METABOLISM AND NUTRITION
Effects of Dietary Conjugated Linoleic Acid and Linoleic:Linolenic Acid Ratio
on Polyunsaturated Fatty Acid Status in Laying Hens1
M. Du, D. U. Ahn,2 and J. L. Sell
Department of Animal Science, Iowa State University, Ames, Iowa 50011-3150
ABSTRACT A study was conducted to determine the
effects of dietary conjugated linoleic acid (CLA) and the
ratio of linoleic:linolenic acid on long-chain polyunsaturated fatty acid status. Thirty-two 31-wk-old White Leghorn hens were randomly assigned to four diets containing 8.2% soy oil, 4.1% soy oil + 2.5% CLA (4.1% CLA
source), 4.1% flax oil + 2.5% CLA, or 4.1% soy oil + 4.1%
flax oil. Hens were fed the diets for 3 wk before eggs and
tissues were collected for the study. Lipids were extracted
from egg yolk and tissues, classes of egg yolk lipids were
separated, and fatty acid concentrations of total lipids,
triglyceride, phosphatidylethanolamine, and phosphatidylcholine were analyzed by gas chromatography.
The concentrations of monounsaturated fatty acids and
non-CLA polyunsaturated fatty acids were reduced after
CLA feeding. The amount of arachidonic acid was decreased after CLA feeding in linoleic acid- and linolenic
acid-rich diets, but amounts of eicosapentaenoic acid and
docosahexaenoic acid were increased in the linolenic-rich
diet, indicating that the synthesis or deposition of longchain n-3 fatty acids was accelerated after CLA feeding.
The increased docosahexaenoic acid and eicosapentaenoic acid contents in lipid may be compensation for the
decreased arachidonic acid content. Dietary supplementation of linoleic acid increased n-6 fatty acid levels in
lipids, whereas linolenic acid increased n-3 fatty acid levels. Results also suggest that CLA might not be elongated
to synthesize long-chain fatty acids in significant
amounts. The effect of CLA in reducing the level of n-6
fatty acids and promoting the level of n-3 fatty acids could
be related to the biological effects of CLA.
(Key words: dietary conjugated linoleic acid, biosynthesis, arachidonic acid, docosahexaenoic acid, egg yolk)
2000 Poultry Science 79:1749–1756
INTRODUCTION
Dietary conjugated linoleic acid (CLA) has anticarcinogenic, antiartherogenic effects and modulates immune
responses (Lee et al., 1995; Ip et al., 1995; Belury et al.,
1996; Ip, 1997). Ip et al. (1997) reported that CLA inhibited the postinitiation phase of carcinogenesis. Visonneau et al. (1997) found that CLA suppressed the growth
of human breast adenocarcinoma cells. Nicolosi et al.
(1997) showed that CLA reduced plasma lipoprotein
content and early development of atherosclerosis in
hamsters. Sugano et al. (1997, 1998) reported that CLA
feeding lowered the concentration of prostaglandin E2
and leukotriene 4 in the serum and spleen of rats. Li
and Watkins (1998) also suggested that CLA changed
fatty acid composition and reduced the prostaglandin
E2 production in rats. Prostaglandin E2 is suspected to
have cancer-promoting effects. Lee et al. (1995) showed
Received for publication September 27, 1999.
Accepted for publication June 30, 2000.
1
1Journal paper No. J-18617 of the Iowa Agriculture and Home Economics Experiment Station (Ames, IA 50011-3150); Project No. 3322,
and supported by the Iowa Egg Council (Ames, IA 50010) and Center
for Designing Foods to Improve Nutrition (CDFIN).
2
To whom correspondence should be addressed: duahn@iastate.edu.
that the content of monounsaturated fatty acids in tissues decreased after CLA feeding. Sugano et al. (1998)
reported a decrease in the concentration of arachidonic
acid and other polyunsaturated fatty acids (PUFA) in the
total lipid of spleen lymphocytes and peritoneal exudate
cells after feeding CLA to mice. Ahn et al. (1999) reported
similar compositional changes in egg yolk lipids after
feeding hens with diets containing CLA.
Long-chain PUFA can be synthesized from either n-3
or n-6 precursors. Bretillon et al. (1999) reported that
dietary cis9, trans11 CLA isomer inhibited the activity
of ∆6-desaturase in rat liver microsomes, which indicates that the decreases in unsaturated fatty acids could
be caused by the competitive inhibition of ∆6-desaturase
by CLA (Belury and Kempa-Steczko, 1997; Du et al.,
1999). If this desatarase inhibition is true, the biosynthesis of arachidonic acid and docosahexaenoic acid (DHA)
should be inhibited, because in birds, ∆6-desaturase is
needed to synthesize both of them. To test the influence
of CLA on the synthesis of long-chain PUFA, linolenic
Abbreviation Key: CLA = conjugated linoleic acid; DHA = docosahexaenoic acid; EPA = eicosapentaenoic acid; GC = gas chromatography;
PC = phosphatidylcholine; PE = phosphatidylethanolamine; PUFA =
polyunsaturated fatty acid; TG = triglyceride.
1749
1750
DU ET AL.
TABLE 1. Percentage composition of diets fed to laying hens
Ingredients
Percentage
Corn
Soy meal
Wheat middlings
Limestone
Dicalcium phosphate
Meat and bone meat
Dehydrated alfalfa meal
Mineral premix1
Vitamin premix2
DL-methionine
Sodium chloride (iodized)
Soybean oil
Flax oil
CLA source
Calculated analysis
Metabolizable energy, kcal/kg
Protein
TSAA
Methionine
Lysine
Calcium
Nonphytate
Sodium
Ether extract
35.29
18.17
22.85
8.77
0.40
3.00
2.50
0.30
0.30
0.14
0.08
8.20–03
4.10–03
0–4.103
2,905
17.00
0.70
0.40
0.90
3.85
0.35
0.20
10.31
1
Mineral premix provides per kilogram of diet: Mn, 80 mg; Zn, 90
mg; Fe, 60 mg; Cu, 12 mg; Se, 0.147 mg; sodium chloride, 2.247 g.
2
Vitamin premix supplies per kilogram of diet: retinyl acetate, 8,065
IU; cholecalciferol, 1,580 IU; 25-hydroxy-vitamin D3, 31.5 µg; dl-α-tocopheryl acetate, 15 IU; vitamin B12, 16 µg; menadcre, 4 mg; riboflavin,
7.8 mg; pantothenic acid, 12.8 mg; niacin, 75 mg; Choline chloride, 509
mg; folic acid, 1.62 mg; biotin, 0.27 mg.
3
In A group, soy oil, 8.20%. In B group, soy oil, 4.10%; conjugated
linoleic acid (CLA) source, 4.10% (equal to 2.5% CLA). In C group, flax
oil, 4.10%; CLA source, 4.10%. In D group, soy oil, 4.10%; flax oil, 4.10%.
acid- or linoleic acid-rich diets were used to analyze the
accumulation of arachidonic acid and DHA after CLA
feeding. Polyunsaturated fatty acids are precursors of
eicosanoids, which relate to the immune response, cancer promotion, and atherosclerosis. Analysis of the influence of CLA on PUFA composition will also help to
illustrate the mechanism of CLA in modulating these
processes. In this study, diets rich in linoleic or linolenic
acid were formulated using soy oil and flax oil to assess
the effects of dietary CLA on PUFA composition in vivo.
MATERIALS AND METHODS
Hen Feeding and Sample Preparation
Thirty-two 31-wk-old White Leghorn hens, kept in
individual cages, were assigned to each of four dietary
treatments that consisted of diets containing 8.2% soy
oil, 4.1% soy oil + 2.5% CLA (4.1% CLA source), 4.1%
flax oil + 2.5% CLA, or 4.1% soy oil + 4.1% flax oil (Table
1). The CLA source used in this study was obtained
from a commercial company3 and contained 61% CLA.
Therefore, 4.1% CLA source added in the diet is equiva-
3
Conlinco, Inc., Detroit Lakes, MN 56502.
Brinkman Instruments, Inc., Westbury, NY 11590-0207.
Sigma-Aldrich, 89552 Steinheim, Germany.
4
5
lent to 2.5% CLA, and the actual amount of CLA (2.5%)
instead of the CLA source (4.1%) was used in the text.
Soybean oil, flax oil, and the CLA source were substituted on a weight:weight basis in different diets. Compositions of experimental diets are presented in Table 1.
After feeding hens for 3 wk, eggs were collected for 4
consecutive d, and four eggs (from different hens) per
treatment were randomly selected and analyzed. After
their eggs were collected, four hens from each group
were sacrificed, and liver and leg muscles were sampled
for fatty acid composition analysis. Tissues were frozen
in liquid nitrogen immediately after sampling. Lipid
classes of egg yolk were separated by thin-layer chromatography. Fatty acid compositions of total egg yolk lipid
and lipid classes were analyzed by gas chromatography (GC).
Lipid Extraction
Two-gram (egg yolk and liver) or 4-g (muscle) samples
were weighed into a test tube with 10 volumes of Folch
1 (chloroform:methanol = 2:1, wt/vol; Folch et al., 1957),
and homogenized with a Brinkman polytron4 (Type PT
10/35) for 10 s at high speed. Twenty-five micrograms
of butylated hydroxyanisole (10%) dissolved in 98% ethanol was added to each sample prior to homogenization.
The homogenate was filtered through a Whatman #1
filter paper into a 100-mL graduated cylinder and 1/4
volume (on the basis of Folch 1) of 0.88% NaCl solution
was added. After the cylinder was capped with a glass
stopper, the filtrate was mixed well. The inside of the
cylinder was washed twice with 10 mL of Folch 2
(3:47:48/CHCl3:CH3OH:H2O), and the contents were
stored until the aqueous and organic layers clearly separated. The upper layer was siphoned off, and the lower
layer was moved to a glass scintillation vial and dried
at 50 C under nitrogen.
Separation of Lipid Classes
The dried lipids of egg yolk and tissue were redissolved with chloroform to set the final concentration of
lipid at 0.2 g/mL. The lipid-chloroform solution (150
µL) was loaded onto an activated (120 C for 2 h) silica
gel plate5 (20 × 20 cm). The plate was developed first
in Solvent I, composed of chloroform:methanol:water
(65:25:4, vol/vol/vol), until the solvent line reached the
middle of the plate. The plate was air dried and then
redeveloped in Solvent II, composed of hexane:diethyl
ether (4:1, vol/vol), until the solvent front reached 1"
below the top of the plate. After air drying for 10 min
at room temperature (22 C), the plates were sprayed
with 0.1% 2′,7′-dichlorofluororescein in ethanol. Lipid
classes were identified under UV light, and the lanes
corresponding to triglyceride (TG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were scraped
into separate test tubes (Ahn et al., 1995), and methylated.
CONJUGATED LINOLEIC ACID AND POLYUNSATURATED FATTY ACIDS
Analysis of Fatty Acid Composition
One milliliter of methylating reagent (anhydrous
methanolic-HCl-3N) was added to the test tube containing total lipid, TG, PE, or PC, capped tightly, and
incubated in a water bath at 60 C for 40 min. After
cooling to room temperature, 2 mL of hexane and 5 mL
of water were added, mixed thoroughly, and left at room
temperature overnight for phase separation. The top
hexane layer containing methylated fatty acids was used
for GC analysis (Chin et al., 1992). Analysis of fatty
acid composition was performed with a GC6 (HP 6890)
equipped with an autosample injector6 and flame ionization detector. A capillary column7 (HP-5, 0.32 mm inside
diameter, 30 m, 0.25 µm film thickness) was used. A
splitless inlet was used to inject samples (1 µL) into the
capillary column. Ramped oven temperature conditions
(180 C for 2.5 min, increased to 230 C at 2.5 C/min, then
held at 230 C for 7.5 min) were used.
Temperatures of both inlet and detector were 280 C.
Helium was used as a carrier gas, and a constant column
flow of 1.1 mL/min was used. Flame ionization detector
air, H2, and make-up gas (helium) flows were 350 mL/
min, 35 mL/min, and 43 mL/min, respectively. Fatty
acids were identified using a Mass Selective detector6
(Model 5973). The GC-Mass Selective detector procedure
was performed with the same column and oven temperature conditions described previously. The ionization potential of the Mass Selective detector was 70 eV, and the
scan range was 45 to 450 m/z. Identification of fatty
acids was achieved by comparing mass spectral data
with those of the Wiley library.7 The CLA isomers in
egg yolk lipids were identified by comparing against
CLA standards purchased from Matreya8 and Nuchek9,
and CLA standards according to the report of Christie
et al. (1997). The compositions of CLA isomers and fatty
acids were reported as percentages of composition of
total lipids, and total peak area (pA*s) was used to calculate fatty acid composition.
Statistical Analysis
The effect of dietary CLA on the fatty acid composition
of egg yolk and tissue lipids was analyzed using SAS
software (SAS Institute Inc., 1985). Student-NewmanKeul’s multiple range test was used to compare differences among mean values (P < 0.05). Mean values and
standard errors of the mean are reported.
RESULTS AND DISCUSSION
The control diet (8.2% soy oil) was rich in linoleic acid,
the 4.1% soy oil + 2.5% CLA diet was high in CLA and
6
Sigma-Aldrich, St. Louis, MO 63178.
Hewlett Packard Co., Wilmington, DE 16808-1610.
8
Matreya, Inc., Pleasant Gap, PA 16823.
9
Nuchek, Elysian, MN 56028.
7
1751
linoleic acid, the 4.1% flax oil + 2.5% CLA diet contained
CLA and was supplemented with linolenic acid, and the
4.1% soy oil + 4.1% flax oil treatment was rich in linoleic
and linolenic acids (Tables 1 and 2). Calculated contributions of the supplemented oils to the dietary fatty acid
levels for each diet are shown in Table 2. The control
diet had 40.22% linoleic acid, the 4.1% soy oil + 2.5%
CLA diet had 21.32% CLA and 22.76% linoleic acid, the
4.1% flax oil + 2.5% CLA diet had 21.32% CLA and
20.28% linolenic acid, and the 4.1% soy oil + 4.1% flax
oil diet contained 26.02% linoleic and 23.50% linolenic
acids. After 3 wk of feeding, there were significant differences in yolk lipid composition among the four groups
(Table 3). The percentage content of arachidonic acid in
egg yolk lipids from hens fed diets containing CLA was
lower than that from the control diet, which may be due
to the reduced linoleic acid level in CLA diets after soy
oil was substituted with flax or the CLA source (Du et
al., 1999). Sugano et al. (1998) also reported a decrease
in the concentration of arachidonic acid after feeding
CLA to mice.
The concentration of linoleic acid in chicken yolk and
tissue lipids was, in order: 8.2% soy oil > 4.1% soy oil +
4.1% flax oil > 4.1% soy oil + 2.5% CLA > 4.1% flax oil
+ 2.5% CLA group, which appeared to be proportional
to the content of dietary linoleic acid (Tables 2 and 3).
The concentrations of linolenic acid in egg yolk lipids
from hens fed diets containing 4.1% flax oil + 2.5% CLA
or 4.1% soy oil + 4.1% flax oil were significantly (P <
0.01) higher than those of the 8.2% soy oil and 4.1% soy
oil + 2.5% CLA treatments, which may be due to the
addition of flax oil. Egg yolk lipids from hens fed diets
containing 4.1% soy oil + 2.5% CLA and 4.1% flax oil +
2.5% CLA had high amounts of stearic and palmitic
acids, indicating that feeding CLA reduced the overall
deposition of unsaturated fatty acid.
There were significant differences in the arachidonic
acid concentrations in egg yolk and tissues from the
four dietary groups; the amount of arachidonic acid was
highest with the 8.2% soy oil diet and lowest with the
4.1% flax oil + 2.5% CLA treatment. This concentration
difference was related to the dietary linoleic acid that is
a substrate for arachidonic acid synthesis, and could
also be related to the inhibiting effects of CLA on ∆6desaturase activity (Bretillon et al., 1999). Egg yolk lipids
from hens fed a diet containing 4.1% flax oil + 2.5% CLA
contained significantly higher amounts of DHA than the
4.1% soy oil + 4.1% flax oil treatment, in spite of the
same dietary content of linolenic acid (Table 3). Because
the DHA content in the diet should be very low and
about the same for all four groups (Tables 1 and 2), the
increase of DHA content in egg yolk in CLA feeding
groups indicated that CLA promoted synthesis of n-3
long-chain PUFA. Alternatively, CLA could alter DHA
metabolism to increase its use for egg yolk accretion.
Turek et al. (1998) fed a diet containing CLA or soybean
oil (control) to rats and found that DHA content in rat
tissue from the CLA diet was significantly higher than
that from the soybean diet; Li and Watkins (1998) also
1752
DU ET AL.
TABLE 2. The fatty acid (FA) composition of soy oil, flax oil, and oil and conjugated linoleic acid (CLA)
source, and the calculated contribution of oil sources to the fatty acid composition of diets
FA composition of oil sources
Calculated FA contribution to diets
Fatty acid
CLA source
Flax oil
Soybean oil
Soy
Soy + CLA1
Flax + CLA1
Soy + flax1
Palmitic
Stearic
Oleic
Linoleic
Linolenic
Others
CLA (cis9, trans11)
CLA (cis10, trans12)
CLA (trans9, trans11)
Other CLA isomers
CLA total
PUFA
Non-CLA PUFA
n6/n3 fatty acid ratio
4.16
2.10
16.97
6.65
ND2
8.60
17.94
20.27
15.34
7.97
61.52
7.15
2.24
15.30
14.83
50.96
0.52
...
...
...
...
...
14.64
4.56
21.56
50.54
8.07
0.63
...
...
...
...
...
11.64
3.63
17.15
40.19
6.41
0.50
...
...
...
...
...
46.60
46.60
6.27
7.47
2.65
15.32
22.74
3.20
3.67
7.14
8.06
6.10
3.17
24.47
50.41
25.94
7.11
4.50
1.72
12.83
8.54
20.26
3.63
7.13
8.06
6.11
3.17
24.47
53.27
28.80
5.58
8.67
2.70
14.66
25.99
23.47
1.45
...
...
...
...
...
49.46
49.46
1.11
1
Based on the calculated value of oils added in diet.
ND = not detected.
2
showed that the concentrations of n-3 fatty acids eicosapentaenoic acid (EPA) and DHA increased after CLA
feeding.
The DHA contents of egg yolk from hens fed a diet
containing 4.1% flax oil + 2.5% CLA and 4.1% soy oil +
4.1% flax oil were much higher than those of the hens
fed diets containing 8.2% soy oil and 4.1% soy oil + 2.5%
CLA, indicating that dietary linolenic acid enhanced the
biosynthesis of DHA. Another possibility is that CLA
influenced the partitioning or transport of DHA to the
uptake of VLDL by the follicle. Arachidonic acid contents in diets with 8.2% soy oil and 4.1% soy oil +2.5%
CLA were higher than those of diets without soy oil,
indicating that dietary linoleic acid increased arachidonic acid level in yolk lipid.
The concentration of oleic acid in yolk lipid from hens
fed a diet containing 4.1% soy oil + 2.5% CLA was much
lower than that of hens fed the 4.1% flax oil + 2.5% CLA
treatment, although the 4.1% soy oil + 2.5% CLA diet
contained more oleic acid than the 4.1% flax oil + 2.5%
CLA treatment (Tables 2, 3, 4, and 5). The reason for the
discrepancy between diet and deposition is not clear. Li
and Watkins (1998) speculated that CLA reduced the
concentration of oleic acid by inhibiting liver ∆9-desaturase activity and found that dietary CLA decreased
the concentrations of palmitoleic and oleic acid, which
agrees with the present study. Lee et al. (1998) also
showed that CLA inhibited stearoyl-coenzyme A desaturase mRNA expression.
Phospholipids, which mainly exist in the membrane
system, were expected to have a more important biofunction than neutral lipids. Therefore, egg yolk lipid
was further separated into PC, PE, and TG parts using
thin-layer chromatography. The concentration of arachi-
TABLE 3. Influence of dietary fat on the fatty acid composition of egg yolk lipid
Fatty acid
Soy oil
Soy oil + CLA
Flax oil + CLA
Flax oil + soy
SEM
Palmitic
Palmitoleic
Stearic
Oleic
Linoleic
Linolenic
CLA1 (cis9, trans11)
CLA (trans10, cis12)
CLA (trans9, trans11)
Other CLA isomers
Arachidonic
EPA2
DHA2
SAFA2
MUFA2
PUFA2
Non-CLA PUFA
n6/n3 fatty acid ratio
22.82b
1.08b
11.84c
31.05a
23.56a
1.21b
0.00b
0.00b
0.00b
0.00c
3.95a
0.22b
2.72c
34.66
32.13
31.66
31.66
6.63
24.80a
0.49d
16.25a
23.22c
18.30c
0.82c
2.91a
2.31a
1.29a
0.99b
2.96b
0.21b
1.37d
41.05
23.71
31.16
23.66
8.86
25.11a
0.60c
14.57b
27.19b
11.98d
4.89a
2.80a
2.33a
1.30a
1.18a
2.06c
0.32a
3.36a
39.68
27.79
30.22
22.61
1.64
22.88b
1.13a
11.78c
31.86a
20.76b
4.87a
0.00b
0.00b
0.00b
0.00c
2.97b
0.30a
3.18b
34.66
32.99
32.08
32.08
2.77
0.256
0.019
0.271
0.324
0.321
0.060
0.040
0.065
0.060
0.018
0.047
0.010
0.076
Means within a row with no common superscript differ significantly (P < 0.05); n = 4.
CLA = conjugated linoleic acid.
2
EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid.
a–d
1
1753
CONJUGATED LINOLEIC ACID AND POLYUNSATURATED FATTY ACIDS
TABLE 4. Influence of dietary fat on the fatty acid composition of phosphatidylcholine of egg yolk lipid
Fatty acid
Soy oil
Soy oil + CLA
Flax oil + CLA
Flax oil + soy
SEM
Palmitic
Palmitoleic
Stearic
Oleic
Linoleic
Linolenic
CLA1 (cis9, trans11)
CLA (cis10, trans12)
CLA (trans11, trans11)
Other CLA isomers
Arachidonic
DHA2
SAFA2
MUFA2
PUFA2
Non-CLA PUFA
n6/n3 fatty acid ratio
27.62a
0.45b
15.76b
25.07c
20.76a
0.70b
0.00b
0.00b
0.00b
0.00b
4.98a
3.20b
25.69a
0.24d
18.69a
22.55d
17.80c
0.10c
1.93a
1.19a
1.59a
1.03a
3.58b
1.49c
26.65a
0.33cd
18.26a
26.02b
12.42d
2.15a
1.96a
1.27a
1.62a
1.10a
2.40c
3.88a
26.64a
0.70a
15.42b
28.57a
19.27b
2.09a
0.00b
0.00b
0.00b
0.00b
3.28b
3.30b
0.614
0.030
0.621
0.219
0.383
0.031
0.042
0.038
0.063
0.017
0.203
0.188
43.38
25.52
29.64
29.64
6.60
44.38
22.79
28.71
22.97
13.45
44.91
26.35
26.80
20.85
2.46
42.06
29.27
27.94
27.94
4.18
Means within a row with no common superscript differ significantly (P < 0.05); n = 4.
CLA = conjugated linoleic acid.
2
DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA =
polyunsaturated fatty acid.
a–d
1
donic acid in PC was closely related to the dietary content of linoleic acid, which was highest with 8.2% soy
oil and lowest with the 4.1% flax oil + 2.5% CLA treatment, and was consistent with the result of total lipids
(Table 4). The DHA content in PC was higher in eggs
from hens fed a 4.1% flax oil + 2.5% CLA diet than in
eggs from hens fed a 4.1% soy oil + 4.1% flax oil diet.
Phosphatidylethanolamine of egg yolk contained a
much higher amount of arachidonic acid and DHA than
other lipid classes (Table 5). However, their relative
changes were still similar to total lipid and PC. The
concentration of eicosapentaenoic acid in PE of egg yolk
from hens fed a 4.1% flax oil + 2.5% CLA diet was very
high compared with other groups. The concentration
changes of arachidonic acid, eicosapentaenoic acid, and
DHA in phospholipids may have significant physiological effects in vivo. Because these PUFA are the precursors
for the biosynthesis of eicosanoids, they could be closely
related to immune response and carcinogenesis in
animals.
The fatty acid composition of TG of egg yolk was
different from those of the PC and PE (Table 6). Very
low levels of arachidonic acid and DHA in TG indicate
that arachidonic acid and DHA are mainly deposited to
PC and PE. The concentrations of linolenic acid in TG
from the 4.1% flax oil + 2.5% CLA and 4.1% soy oil +
4.1% flax oil diets were high compared with those from
the 8.2% soy oil and 4.1% flax oil + 2.5% CLA diets. The
linoleic acid concentration and other fatty acid compositions of TG were similar to those of the dietary sources.
TABLE 5. Influence of dietary fat on the fatty acid composition
of phosphatidylethanolamine of egg yolk lipid
Fatty acid
Palmitic
Stearic
Oleic
Linoleic
Linolenic
CLA1 (cis9, trans11)
CLA (cis10, trans12)
CLA (trans9, trans11)
Other CLA isomers
Arachidonic
EPA2
DHA2
SAFA2
MUFA2
PUFA2
Non-CLA PUFA
n6/n3 fatty acid ratio
Soy oil
b
12.41
30.27a
15.71b
14.37b
1.28cd
0.00b
0.00c
0.00b
0.00b
14.02a
0.69b
9.39b
42.68
15.71
39.75
39.75
2.50
Soy oil + CLA
a
14.70
26.88b
15.64b
16.17a
1.19d
2.06a
1.32a
1.76a
1.25a
10.26b
0.88b
4.85c
41.58
15.64
39.74
33.35
3.82
Flax oil + CLA
a
15.54
26.63b
18.37a
9.16c
1.90a
1.95a
1.15b
1.64a
1.26a
7.10c
3.49a
11.24a
42.17
18.37
38.89
32.89
0.98
Flax oil + soy
b
11.60
30.26a
19.16a
13.14b
1.46b
0.00b
0.00c
0.00b
0.00b
11.54b
0.84b
10.76a
41.86
19.16
37.74
37.74
1.89
SEM
0.472
0.661
0.423
0.433
0.029
0.085
0.040
0.064
0.033
0.412
0.186
0.260
Means within a row with no common superscript differ significantly (P < 0.05); n = 4.
CLA = conjugated linoleic acid.
2
EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid.
a–d
1
1754
DU ET AL.
TABLE 6. Influence of dietary fat on the fatty acid composition of triglycerides of egg yolk lipid
Fatty acid
Soy oil
Soy oil + CLA
Flax oil + CLA
Flax oil + soy
SEM
Palmitic
Palmitoleic
Stearic
Oleic
Linoleic
Linolenic
CLA1 (cis9, trans11)
CLA (cis10, trans12)
CLA (trans9, trans11)
Other CLA isomers
Arachidonic
DHA2
SAFA2
MUFA2
PUFA2
Non-CLA PUFA
n6/n3 fatty acid ratio
21.03b
2.28b
4.61c
38.18a
31.37a
3.05c
0.00c
0.00b
0.00c
0.00c
0.35a
0.23b
26.43a
0.95d
9.08a
23.99d
26.89b
1.22d
3.45a
3.07a
1.36b
1.50b
0.12c
0.00c
21.89b
1.20c
8.37b
30.23c
13.71d
12.88b
3.24b
2.94a
1.63a
1.64a
0.12c
0.45a
18.82c
2.99a
3.61d
32.70b
24.89c
15.15a
0.00c
0.00b
0.00c
0.00c
0.21b
0.22b
0.326
0.050
0.035
0.456
0.475
0.209
0.043
0.073
0.025
0.021
0.021
0.017
25.64
40.46
35.00
35.00
6.97
35.51
24.94
37.61
28.23
22.14
30.26
31.43
36.61
27.16
1.04
22.43
35.69
40.47
40.47
1.63
Means within a row with no common superscript differ significantly (P < 0.05); n = 4.
CLA = conjugated linoleic acid.
2
DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA =
polyunsaturated fatty acid.
a–d
1
This similarity indicated that fatty acid composition of
TG in yolk lipid mainly reflected the fatty acid composition of the diet.
Both the 4.1% soy oil + 2.5% CLA and 4.1% flax oil +
2.5% CLA diets were supplemented with the same level
of CLA, but the amounts of linoleic and linolenic acids
in the diets were different. The hens fed a linoleic acidrich diet (4.1% soy oil + 2.5% CLA) had higher arachidonic acid concentrations in egg yolk lipids than those
fed a linolenic acid-rich diet (4.1% flax oil + 2.5% CLA),
but the concentration of DHA was higher in linolenic
acid-rich diets than in linoleic acid-rich diets (Tables 3,
4, and 5). These results indicate that the fractional content of arachidonic acid, EPA, and DHA of egg yolk
varies directly with the dietary concentration of their
precursor fatty acids under the dietary conditions employed in this study. Liu and Belury (1998) reported that
adding linoleic acid to a CLA diet enhanced arachidonic
acid synthesis.
In the 4.1% soy oil + 2.5% CLA and 4.1% flax oil +
2.5% CLA treatments (Tables 3, 4, and 5), high amounts
of CLA isomers were incorporated into lipid. If CLA
could be elongated to synthesize long-chain unsaturated
fatty acids in significant amounts, there would be more
long-chain fatty acids detected by the GC-Mass Selective
detector procedure. However, GC results did not indicate that there were significant amounts of long-chain
fatty acids available, except arachidonic acid, DHA, and
EPA. Therefore, CLA may not be a favorable substrate
for enzymes (desaturase and elongase) involved in the
TABLE 7. Influence of dietary fat on the fatty acid composition of liver tissues
Fatty acid
Soy oil
Soy oil + CLA
Flax oil + CLA
Flax oil + soy
SEM
Palmitic
Palmitoleic
Stearic
Oleic
Linoleic
Linolenic
CLA1 (cis9, trans11)
CLA (cis10, trans12)
CLA (trans9, trans11)
Other CLA isomers
Arachidonic
EPA2
DHA2
SAFA2
MUFA2
PUFA2
Non-CLA PUFA
n6/n3 fatty acid ratio
20.79ab
0.73a
18.12b
29.80a
19.43a
1.03c
0.00b
0.00c
0.00b
0.00b
8.09a
0.17b
2.25c
38.91
30.53
30.97
30.97
7.98
22.32a
0.29c
19.85a
26.96b
15.49c
0.72c
1.78a
2.25a
0.91a
0.83a
6.13c
0.20b
2.01c
42.71
27.25
30.32
24.55
7.38
21.90a
0.57b
17.06b
26.89b
12.78d
2.73b
1.86a
1.99b
0.75a
0.61a
5.19d
0.52a
5.93a
38.96
27.46
32.36
27.15
1.96
19.98b
0.76a
14.56c
28.85ab
17.83b
3.58a
0.00b
0.00c
0.00b
0.00b
7.29b
0.49a
5.19b
34.54
29.61
34.38
34.38
2.71
0.464
0.046
0.368
0.542
0.350
0.101
0.044
0.069
0.060
0.031
0.219
0.025
0.110
Means within a row with no common superscript differ significantly (P < 0.05); n = 4.
CLA = conjugated linoleic acid.
2
EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid.
a–d
1
1755
CONJUGATED LINOLEIC ACID AND POLYUNSATURATED FATTY ACIDS
TABLE 8. Influence of dietary fat on the fatty acid composition of muscle tissues
Fatty acid
Soy oil
Soy oil + CLA
Flax oil + CLA
Flax oil + soy
SEM
Palmitic
Palmitoleic
Stearic
Oleic
Linoleic
Linolenic
CLA1 (cis9, trans11)
CLA (cis10, trans12)
CLA (cis11, trans13)
Other CLA isomers
Arachidonic
EPA2
DHA2
19.39
1.56bc
10.25
34.24a
28.38a
1.40c
0.00c
0.00c
0.00c
0.00c
6.40a
0.19b
0.56c
29.64
35.80
36.93
36.93
16.18
19.94
1.02c
12.20
31.42b
23.22c
1.03d
1.84a
2.20a
0.93a
0.96a
4.79b
0.14b
0.46c
32.14
32.44
35.57
29.64
17.18
18.36
1.45a
10.60
30.50b
22.00c
2.79a
1.13b
1.22b
0.55b
0.51b
2.87c
0.39a
1.63a
28.96
31.95
33.09
29.68
5.17
19.24
2.06ab
9.96
31.44b
25.89b
2.29b
0.00c
0.00c
0.00c
0.00c
3.58c
0.33a
1.06b
29.20
33.50
33.15
33.15
8.01
0.537
0.189
0.580
0.587
0.518
0.109
0.044
0.068
0.044
0.029
0.264
0.030
0.094
SAFA2
MUFA2
PUFA2
Non-CLA PUFA
n6/n3 fatty acid ratio
Means within a row with no common superscript differ significantly (P < 0.05); n = 4.
CLA = conjugated linoleic acid.
2
EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid.
a–d
1
synthesis of long-chain fatty acids. However, Sebedio et
al. (1997) showed that CLA isomers could be elongated
to C20:4 because there were higher quantities of C20:4
∆-5,8,12,14, and C20:4 ∆-5,8,11,13 in liver lipids of rats
fed CLA than in liver lipids of controls.
In liver (Table 7) and muscle (Table 8) lipids, the concentration of arachidonic acid was much higher in 8.2%
soy oil and 4.1% soy oil + 2.5% CLA than in 4.1% soy
oil + 2.5% CLA and 4.1% flax oil + 2.5% CLA. This result
could be caused by the high amount of linoleic acid in
the diets with added soy oil. The concentrations of EPA
and DHA in the liver and muscle of hens fed 4.1% flax
oil + 2.5% CLA and 4.1% soy oil + 4.1% flax oil were
higher than those of hens fed the 8.2% soy oil and 4.1%
soy oil + 2.5% CLA diets. Significantly higher amounts
of DHA in the liver and muscle of hens fed 4.1% flax
oil + 2.5% CLA than in those of hens fed the 4.1% soy
oil + 4.1% flax oil diet indicate that CLA promoted the
synthesis or deposition of DHA and EPA. This finding
illustrates that CLA increases the level of n-3 long-chain
PUFA. Ward et al. (1998) showed that arachidonic acid
and DHA could affect each other’s levels. The increased
DHA level could be caused by the reduced arachidonic
acid concentration, which might have some feedback
effects and induce more DHA synthesis or incorporation
in compensation (Ward et al., 1998).
This study indicated that CLA feeding reduced monounsaturated fatty acid and non-CLA PUFA content in
egg yolk and tissue lipids. The concentration of DHA in
lipid was increased by dietary CLA, which could be
related to the decreased arachidonic acid content. Results show that dietary supplementation of linoleic or
linolenic acid can enhance the level of arachidonic acid
or DHA in yolk and tissue lipids dramatically.
REFERENCES
Ahn, D. U., J. L. Sell, C. Jo, M. Chamruspollert, and M. Jeffery,
1999. Effect of dietary conjugated linoleic acid on the quality
characteristics of chicken eggs during refrigerated storage.
Poultry Sci. 78:922–928.
Ahn, D. U., F. H. Wolfe, and J. S. Sim, 1995. Dietary α-linolenic
acid and mixed tocopherols, and packaging influences on
lipid stability in broiler chicken breast and leg muscle. J.
Food Sci. 60:1013–1018.
Belury, M. A., and A. Kempa-Steczko, 1997. Conjugated linoleic acid modulates hepatic lipid composition in mice. Lipids 32:199–204.
Belury, M. A., K. P. Nickel, C. E. Bird, and Y. Wu, 1996. Dietary
conjugated linoleic acid modulation of phorbol ester skin
tumor promotion. Nutr. Cancer 26:149–157.
Bretillon, L., J. M. Chardigny, S. Gregoire, O. Berdeaux, and
J. L. Sebedio, 1999. Effect of conjugated linoleic acid isomers
on the hepatic microsomal desaturation activities in vitro.
Lipids 34:965–969.
Chin, S. F., W. Liu, J. M. Storkson, Y. L. Ha, and M. W. Pariza,
1992. Dietary sources of conjugated dienoic isomers of linoleic acid. A newly recognized class of anticarcinogens. J.
Food Comp. Anal. 5:185–197.
Christie, W. W., G. Dobson, and F. D. Gunstone, 1997. Isomers
in commercial samples of conjugated linoleic acid. Lipids
32:1231.
Du, M., D. U. Ahn, and J. L. Sell, 1999. Effect of dietary conjugated linoleic acid on the composition of egg yolk lipids.
Poultry Sci. 78:1639–1645.
Folch, J., M. Less, and G. M. Slaone-Stanley, 1957. A simple
method for the isolation and purification of total lipids from
animal tissues. J. Biol. Chem. 226:497–509.
Ip, C., 1997. Review of the effects of trans fatty acid, oleic acid,
n-3 polyunsaturated fatty acids, and conjugated linoleic acid
on mammary carcinogenesis in animals. Am. J. Clin. Nutr.
66:S1523–S1529.
Ip, C., C. Jiang, H. J. Thompson, and J. A. Scimeca, 1997. Retention of conjugated linoleic acid in the mammary gland is
associated with tumor inhibition during the post-initiation
phase of carcinogenesis. Carcinogenesis 18:755–759.
Ip, C., J. A. Scimeca, and H. Thompson, 1995. Effect of timing
and duration of dietary conjugated linoleic acid on mammary cancer prevention. Nutr. Cancer 24:241–247.
Lee, K. N., M. W. Pariza, and J. M. Ntambi, 1998. Conjugated
linoleic acid decreases hepatic stearoyl-CoA desaturase
mRNA expression. Biochem. Biophys. Res. Commun.
248:817–821.
1756
DU ET AL.
Lee, K. N., J. M. Storkson, and M. W. Pariza, 1995. Dietary
conjugated linoleic acid changes fatty acid composition in
different tissues by decreasing monounsaturated fatty acids.
Page 183 in: IFT Annual Meeting. Book of Abstracts. Anaheim, CA.
Li, Y., and B. A. Watkins, 1998. Conjugated linoleic acids alter
bone fatty acid composition and reduce ex vivo prostaglandin E-2 biosynthesis in rats fed n-6 or n-3 fatty acid. Lipids
33:417–425.
Liu, K. L., and M. A. Belury, 1998. Conjugated linoleic acid
reduces arachidonic acid content and PGE2 synthesis in
murine keratinocytes. Cancer Lett. (CMX) 127:15–22.
Nicolosi, R. J., E. J. Rogers, D. Kritchevsky, J. A. Scimeca, and
P. J. Huth, 1997. Dietary conjugated linoleic acid reduces
plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery 22:266–277.
SAS Institute Inc., 1985. SAS User’s Guide. SAS Institute Inc.,
Cary, NC.
Sebedio, J. L., P. Juaneda, G. Dobson, I. Ramilison, J. C. Martin,
J. M. Chardigny, and W. W. Christie, 1997. Metabolites of
conjugated isomers of linoleic acid (CLA) in the rat. Biochim. Biophys. Acta 1345:5–10.
Sugano, M., A. Tsujita, M. Yamasaki, M. Noguchi, and K. Yamada, 1998. Conjugated linoleic acid modulates tissue levels
of chemical mediators and immunoglobulins in rats. Lipids
33:521–527.
Sugano, M., A. Tsujita, M. Yamasaki, K. Yamada, I. Ikeda, and
D. Kritchevsky, 1997. Lymphatic recovery, tissue distribution, and metabolic effects of conjugated linoleic acid in
rats. J. Nutr. Biochem. 8:38–43.
Turek, J. J., Y. Li, I. A. Schoenlein, K.G.D. Allen, and B. A.
Watkins, 1998. Modulation of macrophage cytokine production by conjugated linoleic acids is influenced by the dietary
n-6:n-3 fatty acid ratio. Nutr. Biochem. 9:258–266.
Visonneau, S., A. Cesano, S. A. Tepper, J. A. Scimeca, D. Santoli,
and D. Kritchevsky, 1997. Conjugated linoleic acid suppresses the growth of human breast adenocarcinoma cells
in SCID mice. Anticancer Res. 17:969–973.
Ward, G. R., Y. S. Huang, E. Bobik, H. C. Xing, L. Mutsaers,
N. Auestad, M. Montalto, and P. Wainwright, 1998. Longchain polyunsaturated fatty acid levels in formulae influence deposition of docosahexaenoic acid and arachidonic
acid in brain and red blood cells of artificially reared neonatal rats. J. Nutr. 128:2473–2487.