Eur J Nutr (2014) 53:989–993 DOI 10.1007/s00394-013-0618-3
SHORT COMMUNICATION
Incorporation of conjugated linoleic acid isomers into porcine erythrocytes Tomazˇ Malovrh • Enver Melkic´ • Drago Kompan Alenka Levart • Lidija Kompan
•
Received: 9 July 2013 / Accepted: 31 October 2013 / Published online: 16 November 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose The aim of the current study was to determine the incorporation of cis (c) 9, trans (t) 11-conjugated linoleic acid (CLA) and t10, c12-CLA into porcine erythrocytes—both isomers were supplemented in equal proportions. Methods The study group consisted of 16 piglets randomly assigned into experimental and control group. For the period of 5 weeks, the piglets from the experimental group were receiving a 1.2 % CLA supplement while the controls were supplemented with the same amount of sunflower oil. For the remaining 7 weeks, the piglets were fed without a supplement. Blood samples to evaluate incorporation of CLA into erythrocyte membranes were taken from all animals on weekly basis. Results Compared to t10, c12-CLA isomer, proportion of c9, t11-CLA isomer in the membrane of erythrocytes was higher for the whole time of the study period. After 4 weeks of feeding, it approaches the plateau. The peak
T. Malovrh E. Melkic´ University Medical Centre Ljubljana, Ljubljana, Slovenia e-mail: [email protected] E. Melkic´ e-mail: [email protected] D. Kompan A. Levart Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia e-mail: [email protected] A. Levart e-mail: [email protected] L. Kompan (&) Institute of Oncology, Ljubljana, Slovenia e-mail: [email protected]
value for both isomers was measured at the end of week 5, with a value of 3.24 g c9, t11-CLA/100 g of fatty acids and a 1.09 g t10, c12-CLA/100 g of fatty acids (p \ 0.0001). After cessation of supplementation, the proportion of both isomers gradually decreased to be almost completely washed out—in 7 weeks. Conclusions During supplementation with equivalent amounts of CLA isomers, their proportion in membranes of porcine erythrocytes increases with time, with higher proportion of c9, t11-CLA. CLA isomers probably differently incorporate into different cell membranes at different species which could explain its various biological functions. Keywords Conjugated linoleic acid Porcine erythrocytes Fatty acid methyl esters
Introduction Conjugated linoleic acids (CLA) are naturally occurring fatty acids found in dairy products and meat of ruminants that are known to possess broad biological activity [1, 2]. CLA isomers are formed in the rumen of ruminants as intermediates in the hydrogenation of linoleic to vaccenic acid with a predominance of cis-9, trans-11 (c9, t11) CLA isomer together with a small amounts of t7, c9-CLA, c11, t13-CLA, t10, c12-CLA and trace amounts of some others [1]. Different CLA isomers have different biological activity [2]. This can be explained also by differences in the intake and incorporation of isomers that should be dependent on the doses of the particular isomers in the supplement [3]. It is also possible that tissues differ in their ability to assimilate CLA isomers. The effect of supplementation with enriched preparations of both commercially available CLA isomers to their incorporation into
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peripheral blood mononuclear cell membranes (PBMC) and erythrocyte membranes had been studied in humans and found that PBMC incorporate both CLA isomers in the same proportions, but less readily, while erythrocytes prefer to build in c9, t11-CLA in comparison with t10, c12CLA [3, 4]. However, the effect of differential incorporation of CLA isomers into the pig cell membranes has not been studied. It has been reported that erythrocyte fatty acid status reflects the integration of polyunsaturated fatty acids into cellular membranes and is therefore a reflection of dietary fat intake [5]. We precluded that a similar differences in the erythrocyte membrane fatty acid status would be observed also with a dietary intake of CLA isomers. Therefore, in the current study, we evaluated the incorporation of c9, t11CLA and t10, c12-CLA given in equal proportions into the membrane of porcine erythrocyte.
Materials and methods Animals and diet The study group consisted of 16 piglets of the Slovenian Landrace breed, randomly assigned into experimental and control group. Until the age of 4 weeks, the piglets were fed ‘‘ad libitum’’ with an experimental feed which consisted of wheat (60 %), maize (8 %), barley (5 %), extracted soya bean meal (10 %), fish meal (3 %) and other special protein concentrates (10 %). The feed contained 13.5 MJ of metabolic energy (ME), 18 % of crude proteins with 12.5 g of Lysine per kg of feed, 2.95 % crude fiber and 3.7 % crude fats. Following that the animals were weighted and the average maintenance energy was calculated according to the average body weight using the equation MaE ¼ 754 5:9BM þ 0:025BM2 BM0:75 ½kJ/day (MaE = maintenance energy in kJ/day, BM = average body mass in kg). For 2 weeks, the piglets were fed 1.8 times of maintenance energy according to the average weight.
Eur J Nutr (2014) 53:989–993
50:50 mixture of c9, t11-CLA and t10, c12-CLA isomers. Average daily dose of CLA ranged from 8.4 g in the first week to 13 g in the last week of supplementation. The control group received an equivalent supplement of sunflower oil. After 5 weeks of supplementation, CLA isomer feeding was stopped and the piglets were on the same feed for remaining 7 weeks. Sample preparation and fatty acid analysis Blood samples were taken into 4.5-ml tubes with K3-EDTA on a weekly basis. Red blood cells were isolated by adding 3 ml of whole blood to 9 ml of isotonic buffer (0.9 % NaCl in 5 mM Na2HPO4 buffer (pH = 8). Then, cells were centrifuged at 1,0009g; for 10 min at 4 °C. The aliquot of supernatant with plasma proteins, and platelets was then removed. This procedure was repeated three times. Fatty acid methyl esters (FAME-s) from the isolated red blood cell samples were prepared using the method of Park and Goins [6]. Extracted FAME-s (2 ll) were separated using an Agilent 6890 series GC instrument equipped with an Agilent 7683 Automatic Liquid Sampler, a split/splitless injector (T = 250 °C, split ratio = 30:1), a flame-ionization detector (FID, T = 250 °C) and a capillary column Omegawax 320 (30 m 9 0.32 mm i.d.), and helium was used as carrier gas. The oven temperature was programmed from 185 to 215 °C at 1 °C/min (final time 9 min). Agilent GC ChemStation software was used for data acquisition and processing. Separated FAME-s were identified by comparison of retention times, and results were calculated using response factors derived from chromatographic standards of known composition (GLC 411, GLC 85, GLC 68A, GLC 423, GLC 80 Nu Chek Prep) (Fig. 1). Beside blood samples, we also made an analysis of fatty acid composition of the basic feed and supplements (Table 1). Applied GC method for FAME separation enables identification, separation and
CLA supplementation From the age of 6 weeks, the piglets were fed 1.8 times of maintenance energy on an individual basis for 5 weeks. The animals were weighed once per week and the maintenance energy individually calculated. Piglets in experimental group received a 1.2 % (1.2 g per 100 g of feed) supplement of CLA, 50:50 mixture of c9, t11-CLA and t10, c12-CLA isomers (TAG-esterified preparation, Larodan ABLimhamnsga˚rdens alle 9, S-216 16 Malmo¨ Sweden). Because a purity of CLA was only 80 %, the amount of CLA supplement was actually 1.5 % on a feed basis of a
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Fig. 1 Partial gas chromatographic separation of erythrocyte FAME in C18:2 to C20:1 regions. Line A—erythrocyte FAME from control group, line B—erythrocyte FAME from CLA group
Eur J Nutr (2014) 53:989–993
991
Table 1 Fatty acid composition of the basic feed and supplements Fatty acid (wt%)
a
Basic feed
CLA supplement
Sunflower oil
C14:0
1.82
0.02
0.07
C15:0
0.09
b
0.02
C16:0
15.99
2.05
6.46
C16:1 n-7
1.20
0.03
0.08
C17:0
0.11
0.02
0.05
C18:0
3.54
2.24
3.98
C18:1 n-9
18.38
11.22
22.25
C18:1 n-7
1.58
0.70
0.68
C18:2 n-6
43.94
2.28
64.46
C16:1 n-9
C18:3 n-3 c9, t11 CLA
3.81
0.51 37.89
t10, c12 CLA
38.60
C20:0
0.32
0.33
0.27
C20:1 n-9
1.34
0.24
0.17
C20:2 n-6
0.07
0.17
0.67
C20:3 n-6 C20:4 n-6
0.07
C20:5 n-3
1.09
C22:0
0.28
C22:4 n-6 C22:5 n-3
0.13
C22:6 n-3
1.88
C24:0
0.20
a
wt%, weight%; g of fatty acid/100 g of all fatty acids
b
empty fields—wt% \0.01
0.23
quantification of both main CLA (c9, t11 and t10, c12) isomers. Minor CLA isomers are not fully resolved and were not assessed in present study. For detailed analysis of minor CLA isomers, additional GC–MS methods, employing more efficient chromatographic columns or complementary AgHPLC techniques, are needed. Statistics
t11-CLA/100 g of total fatty acids (i.e., weight%–wt%) and no trace of t10, c12-CLA. During the first 28 days of CLA supplementation, increased proportion of both CLA isomers in membranes of erythrocytes was observed, with the weekly measured values always being higher than the previous ones for both isomers and c9, t11-CLA being significantly higher in all samples (p \ 0.0001) in comparison with proportion of t10, c12-CLA isomer. Incorporation of both isomers clearly slowed down after day 28, when the signs of a plateau appeared. The highest mean value was measured at the end of the feeding period on day 35, at 3.24 ± 0.3 wt% for c9, t11-CLA and 1.09 ± 0.15 wt% for t10, c12-CLA (Fig. 2). CLA isomers incorporated in membranes mostly replaced C18 and to some extent C20 fatty acids (Table 2). The washout of the isomers started immediately after supplementation was discontinued. Both isomers were almost completely washed out in 7 weeks after cessation of CLA supplementation (Fig. 2). In samples of the control group, mostly no detectable levels of either CLA isomer were found.
Discussion Most studies on the incorporation of CLA isomers have been conducted on humans. Similar results as ours, regarding preferential uptake of c9, t11-CLA, were reported at healthy humans supplemented with c9, t11-CLA or t10, c12-CLA for 8 weeks [7]. Burdge and co-authors studied concentrations of CLA isomers in membranes of human erythrocyte, as we did in the porcine [4]. Concentrations of two isomers in human erythrocyte total lipids were lower than in plasma lipids, but incorporation of both isomers into erythrocytes was approximately twofold greater than that into PBMC [3, 4]. CLA incorporation into the erythrocyte membranes at our pork model probably resembles incorporation in humans, because both species preferentially build in c9, t11-CLA [4]. In PBMCs, they
Results are expressed as a percentage of the total fatty acids (wt%). The data are presented as means and standard deviations values. Paired and unpaired samples t tests were used for comparing mean values. A p B 0.001 value was considered statistically significant because of multiple comparisons.
Results Before supplementation, CLA proportion in membranes was very low, on average 0.01 ± 0.02 g of c9,
Fig. 2 Incorporation and washout of c9, t11 and t10, c12-CLA from the membrane of erythrocytes. Mean incorporated values (g/100 g of FA) for each isomer with standard error bars are presented
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Table 2 Fatty acid composition of the red cell membrane on week 1, 3, 5 and 12 of the experiment in the CLA group (CLA) and control group (C) Fatty acid (wt%)a
Week 1 CLA
Week 5c
Week 3 C
b
p
CLA
C
C14:0
0.39
0.35
0.32
0.26
C15:0
0.34
0.32
0.29
0.25
C16:0 C16:1 n-9
19.88 0.25
19.74 0.28
19.92 0.14
19.85 0.20
C16:1 n-7
0.84
1.02
0.71
0.73
0.017
p
CLA 0.022
\0.001
Week 12 C
0.28
0.25
0.24
0.22
19.75 0.10
19.71 0.21
0.60
0.64
p
CLA 0.004
\0.001
C
0.28
0.24
0.28
0.23
25.30 0.22
20.45 0.20
0.68
0.63
C17:0
1.44
1.43
1.46
1.36
1.31
1.22
1.77
1.47
C18:0
14.16
13.84
16.20
15.34
0.004
17.06
15.72
\0.001
21.94
18.24
C18:1 n-9
25.21
26.37
23.78
26.80
0.049
21.57
25.70
0.015
30.48
30.29
\0.001
\0.001
0.011
p 0.041 0.001
0.002
C18:1 n-7
2.19
2.36
1.80
2.21
1.52
2.15
2.24
2.36
C18:2 n-6
19.32
20.17
18.72
20.00
19.61
20.66
11.45
16.74
\0.001
C18:3 n-3
0.52
0.46
0.50
0.40
0.55
0.40
0.18
0.36
0.005
c9, t11 CLA
1.35
0.03
\0.001
2.49
0.00
\0.001
3.24
0.04
\0.001
0.40
0.00
\0.001
t10, c12 CLA
0.66
0.00
\0.001
0.86
0.00
\0.001
1.09
0.00
\0.001
0.02
0.00
\0.001
0.21
0.14
\0.001
0.22
0.14
0.04
0.10
0.009
0.11
0.08
\0.001
0.27
0.37
\0.001
0.23
0.34
0.51
0.58
0.049
0.33
0.56
0.003
\0.001 \0.001
3.93 1.06
5.60 0.66
\0.001 \0.001
2.24 0.02
4.40 0.21
\0.001 \0.001
C20:0
0.14
0.12
0.18
0.13
C20:1 n-9
0.12
0.15
0.07
0.12
C20:2 n-6
0.29
0.35
0.27
0.35
C20:3 n-6
0.52
0.57
0.51
0.54
C20:4 n-6 C20:5 n-3
4.93 0.80
5.36 0.65
4.15 0.90
5.11 0.58
C22:0
0.00
0.00
0.01
0.02
0.05
0.01
0.03
0.04
C22:4 n-6
0.38
0.38
0.34
0.35
0.30
0.41
0.001
0.27
0.53
C22:5 n-3
1.52
1.36
1.77
1.43
0.002
1.95
1.48
\0.001
0.40
1.01
\0.001
C22:6 n-3
3.18
3.03
3.39
3.08
0.024
3.58
2.96
0.001
0.23
0.95
\0.001
C24:0
0.12
0.12
0.15
0.14
0.20
0.17
0.20
0.19
a
0.002
0.001
0.043
0.018 0.005
\0.001
wt%, weight%; g of fatty acid/100 g of all fatty acids, mean values are presented
b
t test for unpaired samples was performed, p \ 0.05 are shown, p B 0.001 were considered significant
c
group-specific supplementation with CLA or sunflower oil stopped after 5th week
found no isomer-specific incorporation [3]. We did not analyse CLA in plasma lipids; therefore, we have no direct evidence that both isomers were present in equimolar concentration in plasma. However, Burdge and co-authors found no difference in concentration of both isomers in plasma [3]. In our experiment, the highest proportion of c9, t11-CLA and t10, c12-CLA in porcine erythrocytes were 3.24 wt% and 1.09 wt%, respectively, while they reported lower proportions of those CLA isomers (0.42, 0.19 wt% respectively) in human erythrocytes [4]. Although we do not know total fat intake in their study, porcine erythrocytes seem to incorporate CLA more easily than human. Erythrocyte fatty acid status is considered to be a reflection of polyunsaturated fatty acid [5], but this is not so in CLA, because they preferentially concentrate c9, t11-CLA. Thus, we cannot use them for the same purpose when investigating CLA.
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Washout of CLA has mostly been studied in connection to its immune influence, which lasted 5–12 weeks after end of supplementation [8, 9]. We found almost complete washout of CLA in 7 weeks after supplementation stopped. Burdge and co-authors increased input of individual isomers during the supplementation time, while we kept the same dose for the whole time of feeding, as it usually occurs in practice [4]. We showed that also with the constant intake, incorporation increases with time, and after 4 weeks reaches the plateau. It is therefore evident that CLA in porcine model is not incorporated only in a dosedependent, but also in a time-dependent manner. According to differential immunologic, anti-inflammatory and anti-adipose effect of two CLA isomers, which could also be influenced by different lipid membrane composition in different species [1, 10], further studies on incorporation of CLA isomers into different cell
Eur J Nutr (2014) 53:989–993
membranes at different species can also help to explain various biological CLA functions. Acknowledgment This study was supported by Slovenian government P4-0092 research grant. The ethicalness of the study protocol was approved by the Slovenian Veterinary Administration (4.4.-23/ 05). We are grateful to Dr. Gaj Vidmar from the Institute of Rehabilitation, Republic of Slovenia, for help with statistical analyses. Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
References 1. O’Shea M, Bassaganya-Riera J, Mohede IC (2004) Immunomodulatory properties of conjugated linoleic acid. Am J Clin Nutr 79(6 Suppl):1199S–1206S 2. Pariza MW, Park Y, Cook ME (2001) The biologically active isomers of conjugated linoleic acid. Prog Lipid Res 40(4):283–298 3. Burdge GC, Lupoli B, Russell JJ, Tricon S, Kew S, Banerjee T, Shingfield KJ, Beever DE, Grimble RF, Williams CM, Yaqoob P, Calder PC (2004) Incorporation of cis-9, trans-11 or trans-10, cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men. J Lipid Res 45(4):736–741
993 4. Burdge G, Derrick P, Russell J, Tricon S, Kew S, Banerjee T, Grimble R, Williams C, Yaqoob P, Calder P (2005) Incorporation of cis-9, trans-11 or trans-10, cis-12 conjugated linoleic acid into human erythrocytes in vivo. Nutr Res 25(1):13–19 5. Zamaria N (2004) Alteration of polyunsaturated fatty acid status and metabolism in health and disease. Reprod Nutr Dev 44(3):273–282 6. Park PW, Goins RE (1994) In situ preparation of fatty acid methyl esters for analysis of fatty acid composition in foods. J Food Sci 59:1262–1266 7. Noone EJ, Roche HM, Nugent AP, Gibney MJ (2002) The effect of dietary supplementation using isomeric blends of conjugated linoleic acid on lipid metabolism in healthy human subjects. Br J Nutr 88(3):243–251. doi:10.1079/BJN2002615 8. Bassaganya-Riera J, Hontecillas R, Zimmerman DR, Wannemuehler MJ (2002) Long-term influence of lipid nutrition on the induction of CD8(?) responses to viral or bacterial antigens. Vaccine 20(9–10):1435–1444 9. Song HJ, Grant I, Rotondo D, Mohede I, Sattar N, Heys SD, Wahle KW (2005) Effect of CLA supplementation on immune function in young healthy volunteers. Eur J Clin Nutr 59(4):508–517 10. Tricon S, Burdge GC, Kew S, Banerjee T, Russell JJ, Grimble RF, Williams CM, Calder PC, Yaqoob P (2004) Effects of cis-9, trans11 and trans-10, cis-12 conjugated linoleic acid on immune cell function in healthy humans. Am J Clin Nutr 80(6):1626–1633
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SHORT COMMUNICATION
Incorporation of conjugated linoleic acid isomers into porcine erythrocytes Tomazˇ Malovrh • Enver Melkic´ • Drago Kompan Alenka Levart • Lidija Kompan
•
Received: 9 July 2013 / Accepted: 31 October 2013 / Published online: 16 November 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose The aim of the current study was to determine the incorporation of cis (c) 9, trans (t) 11-conjugated linoleic acid (CLA) and t10, c12-CLA into porcine erythrocytes—both isomers were supplemented in equal proportions. Methods The study group consisted of 16 piglets randomly assigned into experimental and control group. For the period of 5 weeks, the piglets from the experimental group were receiving a 1.2 % CLA supplement while the controls were supplemented with the same amount of sunflower oil. For the remaining 7 weeks, the piglets were fed without a supplement. Blood samples to evaluate incorporation of CLA into erythrocyte membranes were taken from all animals on weekly basis. Results Compared to t10, c12-CLA isomer, proportion of c9, t11-CLA isomer in the membrane of erythrocytes was higher for the whole time of the study period. After 4 weeks of feeding, it approaches the plateau. The peak
T. Malovrh E. Melkic´ University Medical Centre Ljubljana, Ljubljana, Slovenia e-mail: [email protected] E. Melkic´ e-mail: [email protected] D. Kompan A. Levart Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia e-mail: [email protected] A. Levart e-mail: [email protected] L. Kompan (&) Institute of Oncology, Ljubljana, Slovenia e-mail: [email protected]
value for both isomers was measured at the end of week 5, with a value of 3.24 g c9, t11-CLA/100 g of fatty acids and a 1.09 g t10, c12-CLA/100 g of fatty acids (p \ 0.0001). After cessation of supplementation, the proportion of both isomers gradually decreased to be almost completely washed out—in 7 weeks. Conclusions During supplementation with equivalent amounts of CLA isomers, their proportion in membranes of porcine erythrocytes increases with time, with higher proportion of c9, t11-CLA. CLA isomers probably differently incorporate into different cell membranes at different species which could explain its various biological functions. Keywords Conjugated linoleic acid Porcine erythrocytes Fatty acid methyl esters
Introduction Conjugated linoleic acids (CLA) are naturally occurring fatty acids found in dairy products and meat of ruminants that are known to possess broad biological activity [1, 2]. CLA isomers are formed in the rumen of ruminants as intermediates in the hydrogenation of linoleic to vaccenic acid with a predominance of cis-9, trans-11 (c9, t11) CLA isomer together with a small amounts of t7, c9-CLA, c11, t13-CLA, t10, c12-CLA and trace amounts of some others [1]. Different CLA isomers have different biological activity [2]. This can be explained also by differences in the intake and incorporation of isomers that should be dependent on the doses of the particular isomers in the supplement [3]. It is also possible that tissues differ in their ability to assimilate CLA isomers. The effect of supplementation with enriched preparations of both commercially available CLA isomers to their incorporation into
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peripheral blood mononuclear cell membranes (PBMC) and erythrocyte membranes had been studied in humans and found that PBMC incorporate both CLA isomers in the same proportions, but less readily, while erythrocytes prefer to build in c9, t11-CLA in comparison with t10, c12CLA [3, 4]. However, the effect of differential incorporation of CLA isomers into the pig cell membranes has not been studied. It has been reported that erythrocyte fatty acid status reflects the integration of polyunsaturated fatty acids into cellular membranes and is therefore a reflection of dietary fat intake [5]. We precluded that a similar differences in the erythrocyte membrane fatty acid status would be observed also with a dietary intake of CLA isomers. Therefore, in the current study, we evaluated the incorporation of c9, t11CLA and t10, c12-CLA given in equal proportions into the membrane of porcine erythrocyte.
Materials and methods Animals and diet The study group consisted of 16 piglets of the Slovenian Landrace breed, randomly assigned into experimental and control group. Until the age of 4 weeks, the piglets were fed ‘‘ad libitum’’ with an experimental feed which consisted of wheat (60 %), maize (8 %), barley (5 %), extracted soya bean meal (10 %), fish meal (3 %) and other special protein concentrates (10 %). The feed contained 13.5 MJ of metabolic energy (ME), 18 % of crude proteins with 12.5 g of Lysine per kg of feed, 2.95 % crude fiber and 3.7 % crude fats. Following that the animals were weighted and the average maintenance energy was calculated according to the average body weight using the equation MaE ¼ 754 5:9BM þ 0:025BM2 BM0:75 ½kJ/day (MaE = maintenance energy in kJ/day, BM = average body mass in kg). For 2 weeks, the piglets were fed 1.8 times of maintenance energy according to the average weight.
Eur J Nutr (2014) 53:989–993
50:50 mixture of c9, t11-CLA and t10, c12-CLA isomers. Average daily dose of CLA ranged from 8.4 g in the first week to 13 g in the last week of supplementation. The control group received an equivalent supplement of sunflower oil. After 5 weeks of supplementation, CLA isomer feeding was stopped and the piglets were on the same feed for remaining 7 weeks. Sample preparation and fatty acid analysis Blood samples were taken into 4.5-ml tubes with K3-EDTA on a weekly basis. Red blood cells were isolated by adding 3 ml of whole blood to 9 ml of isotonic buffer (0.9 % NaCl in 5 mM Na2HPO4 buffer (pH = 8). Then, cells were centrifuged at 1,0009g; for 10 min at 4 °C. The aliquot of supernatant with plasma proteins, and platelets was then removed. This procedure was repeated three times. Fatty acid methyl esters (FAME-s) from the isolated red blood cell samples were prepared using the method of Park and Goins [6]. Extracted FAME-s (2 ll) were separated using an Agilent 6890 series GC instrument equipped with an Agilent 7683 Automatic Liquid Sampler, a split/splitless injector (T = 250 °C, split ratio = 30:1), a flame-ionization detector (FID, T = 250 °C) and a capillary column Omegawax 320 (30 m 9 0.32 mm i.d.), and helium was used as carrier gas. The oven temperature was programmed from 185 to 215 °C at 1 °C/min (final time 9 min). Agilent GC ChemStation software was used for data acquisition and processing. Separated FAME-s were identified by comparison of retention times, and results were calculated using response factors derived from chromatographic standards of known composition (GLC 411, GLC 85, GLC 68A, GLC 423, GLC 80 Nu Chek Prep) (Fig. 1). Beside blood samples, we also made an analysis of fatty acid composition of the basic feed and supplements (Table 1). Applied GC method for FAME separation enables identification, separation and
CLA supplementation From the age of 6 weeks, the piglets were fed 1.8 times of maintenance energy on an individual basis for 5 weeks. The animals were weighed once per week and the maintenance energy individually calculated. Piglets in experimental group received a 1.2 % (1.2 g per 100 g of feed) supplement of CLA, 50:50 mixture of c9, t11-CLA and t10, c12-CLA isomers (TAG-esterified preparation, Larodan ABLimhamnsga˚rdens alle 9, S-216 16 Malmo¨ Sweden). Because a purity of CLA was only 80 %, the amount of CLA supplement was actually 1.5 % on a feed basis of a
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Fig. 1 Partial gas chromatographic separation of erythrocyte FAME in C18:2 to C20:1 regions. Line A—erythrocyte FAME from control group, line B—erythrocyte FAME from CLA group
Eur J Nutr (2014) 53:989–993
991
Table 1 Fatty acid composition of the basic feed and supplements Fatty acid (wt%)
a
Basic feed
CLA supplement
Sunflower oil
C14:0
1.82
0.02
0.07
C15:0
0.09
b
0.02
C16:0
15.99
2.05
6.46
C16:1 n-7
1.20
0.03
0.08
C17:0
0.11
0.02
0.05
C18:0
3.54
2.24
3.98
C18:1 n-9
18.38
11.22
22.25
C18:1 n-7
1.58
0.70
0.68
C18:2 n-6
43.94
2.28
64.46
C16:1 n-9
C18:3 n-3 c9, t11 CLA
3.81
0.51 37.89
t10, c12 CLA
38.60
C20:0
0.32
0.33
0.27
C20:1 n-9
1.34
0.24
0.17
C20:2 n-6
0.07
0.17
0.67
C20:3 n-6 C20:4 n-6
0.07
C20:5 n-3
1.09
C22:0
0.28
C22:4 n-6 C22:5 n-3
0.13
C22:6 n-3
1.88
C24:0
0.20
a
wt%, weight%; g of fatty acid/100 g of all fatty acids
b
empty fields—wt% \0.01
0.23
quantification of both main CLA (c9, t11 and t10, c12) isomers. Minor CLA isomers are not fully resolved and were not assessed in present study. For detailed analysis of minor CLA isomers, additional GC–MS methods, employing more efficient chromatographic columns or complementary AgHPLC techniques, are needed. Statistics
t11-CLA/100 g of total fatty acids (i.e., weight%–wt%) and no trace of t10, c12-CLA. During the first 28 days of CLA supplementation, increased proportion of both CLA isomers in membranes of erythrocytes was observed, with the weekly measured values always being higher than the previous ones for both isomers and c9, t11-CLA being significantly higher in all samples (p \ 0.0001) in comparison with proportion of t10, c12-CLA isomer. Incorporation of both isomers clearly slowed down after day 28, when the signs of a plateau appeared. The highest mean value was measured at the end of the feeding period on day 35, at 3.24 ± 0.3 wt% for c9, t11-CLA and 1.09 ± 0.15 wt% for t10, c12-CLA (Fig. 2). CLA isomers incorporated in membranes mostly replaced C18 and to some extent C20 fatty acids (Table 2). The washout of the isomers started immediately after supplementation was discontinued. Both isomers were almost completely washed out in 7 weeks after cessation of CLA supplementation (Fig. 2). In samples of the control group, mostly no detectable levels of either CLA isomer were found.
Discussion Most studies on the incorporation of CLA isomers have been conducted on humans. Similar results as ours, regarding preferential uptake of c9, t11-CLA, were reported at healthy humans supplemented with c9, t11-CLA or t10, c12-CLA for 8 weeks [7]. Burdge and co-authors studied concentrations of CLA isomers in membranes of human erythrocyte, as we did in the porcine [4]. Concentrations of two isomers in human erythrocyte total lipids were lower than in plasma lipids, but incorporation of both isomers into erythrocytes was approximately twofold greater than that into PBMC [3, 4]. CLA incorporation into the erythrocyte membranes at our pork model probably resembles incorporation in humans, because both species preferentially build in c9, t11-CLA [4]. In PBMCs, they
Results are expressed as a percentage of the total fatty acids (wt%). The data are presented as means and standard deviations values. Paired and unpaired samples t tests were used for comparing mean values. A p B 0.001 value was considered statistically significant because of multiple comparisons.
Results Before supplementation, CLA proportion in membranes was very low, on average 0.01 ± 0.02 g of c9,
Fig. 2 Incorporation and washout of c9, t11 and t10, c12-CLA from the membrane of erythrocytes. Mean incorporated values (g/100 g of FA) for each isomer with standard error bars are presented
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992
Eur J Nutr (2014) 53:989–993
Table 2 Fatty acid composition of the red cell membrane on week 1, 3, 5 and 12 of the experiment in the CLA group (CLA) and control group (C) Fatty acid (wt%)a
Week 1 CLA
Week 5c
Week 3 C
b
p
CLA
C
C14:0
0.39
0.35
0.32
0.26
C15:0
0.34
0.32
0.29
0.25
C16:0 C16:1 n-9
19.88 0.25
19.74 0.28
19.92 0.14
19.85 0.20
C16:1 n-7
0.84
1.02
0.71
0.73
0.017
p
CLA 0.022
\0.001
Week 12 C
0.28
0.25
0.24
0.22
19.75 0.10
19.71 0.21
0.60
0.64
p
CLA 0.004
\0.001
C
0.28
0.24
0.28
0.23
25.30 0.22
20.45 0.20
0.68
0.63
C17:0
1.44
1.43
1.46
1.36
1.31
1.22
1.77
1.47
C18:0
14.16
13.84
16.20
15.34
0.004
17.06
15.72
\0.001
21.94
18.24
C18:1 n-9
25.21
26.37
23.78
26.80
0.049
21.57
25.70
0.015
30.48
30.29
\0.001
\0.001
0.011
p 0.041 0.001
0.002
C18:1 n-7
2.19
2.36
1.80
2.21
1.52
2.15
2.24
2.36
C18:2 n-6
19.32
20.17
18.72
20.00
19.61
20.66
11.45
16.74
\0.001
C18:3 n-3
0.52
0.46
0.50
0.40
0.55
0.40
0.18
0.36
0.005
c9, t11 CLA
1.35
0.03
\0.001
2.49
0.00
\0.001
3.24
0.04
\0.001
0.40
0.00
\0.001
t10, c12 CLA
0.66
0.00
\0.001
0.86
0.00
\0.001
1.09
0.00
\0.001
0.02
0.00
\0.001
0.21
0.14
\0.001
0.22
0.14
0.04
0.10
0.009
0.11
0.08
\0.001
0.27
0.37
\0.001
0.23
0.34
0.51
0.58
0.049
0.33
0.56
0.003
\0.001 \0.001
3.93 1.06
5.60 0.66
\0.001 \0.001
2.24 0.02
4.40 0.21
\0.001 \0.001
C20:0
0.14
0.12
0.18
0.13
C20:1 n-9
0.12
0.15
0.07
0.12
C20:2 n-6
0.29
0.35
0.27
0.35
C20:3 n-6
0.52
0.57
0.51
0.54
C20:4 n-6 C20:5 n-3
4.93 0.80
5.36 0.65
4.15 0.90
5.11 0.58
C22:0
0.00
0.00
0.01
0.02
0.05
0.01
0.03
0.04
C22:4 n-6
0.38
0.38
0.34
0.35
0.30
0.41
0.001
0.27
0.53
C22:5 n-3
1.52
1.36
1.77
1.43
0.002
1.95
1.48
\0.001
0.40
1.01
\0.001
C22:6 n-3
3.18
3.03
3.39
3.08
0.024
3.58
2.96
0.001
0.23
0.95
\0.001
C24:0
0.12
0.12
0.15
0.14
0.20
0.17
0.20
0.19
a
0.002
0.001
0.043
0.018 0.005
\0.001
wt%, weight%; g of fatty acid/100 g of all fatty acids, mean values are presented
b
t test for unpaired samples was performed, p \ 0.05 are shown, p B 0.001 were considered significant
c
group-specific supplementation with CLA or sunflower oil stopped after 5th week
found no isomer-specific incorporation [3]. We did not analyse CLA in plasma lipids; therefore, we have no direct evidence that both isomers were present in equimolar concentration in plasma. However, Burdge and co-authors found no difference in concentration of both isomers in plasma [3]. In our experiment, the highest proportion of c9, t11-CLA and t10, c12-CLA in porcine erythrocytes were 3.24 wt% and 1.09 wt%, respectively, while they reported lower proportions of those CLA isomers (0.42, 0.19 wt% respectively) in human erythrocytes [4]. Although we do not know total fat intake in their study, porcine erythrocytes seem to incorporate CLA more easily than human. Erythrocyte fatty acid status is considered to be a reflection of polyunsaturated fatty acid [5], but this is not so in CLA, because they preferentially concentrate c9, t11-CLA. Thus, we cannot use them for the same purpose when investigating CLA.
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Washout of CLA has mostly been studied in connection to its immune influence, which lasted 5–12 weeks after end of supplementation [8, 9]. We found almost complete washout of CLA in 7 weeks after supplementation stopped. Burdge and co-authors increased input of individual isomers during the supplementation time, while we kept the same dose for the whole time of feeding, as it usually occurs in practice [4]. We showed that also with the constant intake, incorporation increases with time, and after 4 weeks reaches the plateau. It is therefore evident that CLA in porcine model is not incorporated only in a dosedependent, but also in a time-dependent manner. According to differential immunologic, anti-inflammatory and anti-adipose effect of two CLA isomers, which could also be influenced by different lipid membrane composition in different species [1, 10], further studies on incorporation of CLA isomers into different cell
Eur J Nutr (2014) 53:989–993
membranes at different species can also help to explain various biological CLA functions. Acknowledgment This study was supported by Slovenian government P4-0092 research grant. The ethicalness of the study protocol was approved by the Slovenian Veterinary Administration (4.4.-23/ 05). We are grateful to Dr. Gaj Vidmar from the Institute of Rehabilitation, Republic of Slovenia, for help with statistical analyses. Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
References 1. O’Shea M, Bassaganya-Riera J, Mohede IC (2004) Immunomodulatory properties of conjugated linoleic acid. Am J Clin Nutr 79(6 Suppl):1199S–1206S 2. Pariza MW, Park Y, Cook ME (2001) The biologically active isomers of conjugated linoleic acid. Prog Lipid Res 40(4):283–298 3. Burdge GC, Lupoli B, Russell JJ, Tricon S, Kew S, Banerjee T, Shingfield KJ, Beever DE, Grimble RF, Williams CM, Yaqoob P, Calder PC (2004) Incorporation of cis-9, trans-11 or trans-10, cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men. J Lipid Res 45(4):736–741
993 4. Burdge G, Derrick P, Russell J, Tricon S, Kew S, Banerjee T, Grimble R, Williams C, Yaqoob P, Calder P (2005) Incorporation of cis-9, trans-11 or trans-10, cis-12 conjugated linoleic acid into human erythrocytes in vivo. Nutr Res 25(1):13–19 5. Zamaria N (2004) Alteration of polyunsaturated fatty acid status and metabolism in health and disease. Reprod Nutr Dev 44(3):273–282 6. Park PW, Goins RE (1994) In situ preparation of fatty acid methyl esters for analysis of fatty acid composition in foods. J Food Sci 59:1262–1266 7. Noone EJ, Roche HM, Nugent AP, Gibney MJ (2002) The effect of dietary supplementation using isomeric blends of conjugated linoleic acid on lipid metabolism in healthy human subjects. Br J Nutr 88(3):243–251. doi:10.1079/BJN2002615 8. Bassaganya-Riera J, Hontecillas R, Zimmerman DR, Wannemuehler MJ (2002) Long-term influence of lipid nutrition on the induction of CD8(?) responses to viral or bacterial antigens. Vaccine 20(9–10):1435–1444 9. Song HJ, Grant I, Rotondo D, Mohede I, Sattar N, Heys SD, Wahle KW (2005) Effect of CLA supplementation on immune function in young healthy volunteers. Eur J Clin Nutr 59(4):508–517 10. Tricon S, Burdge GC, Kew S, Banerjee T, Russell JJ, Grimble RF, Williams CM, Calder PC, Yaqoob P (2004) Effects of cis-9, trans11 and trans-10, cis-12 conjugated linoleic acid on immune cell function in healthy humans. Am J Clin Nutr 80(6):1626–1633
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