [21], enzymes
operating in the PtdIns cycle, have been reported to
contribute to the enrichment of arachidonate in PtdIns in
mammals. Further experiments are needed to clarify the
involvement of the PtdIns cycle in the accumulation of
arachidonic acid in PtdIns of fish.
Fig. 3. Effects of docosahexaenoic acid (DHA) on the incorporation of
[
14
C]arachidonic acid (*AA) into exogenously added lysoPtdCho and
lysoPtdIns in microsomes from liver of yellowtail and liver of rat.

Microsomes (0.1 mg protein ) from liver of yellowtail (A) or rat (B)
were incubated at 37 °C for 10 min with 10 nmol labeled arachidonic
acid (*AA) and the indicated amount of unlabeled DHA in the pre-
sence of both 6.4 nmol 1-acyl-2-lyso-PtdIns and 6.4 nmol 1-acyl-
2-lyso-PtdCho. After the incubation, phospholipids were separated by
2D TLC, and radioactivity was measured. Therefore, only the amount
of arachidonic acid incorporated into each lysophospholipid could be
determined. Similar results were obtained in three independent
experiments with microsomes from different yellowtails or rats.
Table 5. Molecular species composition of PtdIns from various tissues of yellowtail. The isolated PtdIns was converted to dinitrobenzoyl derivative as
described in materials and methods and analyzed by HPLC. Values are mol percentages, given as the mean ± SD. Tissues were obtained from
three different yellowtails.
Molecular species Brain Heart Liver Spleen Kidney Ovary
18:1/20:5(n)3) 5.8 ± 1.9 2.5 ± 0.3 3.6 ± 1.0 2.5 ± 1.1 1.6 ± 1.5 1.5 ± 0.4
18:1/22:6(n)3)+16:0/20:5(n)3) 11.5 ± 2.5 3.1 ± 1.0 4.1 ± 1.0 4.6 ± 2.3 4.7 ± 4.4 4.6 ± 1.1
16:0/22:6(n)3) 6.2 ± 1.2 1.0 ± 0.5 1.7 ± 1.5 2.3 ± 1.2 1.4 ± 1.3 2.7 ± 1.4
18:1/20:4(n)6) 9.1 ± 1.6 9.9 ± 0.2 9.4 ± 2.5 6.9 ± 1.3 6.8 ± 0.8 8.8 ± 2.7
16:0/20:4(n)6) 7.3 ± 2.2 4.7 ± 0.6 4.7 ± 1.8 4.9 ± 3.0 3.3 ± 1.6 6.7 ± 2.7
18:0/20:5(n)3) 16.7 ± 0.1 17.6 ± 3.4 18.9 ± 4.8 15.0 ± 6.3 15.5 ± 6.3 9.0 ± 4.6
18:0/22:6(n)3) 11.8 ± 1.8 1.9 ± 0.5 3.0 ± 3.2 9.4 ± 4.8 5.3 ± 1.3 8.6 ± 4.2
18:0/20:4(n)6) 19.8 ± 2.6 54.8 ± 1.2 44.4 ± 5.5 45.4 ± 5.9 31.9 ± 4.1 47.4 ± 6.4
Fig. 2. Incorporation of [
14
C]arachidonic acid or [
14
C]docosahexaenoic
acid (DHA) into exogenously added lysoPtdCho or lysoPtdIns in
microsomes from liver of yellowtail or liver of rat. The incubation was
conducted at 37 °Cfor10minwith0.1mgproteinfrommicrosomes
of yellowtail liver in the presence of 32 nmol 1-acyl-2-lyso-PtdIns (A)

or 32 nmol 1-acyl-2-lyso-PtdCho (B). The same experiments were
conducted with rat liver microsomes in the presence of lysoPtdIns (C)
or lysoPtdCho (D). After the incubation, phospholipids were separ-
ated by 2D TLC, and radioactivity was measured. Values are
means ± SD (three microsomal preparations from different yellow-
tailsorrats).
Ó FEBS 2003 Arachidonate in phosphatidylinositol of yellowtail (Eur. J. Biochem. 270) 1471
It has been widely reported that PtdIns contains abun-
dant arachidonate and is composed mainly of 1-stearoyl-
2-arachidonoyl species in mammals [1–12]. We have
confirmed this characteristic in rat tissues in this study.
This was also the case for tissues of chicken: the arachidonic
acid contents of PtdIns from chicken brain, heart, liver and
kidney were 40.6%, 30.9%, 33.7% and 30.3%, respectively
(T. Tanaka, T. Hiyama & K. Satouchi, unpublished results).
In this study, we have demonstrated that this characteristic
of PtdIns also applies to tissues of yellowtail, a marine fish.
We do not know if all fish species have this feature, but it
has been reported that 1-stearoyl-2-arachidonoyl-PtdIns is
the predominant molecular species in codfish roe [26] and
several tissues of salmon [27–29]. In plants and some insects
[37], arachidonic acid is not a lipid constituent, therefore the
molecular conservation of PtdIns is not a characteristic of
all multicellular organisms. However, in preliminary experi-
ments, PtdIns from liver of Xenopus laevis, a frog, was found
to contain abundant arachidonate compared with other
phospholipids.
Inositolphospholipids are known to be a source of
diacylglycerol, which activates PKC. There is evidence that
PKC activation correlates with the transient accumulation

of diacylglycerol derived from inositolphospholipid [38].
PKC isoforms that can be activated by diacylglycerol have
been reported to exist even in fish cells [39,40]. The
molecular conservation of PtdIns gives rise to the unifica-
tion of diacylglycerol molecular species produced in
response to agonistic stimulation. It is still unclear whether
PKC discriminates the structural difference between
1-stearoyl-2-arachidonoylglycerol and other PUFA-
containing diacylglycerol molecular species. Bell & Sargent
[40] have reported that n)3-rich diacylglycerols prepared
from cod roe have a similar potency to 1-stearoyl-
2-arachidonoylglycerol for increasing PKC activity in vitro.
Similar results have been reported with synthetic 1-stearoyl-
2-docosahexaenoylglycerol [41]. On the other hand, evi-
dence has emerged that activation of PKC is dependent
on the composition of diacylglycerol molecular species
[38,42,43] and that diacylglycerols containing PUFAs, such
as arachidonic acid and mead acid (20:3, D-5c,8c,11c), are
more potent activators of PKC [44]. In addition, 1-stearoyl-
2-arachidonoylglycerol has been reported to be a more
potent activator of PKC than diacylglycerols rich in n)3
series PUFA under certain conditions [45]. It has been
reported that 1-stearoyl-2-arachidonoylglycerol attains a
V-shaped conformation in biological membranes that
facilitates anchoring of PtdSer-requiring proteins [46].
Furthermore, some Ca
2+
channels that mediate the influx
of Ca
2+

across the plasma membrane are directly activated
by 1-stearoyl-2-arachidonoylglycerol [47]. The physiological
significance of the enrichment of arachidonate in PtdIns can
be clarified by investigating the functions of cells in which
the arachidonic acid residue of PtdIns has been replaced
with another fatty acid. We have demonstrated that
polymethylene-interrupted fatty acids, such as sciadonic
acid (20:3, D-5c,11c,14c), mimic arachidonic acid in the
biosynthesis of PtdIns in cells [25]. We are now conducting
experiments to clarify whether such an acyl residue modi-
fication of PtdIns affects the cell response to agonistic
stimulation using Swiss 3T3 cells.
In conclusion, the characteristic that PtdIns con-
tains abundant arachidonate and is composed mainly of
1-stearoyl-2-arachidonoyl species also applies to tissues of
yellowtail. Lysophospholipid acyltransferase systems of the
yellowtail enable PtdIns to accumulate arachidonate in the
presence of large amounts of docosahexaenoic acid and a
limited supply of arachidonic acid. As PtdIns plays an
important role as a source of signaling molecules, the
conserved hydrophobic structure of PtdIns (the 1-stearoyl-
2-arachidonoyl moiety) may have physiological significance
in vertebrates.
Acknowledgements
This study was supported in part by a Grant-in-Aid for Encouragement
of Young Scientists (No. 12771422) from the Ministry of Education,
Science, Sports, and Culture of Japan.
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Ó FEBS 2003 Arachidonate in phosphatidylinositol of yellowtail (Eur. J. Biochem. 270) 1473