Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 223-229© PearsonProfessionalLtd 1996

Effects of 7-1inolenic acid and its
positional isomer pinolenic acid on
immune p a r a m e t e r s of Brown-Norway
rats
N. Matsuo 1, K. Osada 2, T. Kodama 1, B. O. Lim 1, A. Nakao ~, K. Yamada ~, M. S u g a n o 1
1Laboratory of Food Science, Kyushu University Schoo[ of Agriculture, Fukuoka 812-81, Japan.
2Faculty of Agriculture, Hirosaki University, Hirosaki, Aomori 036, Japan.

Summary Male Brown-Norway rats given purified diets containing safflower oil (SFO, linoleic acid, 18:2 n-6), evening

primrose oil (EPO, y-linolenic acid, 6,9,12- 18:3 n-6) or Korean pine seed oil (PSO, 5,9,12- 18:3) at the 10% level were
immunized twice with intraperitoneal ovalbumin, on days 14 and 35 of the feeding diets, and killed one day after thesecond booster. The relative population of CD4 ÷ T-lymphocytes in the spleen was significantly lower in rats fed SFO
than in those fed EPO or PSO, while that of CD8 ÷ subsets remained unchanged. There was a significant increase in
the splenic production of IgG and IgE in the PSO group compared to the SFO group, while EPO significantly increased
IgE. The periodical response patterns of the serum levels of IgG and IgE varied depending on the source of dietary
fats, and the initial rise of total immunoglobulins tended to be higher in the EPO group. The release by peritoneal
exudate cells of histamine was comparable among three groups irrespective of saturation by calcium ionophore
A23187, while PSO significantly increased leukotriene B4 production. These observations not only indicate specificroles of 7-1inolenic acid but also diverse influences of different octadecatrienoic acids in various immune
measurements.

INTRODUCTION

The type of dietary fatty acids, in particular polyunsaturated fatty acids (PUFA), crucially influences various parameters of the immune functions. 1-6 In general,
PUFA of the n-3 family suppress hypersensitivity while
those of the n-6 family tend to exaggerate the responsewhen ingested excessively.7,8 However, not all the n-6
PUFA cause similar effects, and 7-1inolenic acid appears
to reduce hypersensitivity.,9 10
Korean pine seed (Pinus oriental#) contains the peculiar octadecatrienoic acid, 5,9,12- 18:3 called pinolenicacid (PNO), and this acid exerts characteristic effects on
various lipid parameters including tissue lipid levels,
PUFA metabolism and eicosanoid production. 1~'12Considering the specificity of the enzymes involved in the
Received 7 December 1995
Accepted 21 December 1995
Correspondence to: N. Matsou, Tel. 92-641-1101; Fax. 92-632-1962.

metabolism of PUFA and, hence, the production ofeicosanoids, it seems likely that the existence of a doublebond at the 5-position, instead of the 6-position as in
7-1inolenic acid (GLA), potentially influences various
aspects of lipid metabolism. These effects in turn modify
the diverse parameters of the immune functions.
The present study deals with the effects of GLA and
PNO, as compared to linoleic acid, on the production of
immunoglobulins and chemical mediators in immune
sensitive Brown-Norway rats while immunized with
ovalbumin.
MATERIAL AND METHODS
Animals and diets

Male 4 weeks old Brown-Norway rats were purchasedfrom Seiwa Experimental Animals, Fukuoka, and given
commercial pellet (type NMF, Oriental Yeast Co., Tokyo).
After acclimatization for 7 days, the animals were fed
223

224

Matsuo et al

Table 1 Fatty acid compositions of dietary fats
Fatty acid

Safflower oil

Evening
primrose oil(weight %)Pine seed oil

16:0
18:0
18:1
18:2 n-6
18:3 n-3
18:3 (PO)*

13.8
2.7
18.8
64.6
-

17.0
2.5
18.1
55.8
6.5
-

4.9
2.2
28.7
47.1
17.1

*Pinoleic acid (cis-5,cis-9,cis-12-octadecatrienoic acid).

one of the three AIN-76 type purified diets containing
different fats throughout. The diets composition was, byweight percent, casein, 20; fat, 10; vitamin mixture, 1.0; mineral mixture, 3.5; choline bitartrate, 0.2, DL-methionine,0.3, cellulose, 5; corn starch, 15; and sucrose to 100. The
dietary fats used were edible grade safflower oil (Linol Oil
Co., Nagoya), evening primrose oil (Eisai Co., Tokyo), andKorean pine seed oil (Tama Co., Nagoya) (Table 1). Rats
were immunized on days 14 and 35 of the experiment
diets with 10mg of intravenous ovalbumin (Sigma
Chemical Co., St Louis, MO) and blood was withdrawn
from the tail vein every week for measurements ofserum immunoglobulin antibodies. 1 day after the second booster, peritoneal exudate cells (PEC) were collectedunder light diethyl ether anesthesia as described below.
The spleen and liver were then excised immediately.

bicarbonate buffer and each well was treated with 150 glof the solution for I h at 37°C. After blocking with 300 glof the blocking solution for 1 h at 37°C, wells were treatedwith 100 gl of the culture supematant or rat serum(10 x 6 times diluted for IgG and 10 times diluted for IgE)
for 1 h at 37°C. Bound IgE was then detected by reactingwith biotin-conjugated mouse anti-rat IgE (2000 times
diluted, Beryl, Montgomery, TX) followed by PODconjugated avidin (5000 times diluted, Dakopatts) for 1 h
at 4°C. Bound IgG was detected by reacting stepwise with
100 gl of POD-conjugated rabbit anti-rat IgG(Fab')2 (2000
times diluted, Biosoft) for 1 h at 37°C. Wells were rinsed
four times with 0.05% Tween 20 in PBS between eachstep. After incubating at 37°C for 15 min with 100 ~1 ofthe substrate solution, the reaction was stopped byadding 100 gl of 1.5% oxalic acid, and A415was measured
with a MPR-A4i ELISA reader (Tohso, Tokyo).
Isolation of splenic T-lymphocyte subsets

To the spleen lymphocytes suspended at 1 x 106 cells/
100 ml 10% FBS/PBS was added 5 ml of either CD4-FITC
or CD8-PE monoclonal antibodies (Serotec, Kidlington,
Oxford), and incubated at 4°C for 30 min. The lymphocytes were rinsed three times with PBS containing 10%
FBS and centrifuged at 1200 rpm for 5 min. The stained
lymphocytes were fixed by 2% paraformaldehyde andwere counted by flow cytometry (Epics Profile II, Coulter
Electronics, Luton, Beds). 14

Preparation of rat spleen lymphocyte and cell culture

Spleen lymphocytes were squeezed out into the
RPMI1640 medium. 13After incubating the cells at 37°C
for 30 rain to remove fibroblasts, 5 ml of the cell suspension was layered on 4 ml of Lymphocyte-Rat (Cedarlane,
Hornby, Canada) and centrifuged at 300 x g for 30 rain.
The lymphocyte band at the interface was removed,
and the cells were rinsed three times with the RPMI1640
medium. The lymphocytes were cultured in 10%
FBS/RPMI 1640 medium for 6 h (IgE) or 24 h (IgG), andthe Ig contents of the culture supernatants were measured by ELISA.
Measurements of immunoglobulins

Measurements of total and specific Igs were executedusing sandwich ELISA methodsJ 3 To measure total Igs,
goat anti-rat IgE, rabbit anti-rat IgG (Fab')2, (all from
Biosoft, Paris) were used to fix respective Igs. These antibodies were diluted 1000 times with 50 mM carbonatebicarbonate buffer (pH 9.6), and each well of 96-wellplates was treated with 100 gl of the solution for 1 h
at 37°C. To measure specific Igs, ovalbumin was dissolved
at the concentration of 50 mg/ml in 50 mM carbonate-

Preparation of rat peritoneal exudate cells (PEC)

Tyrode buffer (137 mM NaC1, 2.7 mM KC1, 1.8 mM CaC12,
1.1 mM MgC12, l l.9mM Nat-ICO3, 0.4mM NaH2PO4,
5.6raM glucose, pH 7.2) containing 0.1% (w/v) BSA(Boehringer Mannheim GmbH) was injected into the peritoneal cavity and the abdomen was gently massaged for
2 min. The abdomen was then opened, and the fluid
containing the PEG was collected with a Pasteur pipette.
Cells were gently washed with Tyrode buffer and centrifuged at 200 x g for 10 min at 4°C. The cell pellets were
resuspended in a modified ammonium chloride buffer(150mM NH4C1, 10ram KHCO3, 10raM EDTA-2Na, pH
7.4) and then incubated for 5 min at 4°C. The cell suspension was centrifuged at 200 x g for 5 min at 4°C and thecells were resuspended in the Tyrode buffer. Cell viability
was measured by trypan blue staining and mast cells wereidentified by toluidine blue staining. Viability of thispreparation was more than 95% and mast cells occupied
5-10% of the total cells.
Measurement of histamine

PEC (1 × 106 cells) were suspended in 2.25 ml of Tyrode

Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 223-229© Pearson Professional Ltd 1996

Octadecatrienoic acid and immune function

buffer containing 0.9 mM CaC12 in polypropylene tubes
at 4°C. The cell suspension was mixed with 0.25 ml of
50gM A23187, and incubated for 2 0 m i n at 37°C. The
reaction was terminated by incubating for 15 min at 4°C.
The cell suspension was then centrifuged at 300 x g for
10 min and the content of histamine in the supernatants
was measured by fluorometric assay. 15 To the mixture of
2 ml of sample solution, 0.75 g of NaC1 and 0.5 ml of 1N
NaOH was added 5 ml of the mkxture of n-butanol andchloroform (3:2, v/v) and mixed for 5 min. After centrffugation at 270 x g for 5 min, 4 ml of the organic solvent
layer was recovered and mixed with 2 ml n-heptane and1.5 ml 0.1N HC1 for 5 min. After centrifugation at 270 x g
for 5 min, 1 ml of the HC1 layer was recovered, mixed with
0.15 ml of 1N NaOH and 0.1 ml of 0.2% o-phthalaldehyde(OPT), and stood for 5 min at room temperature. The reaction was terminated by adding 0.14 ml of 0.5N H2SO4, andthe fluorescence intensity was measured using a spectrofluorophotometer (RFS00, Simadzu Co., Kyoto) with theexcitation at 360 nm and the emission at 450 nm.
Measurement of leukotriene B4 (LTB4)PEC (2 x 106 cells) were suspended in 45 gl of Tyrode
buffer containing 0.9 mM CaC12 in polypropylene tubes at
4°C. The cell suspensions were mixed with 5 gl of 50 gM
A23187 or 10gg/ml compound 48/80, and incubatedfor 2 0 m i n at 37°C. The reaction was terminated byadding 50gl of the mixture of acetonitrile:methanol(30:25, v/v) and kept at -30°C for 15 min. To measure LTB4,
the internal standard, 50 ng of PGB2 (Sigma ChemicalCo., St Louis, MO) was added to the cell suspensions
and centrifuged at 300 x g for 10 min. The supernatant
was filtrated through a 0.22 gm filter (Millipore, Tokyo),and LTB4 was measured by reversed-phase HPLC (SCL10& Simadzu Co., Kyoto) as described previously. 16
Briefly, 20 gl of the sample was injected on a ODS-A
column (150 x 6.0mm, 5 g m particle size). A mixtureof acetonitrile:methanol:H20 (30:25:45, v/v/v), 5 m M
CH3COONH4 and 1 mM EDTA-2Na (pH 5.6) was used as amobile phase with a flow rate of 1.1 ml/min. LTB4 andPGB2 were detected by absorbance at 280 nm (SPD-10A,
Simadzu Co., Kyoto). Quantification was made by comparing the LTB4 peak areas with that of the known
amount of the standard and correction for recovery.

Statistical analysis

Results were expressed as mean + SE. To establish theexact nature of the differences between the groups,
one-way analysis of variance was followed by Duncan's
new multiple range test, 2°
RESULTS
Food intake, growth and liver weight

On average, a rat weighing 82 g was fed 8.1 g per day andgained 140 g body weight over 7 weeks. There were no
significant differences in these indices among the threegroups. The average relative liver weight was the same inall groups, 3.8 g per 100 g body weight.
Splenic T-lymphocyte subsets

As shown in Table 2, there was a significant reductionof the relative population of CD4 + T cell in rats fed SFO
compared to those fed EPO and PSO. However, the proportion of CD8 + remained unchanged. Consequently, theratio of CD4+/CD8 + in the EPO and PSO groups tended
to be higher than in the SFO group.
Serum immunoglobulin levels

The time courses of the serum immunoglobulin levels
are depicted in Figure 1. In the EPO and SAF groups total
IgG levels increased 1 week after immunization, anddecreased thereafter. The peak level for IgG was highest
in the EPO group followed by SFO, while in rats fed PSO
it did not increase. The contours of specific IgG were,
however, the same among the three groups a n d - i t
increased after the second booster. The contours of total
IgE levels resembled those of IgG, but the peak level ofthe PSO group was between the EPO and SFO groups after
1 week, and decreased to a low level 2 weeks after
immunization. The specific IgE levels of rats fed EPO
reached a peak level 1 week after the immunization andkept at the high level until the second booster. The SFO
and PSO groups depicted similar response patterns inwhich the specific IgE levels remained unchanged until
1 week after the ovalbumin administration, particularly

Table 2 Effect of dietary fats on splenic T-lymphocyte subsets

Analyses of liver fatty acid compositions

Liver lipids were extracted by a mixture of chloroform-methanol 17 and phospholipids were separated into
phosphatidylcholine and phosphatidylethanolamine by
thin-layer chromatography. TM The fatty acid compositionsof these phospholipids were analyzed by gas-liquid
chromatography. 19© Pearson Professional Ltd 1996

225

Dietary fat

CD4 +

CD8 +

CD4÷/CD8 +Safflower oil

36.1 + 0.7"
43.1 + 0.7 b
39.2 _+ 2.7 b

7.73 _ 0.63
7.78 _+ 0.48
6.88 _+ 0.24

4.72 _+0.22
5.66 _+ 0.46
5.74 _+0.49

Evening primrose oil
Pine seed oil

Means + SE for 4 rats. aWalues not sharing a c o m m o n letter are
significantly different at P < 0.05

Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 223-229

226

Matsuo et al

10 8 . ~

'

'

i

•

400

i

T o t a l IgG
300
A

S~)

1°7

200

a

;o

100

f ..I

10 6

i

I

I

f

I

J

0
70

1° 5

60
50

10 4

40

103

30
20

1112

10

lO

=

0

I
10

~

I
20

i

I
30

i

40

0

0

10

20

30

40

Time (day)Fig. 1 The effect of various dietary fats on serum immunoglobulin levels in Brown-Norway rats.
Measurements of total and specific Igs in rat serum were executed using sandwich ELISA
methods as described in Materials and Methods. O, safflower oil; e, evening primrose oil; A, pineseed oil. Data are presented as means + SE for 4 rats. abValues without a common superscript
letter are significantly different at P < 0.05

in the PSO group, and reached the peak values after 3
weeks. The levels then decreased at 4 weeks.
Ig production by spleen lymphocytes

Spleen lymphocytes were cultured in 10% FBS/
RPMI1640 medium for 6 h (IgE) or 2 4 h (IgG), and Ig
contents of culture supernatants were measured by ELISAAs shown in Figure 2, the content of total IgG in the PSO
group was significantly higher than in the other twogroups. However, the specific IgG content in the PSO
group was similar to the SFO group, and was significantly
higher than in the EPO group. On the other hand, thetotal and specific IgE contents were higher in the EPO andPSO groups than in the SFO group although there was nodifference in the two former groups.

parable between SFO and PSO, the desaturation index
was also significantly higher in the former due to alower percentage of linoleic acid. A similar response
pattern was observed in the desaturation index ofphosphatidylethanolamine.
Production of chemical mediators by peritoneal exudatecells

As shown in Figure 3, the peritoneal exudate cells stimulated by calcium ionophore A23187 released a similar
amount of histamine in three groups. In contrast, LTB4
production tended to reduce in the EPO groups, andsignificantly increased in the PSO group compared to the
SFO group.
DISCUSSION

Fatty acid compositions of liver phospholipids

As shown in Table 3, the percentage of linoleic acidin liver phosphatidylcholine was significantly lower andthat of arachidonic acid was significantly higher in rats
fed EPO than in those fed SFO. Consequently, the desaturation index for linoleic acid, the ratio of (20:3 n-6 + 20:4
n-6)/18:2 n-6, was significantly higher in the EPO group.
Although the percentage of arachidonic acid was corn-

Recent studies have stressed a role of dietary PUFA indiverse immunological functions. 1-~ In general, PUFAsof the n-3 family improve hyperreactivity compared ton-6 PUFA. z,8 Eicosapentaenoic acid (EPA, 20:5 n-3) anddocosahexaenoic acid (DHA, 22:6 n-3) in fish oil inhibit
the 5-lipoxygenase pathway and LTB4 generation in neutrophils and monocytes in vitro, 2~resulting in alleviationof rheumatoid arthritis. 22 The effect of linoleic acid, theProstaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 223-229© Pearson Professional Ltd 1996

Octadecatrienoic acid and immune function

20 [ T o t a l IgG

L

a

40

2"51Total IgEHistamine

3o

a

2"0t

227

b J
C

10

,0

0 , 5 ~

E

10

-

g
o.o SFO

EPO

PSO

0

J

-

!

"

LTB4
E
"E

b

a

oc-o
10

b

c

0

///./
,r • ./v/ / / /,/ /,/// / / /

I=.J

/'///

/ / " / ,,,/ .zr . r /

5

b

lO

a

8
6

a

4!-

a

//./',/

/.///

2

0

SFO

EPO

PSO

SFO

EPO

0

PSO

Fig. 2 The effect of various dietary fats on Ig production from rat
spleen lymphocytes. Rat spleen lymphocytes were cultured in 10%
FBS/RPMI 1640 medium for 6 h (IgE) or 24 h (IgG). The Ig
contents of the culture supernatants were measured by ELISA
methods as described in Materials and Methods. SFA, safflower oil;
EPO, evening primrose oil; PSO, pineseed oil. Data are presented
as m e a n s -+ SE for 3 dishes. ~"°Values without a c o m m o nsuperscript letter are significantly different at P < 0.05

EPO

SFO

PSO

Fig. 3 Effect of various dietary fats on histamine and LTB4 releasefrom rat peritoneal exudate cells. Cells were incubated at 37°C for
20 min. Histamine content of s u p e m a t a n t was measured by afluorometric a s s a y and LTB 4 content of supernatant was measured
by reversed-phase HPLC assay, as described in Materials andMethods. SFA, safflower oil; EPO, evening primrose oil; PSO, pineseed oil. Data are presented as m e a n s _+ SE for 4 rats. a"Values
without a common superscript letter are significantly different at
P < 0.05.

Table 3 Fatty acids composition of liver phospholipids
Dietary fat

Fatty acids (weight %)
16:0

16:1

18:0

18:1

18:2
n-6

18:3
(PO)*

20:3
n-6

20:4
n-6

22:5
n-6

22:6
n-3

Ratio
(20:3 + 20:4)/18:2

Phosphatidylcholine
SFO
19.5 + 0.2 ~ 0.7 + 0.0 a 23.0 ± 0.4 4.2 + 0.2 a 17.5 ± 0.5 a 0.6 ± 0.1 a 29.8±0.42 1.1±0.1
EPO
19.6+0.4 a 0.6±0.0 a 23.3+0.4 4.0+0.1 a 14.8±0.4 b 0 . 9 ± 0 . 1 a 3 2 . 0 ± 0 . 3 b 1.0±0.1
PSO
1 7 . 5 + 0 . 4 b 1 . 2 ± 0 . 0 b 2 2 . 9 + 0 . 4 6 . 0 ± 0 . 2 b 1 4 . 2 ± 0 . 5 b 3 . 7 ± 0 . 3 1.7±0.1 b 2 8 . 5 ± 0 . 9 a 1.2±0.1

1.9+0.2
1.6±0.1
1.9±0.2

Phosphatidylethanolamine
SFO
14.7+0.2 a 0.2+0.0 a 26.6+0.2
EPO
14.8+0.5" 0 . 2 ± 0 . 0 ~ 2 7 . 3 ± 0 . 8
PSO
12.9±0,4" 0,5±0.0" 26.7±0,26.4±0.2 a 3.6+0.1"
5.5+0.4 b 4.1±0.2 b
6.0±0.2 a 4.5+0.1 b

3.5±0.1 a 9.2±0.2
3.6±0.18 7 . 7 + 0 . 4 b
4.9±0.1 b 7.6±0.2"

0.4+0.0" 32.5±0.2 3.4±0.3
0.6±0.0 b 32.0±0.72 3.2±0.3
1.6±0.1 1.1_+0.1 ° 3 2 . 4 + 0 . 4 3 . 6 + 0 . 3

1.7±0.1 ~2.2±0.1 ~2.2±0.1 b

Data are presented as means + SE for 8 rats. SFO, safflower oil; EPO, evening primrose oil; PSO, pine seed oil. ab°Values without a common
superscript letter are significantly different at P < 0.05. *Pinoleic acid (5, 9, 12-octadecatrienoic acid),© Pearson Professional Ltd 1996

Prostaglandins, Leukotrienes and Essential Fatty Acids (1996) 55(4), 223-229

228

Matsuo et al

most abundantly occurring n-6 PUFA in the diet, rather
tends to stimulate these responses. 7,23
On the other hand, evening primrose oil (EPO), a raresource of GLA (18:3 n-6), improves atopic eczema
after oral administration, 9,1° although it simultaneously
contains a relatively large amount of linoleic acid. EPO
increases tissue levels of prostaglandin E1 (PGEI) andsuppresses chronical inflammation. 24,25 In rats, PGE1 suppresses the inflammation of adjuvant arthritis, 2~ andi m m u n e complex vasculitis. 27Different GLA pools appear
to occur in the b o d y between endogenously formed
GLA and exogenously absorbed GLA.2s Thus, the supplementation of EPO increases the level of dihomo-ylinolenic acid (DGLA, 20:3 n-6), which is the precursor of
the putative anti-inflammatory substance, PGEI, in neutrophil and epidermal phospholipids, 29and consequently
reduces LTB4 release from h u m a n polymorphonuclear
neutrophil. 3° These observations suggest a possible roleof GLA in the immune regulation.
The present study indicated that LTB4 production in the
exudate cells tended to be suppressed by feeding EPO,
while PSO significantly elevated the production. On theother hand, histamine release from peritoneal exudate
cells was not significantly different a m o n g three dietarygroups. Both histamine and LTB4 are chemical mediatorsfrom mast cells and cause an inflammatory reaction. 31
Since the source of the precursor arachidonic acid for
LTB4 is the m e m b r a n e phospholipids and since the fatty
acid composition of m e m b r a n e phospholipids is readily
modified by the type of dietary fats, it is likely that the
production of LTB4 can be modulated by dietary fat. In
fact, n-3 PUFA decrease LTB4 release from mast cells but
do not inhibit histamine release in the case of n-6 PUFA. 7
Immunoglobulin production is regulated by T lymphocytes, and T cells are classified CD4 and CD8 positive
T cells having helper and suppressive functions, respectively.32 In the present study, the relative proportion ofCD4 ÷ T cells in the spleen lymphocytes significantly
increased in rats fed EPO and PSO compared to SFO, but
the CD8 ÷ T cell population remained unchanged. The
ratio of CD4÷/CD8 ÷T cells in the EPO and PSO groups wastherefore higher than in the SFO group. On the other
hand, the total IgG production by spleen lymphocytes
was significantly higher in the PSO group than in theother two groups, although the specific IgG content in
the PSO group was significantly higher than in the EPO
group. In addition, total and specific IgE productions byspleen lymphocytes were significantly higher in the EPO
and PSO groups than in the SFO group. These results
suggest that the increase of the CD4 ÷ T cell populationand the CD4+/CD8 + ratio affects the IgE production
by spleen lymphocytes. Kemeny et a133 reported thatthe decrease of the CD8 ÷ T cell population and theCD4+/CD8 ÷ ratio enhanced IgE production.

The regulation of T lymphocytes by dietary fatty acids
is related to the development of immune functions.
In human, EPO supplemented in atopic eczema reduced
the ratio of CD4÷/CD8 + T cells. 34 This ratio in children
with atopic eczema was reported to be high. 35The present
study, however, indicated that dietary GLA and its positional isomer, pinolenic acid, tended to elevate the ratio of
CD4+/CD8 +.
Dietary fatty acids also exert potent and pervasive
effects on both specific and non-specific immune reactions. PUFA inhibited proliferation of T lymphocytes 3 orenhanced proliferation of T and B lymphocytes. 4 Dietary
fatty acids modulated immune responses. 5,6Our observations indicated that concentrations in serum and production b y spleen lymphocytes of Igs were different between
the GLA and PNO as dietary GLA increased serum Igs inan early phase after immunization, while PNO increased
the concentration only a little or left it unchanged. In contrast, Ig production b y spleen lymphocytes was increasedby the PNO supplementation. These results suggest that
dietary GLA and its isomer cause different immune
responses including immunoglobulin and inflammatory
mediator production. The mechanisms causing thesedifferent responses deserve further study.

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