Lipids (2007) 42:509–518 DOI 10.1007/s11745-007-3051-7
ORIGINAL ARTICLE
Comparing Subcutaneous Adipose Tissue in Beef and Muskox with Emphasis on trans 18:1 and Conjugated Linoleic Acids Michael E. R. Dugan Æ John K. G. Kramer Æ Wayne M. Robertson Æ William J. Meadus Æ Noelia Aldai Æ David C. Rolland
Received: 3 January 2007 / Accepted: 5 March 2007 / Published online: 10 May 2007 Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food, Canada 2007
Abstract Muskox (Ovibos moschatus) are ruminant animals native to the far north and little is known about their fatty acid composition. Subcutaneous adipose tissue (backfat) from 16 wild muskox was analyzed and compared to backfat from 16 barley fed beef cattle. Muskox backfat composition differed substantially from beef and the most striking difference was a high content of 18:0 (26.8 vs. 9.77%). This was accompanied by higher levels of most other saturated fatty acids except beef had more 16:0. Muskox backfat also had a lower level of cis-18:1 and this was related to a lower expression of steroyl-CoA desaturase mRNA. Beef backfat had a higher level of total trans-18:1 (4.25 vs. 2.67%). The most prominent trans-18:1 isomers in beef backfat were 10t-18:1 (2.13%) and 11t-18:1 (0.77%) whereas the most prominent isomers in muskox backfat were 11t-18:1 (1.41%), 13t/14t- (0.27%) and 16t-18:1 (0.23%). The total conjugated linoleic acid (CLA) content was higher in beef backfat than muskox (0.67 vs. 0.50%) with 9c,11t-18:2 as the most abundant CLA isomer. The second most abundant CLA isomer in beef backfat was 7t,9c-18:2 (0.10%) whereas in muskox it was 11t13c-18:2 (0.04%). Muskox backfat had a higher content of 18:3n-3 and its elongation and desaturation products 20:5n-3, 22:5n3 and 22:6n-3 and a lower n-6/n-3 ratio. Overall, the high forage diet of muskox seemed to produce a healthier fatty acid profile and highlighted the need to develop feeding M. E. R. Dugan (&) W. M. Robertson W. J. Meadus N. Aldai D. C. Rolland Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C&E Trail, Lacombe, AB, Canada T4L 1W1 e-mail:
[email protected] J. K. G. Kramer Food Research Program, Agriculture and Agri-Food Canada, Guelph, ON, Canada
strategies for intensively raising beef that will not negatively impacting fatty acid composition. Keywords Beef Muskox Conjugated linoleic acid trans 18:1 Stearic acid Saturated fatty acids StearylCoA desaturase Abbreviations ACO Acyl CoA oxidase CLA Conjugated linoleic acid FAME Fatty acid methyl esters FAS Fatty acid synthase MUFA Monounsaturated fatty acids PUFA Polyunsaturated fatty acids SFA Saturated fatty acids SCD-1 Stearyl CoA desaturase-1 SFA Saturated fatty acids
Introduction Muskox (Ovibos moschatus) are ruminant animals native to the far north and along with goats and sheep fall into the sub-family Caprinae of the Bovidae family. In Canada, muskox are found mainly on the Arctic islands and the majority of these can be found on Banks Island. Banks Island is the most western island in the Canadian Arctic Archipelago and covers an area of 70,266 km2 and has a muskox population of approximately 65,000, representing about 47% of the total population of the Canadian Northwest Territories [1]. Muskox are harvested for local use, for export of highly valued quiviut and hides and also for developing markets for their meat. The nutritive value of
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the meat is in part related to its fatty acid composition, but little is known about muskox lipid composition and nothing is known about the conjugated linoleic acid (CLA) or trans-18:1 content of its tissues. Adamczewski et al. [2] reported the content of lean, fat and bone in muskox and the percent lipid class distribution, but not their fatty acid compositions. Baker et al. [3] only reported the fatty acid composition of milk fat from muskox but did not include total nor individual CLA or trans-18:1 isomers. Conjugated linoleic acid has many purported roles in the prevention and possible treatment of several diseases including diabetes, obesity and some types of cancer [4, 5]. Rumenic acid (9c,11t-18:2), the major CLA isomer in ruminant fats has been associated with beneficial effects, while the other CLA isomers, notably 10t,12c-18:2, have not [6–8]. In addition, vaccenic acid (11t-18:1), the major trans-18:1 isomer generally present in ruminant fats is equally effective in cancer prevention [9], because of its endogenous conversion to 9c,11t-18:2 in animals [10], humans [11], and human cell lines [12]. The health effects of several trans-18:1 isomers other than 11t-18:1, notably 9tand 10t-18:1, were shown to be associated with negative plasma lipid and lipoprotein profiles [13, 14]. However, butter fat enriched in 11t-18:1 and 9c,11t-18:2 improved the plasma cholesterol profile in hamsters [15] and natural beef fat reduced the growth of human cancer cells [16]. The present communication reports the comprehensive fatty acid composition of subcutaneous adipose tissue (backfat) from wild muskox and compares it to the most commonly consumed adipose tissue of ruminant origin in Canada (i.e., beef) finished on a high barley diet at the Lacombe Research Centre. Additional mRNA analysis of fatty acid metabolizing enzymes was then conducted to help explain differences in the levels of saturated (SFA) and monounsaturated fatty acids (MUFA) between these ruminant species.
Experimental Procedures Animals, Diets and Sample Collection Sixteen muskox and 16 beef animals were used in this study. Beef animals were raised and both beef and muskox were slaughtered in accordance to principles and guidelines of the Canadian Council of Animal Care. The average age of the muskox was estimated to be 2.5 years and the beef animals 1.5 years. Wild muskox were harvested from Banks Island where a high proportion of their diet consists of sedges (Carex aquatilis, Eriophorum spp.; [17]) which are similar to grasses in composition with high fiber levels and relatively low protein contents [18]. Post-slaughter, muskox carcasses were rapidly chilled outdoors (–20 to
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–25 C with a wind chill of approximately –40 C) and frozen carcasses were shipped to the Lacombe Research Centre. The beef animals were raised under typical cow/ calf and feedlot conditions for Western Canada at the Lacombe Research Centre. In the last 75 days prior to slaughter, beef animals had ad libitum access to a finishing diet consisting of 22.0% barley silage, 73.3% barley grain, 1.6% molasses, 3.1% feedlot supplement (32% crude protein) and 22 mg/kg sodium monensin. Beef animals were slaughtered and processed at the Lacombe Research Centre abattoir and adipose tissue samples were collected from both muskox and beef animals between the fifth and sixth ribs at 5 cm off the midline and these samples remained frozen at –80 C until analyzed. Feed Fatty Acid Analysis Fatty acid methyl esters (FAME) from the beef feedlot diet and sedges grab sampled from Banks Island (i.e., muskox diet) were prepared according to Sukhija and Palmquist [19] and using a Varian 3800 GC (Varian, Walnut Creek, CA, USA) equipped with a Varian 8100 autosampler and a 30 m SP2340 capillary column (Supelco, Bellefonte, PA, USA). The system was operated under constant pressure (15 psi) using hydrogen as the carrier gas and a 20:1 split ratio. The injector and detector were held at 250 C and the FAME were quantified using a flame ionization detector. Samples were injected (1 ll, 0.5 lg/ll) and the column temperature was held initially at 50 C for 30 s, increased to 170 C at 25 C/min, held for 3 min, increased to 180 C at 2 C/min, then increased to 230 C at 10 C/ min. Chromatograms were integrated using Varian Star Chromatography Workstation software. Adipose Tissue Analyses Subcutaneous adipose tissue was directly methylated with sodium methoxide and the FAME were analyzed using the GC and Ag+-HPLC equipment and methods outlined by Cruz-Hernandez et al. [20]. The trans-18:1 isomers were, however, analyzed using two complementary GC temperature programs instead of a preparatory Ag+-TLC separation followed by GC separation at 120 C. The first temperature program was as previously described (temperature program A; 45 C held for 4 min, increased at 13 C/min to 175 C and held for 27 min, then increased at 4 C/min to 215 C and held for 35 min) [20]. The second program (temperature program B; 45 C held for 4 min, increased at 13 C/min to 150 C and held for 47 min, then increased at 4 C/min to 215 C and held for 35 min) improved the resolution of some trans-18:1 isomers (6-8tto 11t-18:1) and allowed for analysis of the remaining trans-18:1 isomers by difference.
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For the identification of FAME by GC, the reference standard no. 461 from Nu-Check Prep Inc. (Elysian, MN, USA) was used. Branch-chain FAME were identified using a GC reference standard BC-Mix1 purchased previously from Applied Science (State College, PA, USA). All the geometric isomers of linoleic and linolenic acids were prepared by mild iodine isomerization [20]. The CLA isomers no. UC-59M from Nu-Chek Prep Inc. was used since it contained all four positional CLA isomers, and they were analyzed by GC and Ag+-HPLC [20]. Individual CLA isomers were obtained from Matreya Inc. (Pleasant Gap, PA, USA) and isomerized using iodine to obtain all geometric CLA isomers [20]. All chemicals and solvents were of analytical grade. mRNA Analysis of Fatty Acid Metabolizing Enzymes Total RNA was isolated from muskox (n = 8) and beef (n = 8) adipose tissue in ten volumes of the guanidium thiocyanate based TRI reagent solution in accordance with the manufacturer’s specifications (Sigma, St Louis, MO, USA). A prior clean-up step was required after the adipose samples were dissolved in the TRI reagent to remove excess lipid by centrifugation at 10,000 g at 20 C and removal of the upper lipid fraction prior to addition of chloroform. Pools of cDNA were generated from each sample at 42 C for 60 min (Sigma, St Louis, MO, USA) using: 5 lg of total RNA primed with 300 ng of random hexamers, 200 U of MMLV-reverse transcriptase (RT) enzyme, 1 mM of dATP, dGTP, dTTP and dCTP, 10 mM DTT, and 10 U of RNAse inhibitor in the manufacturer’s RT buffer. The yield of cDNA was measured according to the polymerase chain reaction signal generated from the internal house keeping gene b-actin [21]. To generate PCR primers for the ‘‘comparative gene expression studies’’, new cDNAs were cloned and
sequenced from the muskox adipose RNA for the stearyl CoA desaturase-1 (SCD1), fatty acid synthase (FAS), acyl CoA oxidase (ACO) and b-actin mRNAs. To help standardized the gene expression analysis studies, PCR primers were designed to have 100% homology with their equivalent beef mRNAs, SCD1 (Gb no. AY241932), FAS (Gb no. AF285607), ACO (Gb no. BF230141), and 96% homology with b-actin (Gb no. AY141970) (Table 1). Sequence homologies between muskox and beef cDNAs were analyzed using the ClustalW program [22]. Primer sequences, which matched both muskox and beef cDNAs, were generated using the Primer3 program [23] and validated using the BLASTn program [24]. Gene expression activity in the beef and muskox adipose samples was measured using SYBR green labeled Real-time quantitative RT-PCR analysis method in a Stratagene Mx4000 machine (Stratagene, La Jolla, CA, USA). For each sample, cDNA was added to 1X Brilliant SYBR green QPCR master mix and then aliquoted into 20 lL fractions. The specific gene primers were added to their independent fraction and run simultaneously in the Mx4000 machine. Gene expression activity was estimated by the number of PCR cycles required to reach a minimum threshold for cycle detection (Ct value). The signal for b-actin was used as an internal standard to normalize the estimates of FAS, ACO and SCD1 mRNA levels between species. Statistical Analysis Data were analyzed by ANOVA using the GLM procedure of SAS software (SAS Systems, Release 8.2, SAS Institute Inc., Cary, NC, USA). Results were expressed as mean ± SEM, and a P value of 0.05 was considered significant.
Table 1 Primers, Genbank accession numbers, and the expected product size from PCR amplification of cDNA and genomic DNA templates of muskox and bovine adipose samples Gene
Muskox cDNA (bp)
Muskox genomic DNA (bp)
Bovine cDNA (bp)
Forward reverse
Muskox Genbanka accession number
SCD1
214
214
214
5¢-cctaccaggataaggagggc-3¢
AY530631
5¢- tttgtaggttcggtgactcc-3¢ FAS
393
482
373
ACO
217
519
217
5¢-agcgggaagcgtgtgatgg-3¢
AY768701
5¢-ttcccggctgtgtgccac-3¢ 5¢-gtggaacctaacgtccattg-3¢
AY766151
5¢-cctgggtgatctgagact-3¢ b-actin
231
343
224
5¢-ggacttcgagcaggagatgg-3¢
DQ017256
5¢-gcaccgtgttggcgtagagg-3¢ SCD1 Stearyl CoA desaturase-1, FAS fatty acid synthase, ACO acyl CoA oxidase a
Genbank, National Institute of Health, Bethesda
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512
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Results and Discussion
Table 3 Muskox and beef subcutaneous adipose tissue saturated fatty acid composition (n = 16 in each group)
Diet
Percentage of total fatty acids
The fatty acid composition of the beef diet was typical for barley grain lipids with high levels of linoleic acid (18:2n6; 48.9%), substantial amounts of linolenic acid (18:3n-3; 7.63%) and an 18:2n-6/18:3n-3 ratio of 6.4 (Table 2). The beef diet also contained intermediate amounts of 9c-18:1 and 16:0. The sedge lipids consisted mainly of SFAs (55.9%) that included 16:0 (23.3%), 18:0 (3.8%) and the long-chain SFAs 20:0 (6.4%), 22:0 (14.8%) and 24:0 (4.7%) (Table 2) which would be indicative of a high content of waxy cuticle [25] and a reduced feeding value for this forage. The sedges contained a higher level of 18:3n-3 relative to the beef diet, but the amount was lower than that reported in other leaf lipids [26]. Sedge lipids also contained substantial amounts of 18:2n-6 (15.9%) and had an 18:2n-6/18:3n-3 ratio of 1.4.
10:0
Adipose Tissue SFA Muskox subcutaneous adipose tissue had higher total SFAs than beef including most of the individual SFAs except beef adipose tissue had more 16:0 (P < 0.05; Table 3), which may be indicative of a higher plane of nutrition and
Muskox
SEM
0.05**
0.15
0.01
12:0
0.07
0.13
0.03
14iso
0.03**
0.07
0.00
14:0
3.38*
4.30
0.31
15iso
0.10**
0.33
0.01
15ai
0.15**
0.29
0.02
15:0
0.52
0.53
0.05
16 iso
0.15**
0.21
0.01
16:0
Beef
25.4*
23.4
0.56
17iso
0.33**
0.42
0.01
17ai 17:0
0.59** 1.18*
0.94 1.39
0.03 0.07
18:0 iso
0.15*
0.17
0.01
18:0
9.77**
19:0
0.05**
0.24
0.01
20:0
0.07**
0.64
0.02
21:0
0.01**
0.12
0.01
22:0
0.01**
0.33
0.01
23:0
0.02**
0.12
0.01
24:0
0.01**
0.16
0.01
26:0
0.00**
0.06
Sum SFA Sum iso and anteiso
42.0** 1.49**
26.8
60.9 2.42
0.67
0.00 1.11 0.07
SEM standard error of the mean, SFA saturated fatty acids Table 2 Dietary fatty acid content and percent composition Beef diet Dietary fat (mg FAME/g DM)
29.3
Sedgesa 8.6
Fatty acid (FAME, %) 14:0
0.33
15:0
0.08
16:0
17.7
9c-16:1
0.28
1.35
17:0
0.11
0.58
18:0
1.31
3.80
0.52 23.3
20.5
10.9
11c-18:1 18:2n-6
1.27 48.9
1.57 15.9
20:0
0.28
6.41
18:3n-3
7.63
11.08
11c-20:1
0.72
0.21
22:0
0.23
14.89
13c-22:1
0.13
0.94
24:0
0.12
4.70
15c-24:1
0.08
0.59
FAME fatty acid methyl esters, DM dry matter a
1.65
9c-18:1
Diets were analyzed in duplicate Sedges are a major component of the muskox diet
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*P < 0.05, beef differs from muskox, **P < 0.01, beef differs from muskox
increased rates of de novo fatty acid synthesis in beef fat. Of particular interest, muskox adipose tissue had higher levels of long-chain SFAs (‡18:0; P < 0.05), specifically 18:0 (26.8 vs. 9.8% in beef fat). High 18:0 levels were previously reported in the milk fat of muskox [3]. Finding a high SFA content in muskox adipose tissue was unusual since decreased levels of SFA are generally observed in animals living at low ambient temperatures, as expected in the Arctic, which maintains their fat in a liquid or semiliquid state [27]. The high insulation value of the coat may negate their need for desaturation, as well as their higher content of low melting point branch-chain FAs (noted below). Sheep and goats are more closely related to muskox than beef cattle, and they also have been found to deposit high levels of 18:0 when grazing forages [28], but 18:0 is lower in lambs when fed a high-energy corn diet [29]. Comparably high levels of 18:0 have been found in other wild ruminants (elk, deer and antelope) under natural grazing conditions [30]. The high level of 18:0 in the fat of muskox could be related to the diet, to more extensive biohydrogenation of dietary polyunsaturated fatty acids
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(PUFAs) compared to beef cattle and/or to reduced rates of 18:0 desaturation.
10t 9c 10t
Branched-Chain Fatty Acids
11c
9t
11t
6-8t
11t
6-8t 9t
min 66
65
13t /14t / 6-8c
13c 14c 16t
12c
15c
12t 10c 15t
36
37
38
min
39
Fig. 1 Partial GC chromatograms of the 18:1 region from representative adipose tissue FAME of barley fed beef adipose tissue showing the maximum range of 10t- and 11t-18:1 isomer distribution among samples using GC temperature program A (details in M&M section). Insert shows separation of partial trans-18:1 isomers of the same beef samples using GC temperature program B
11c 9c
11t
12t / 6-8c
Muskox adipose tissue was found to contain higher levels of C14 to C18 branched-chain (iso and anteiso) fatty acids (P < 0.05; Table 3). Short branched-chain fatty acids arise from deamination of branched-chain amino acids by rumen microorganisms [31] or by carboxylation of propionyl-CoA to form methylmalonyl-CoA [32]. These branched-chain fatty acids can then be incorporated during fat synthesis in ruminants to form longer chain branched-chain fatty acids [33]. Higher levels of branch-chain fatty acids typically result when readily fermentable carbohydrate sources are available, causing an increase in proprionate production [27]. Given the relatively poor quality of forage available to muskox and lack of grain in their diet, synthesis of branched-chain fatty acids through carboxylation of propionyl-CoA seems unlikely. Furthermore, muskox may be predisposed to deposit higher levels of branched-chain FAs similar to sheep and goats who were shown to deposit greater levels of branched-chain FAs compared to cattle when fed the same diet [32].
12t / 6-8c
9c
6-8t 9t 10t
The separation of the cis and trans-MUFA, particularly of the 18:1 isomers remains a challenge, as evident from the partial GC chromatograms of beef (Fig. 1) and muskox backfat (Fig. 2). When cis and trans-MUFA are not prefractionated using Ag+-TLC prior to GC, one deals with partial resolution of 10t- and 11t-18:1 because of the large differences in their relative abundance, and overlap of 13tto 16t-18:1 with cis-18:1 isomers. To avoid this, two separate GC temperature programs were employed in this study to improve the resolution of 6-8t- to 11t-18:1 isomers by lowering the temperature during the plateau region from 175 to 150 C (see inserts in Figs. 1, 2), and to determine the remaining trans-18:1 isomers by difference between the two separate GC chromatograms. The most abundant cis-MUFAs in both beef and muskox adipose tissue were 9c-16:1 and 9c-18:1 and the latter contained trace amounts of 10c-18:1 (Table 4). The adipose tissue of beef contained much more 9c-18:1 (38.0 vs. 27.4%) and correspondingly less 18:0 than found in muskox (9.8 vs. 26.8%). Beef adipose tissue also contained significantly higher levels of most of the other cis-MUFA, some arising from D9 desaturation of their SFA precursors, while others are possible intermediates of rumen biohydrogenation of PUFAs [34]. Beef adipose tissue contained more total trans-18:1 relative to muskox (P < 0.01; Table 4) and this was
6-8t
13t /14t / 6-8c
cis- and trans-MUFA
10t 9t
66 min
65 11c
14c 16t 19:0 15c
12c
12t
13c
10c 15t
36
37
38
39
min
Fig. 2 Partial GC chromatograms of the 18:1 region from representative adipose tissue FAME of wild muskox adipose tissue showing the maximum range of 10t- and 11t-18:1 isomer distribution among samples using GC temperature program A (details in M&M section). Insert shows separation of partial trans-18:1 isomers of the same muskox samples using GC temperature program B
primarily due to a higher level of 10t-18:1 and slightly greater levels of 6-8t, 9t and 12t-18:1 (P < 0.01; Fig. 3). Conversely, the most abundant trans-18:1 isomer in muskox adipose tissue was 11t-18:1 and its level was greater than that found in beef fat (P < 0.01). The trans-18:1 content and composition was also more variable in beef than muskox fat as evident from the partial GC chromatograms presented in Figs. 1 and 2 demonstrating the range in trans-18:1 compositions encountered in beef and muskox, respectively. Feeding high levels of concentrate significantly increases 10t-18:1 in adipose tissue of ruminants making it
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Table 4 Muskox and beef subcutaneous adipose tissue monounsaturated fatty acid composition (n = 16 in each group) Percentage of total fatty acids
Beef
Muskox
SEM
9c-14:1
1.27*
0.14
0.08
Sum 16:1t
0.10
0.11
0.00
Total 16:1c
5.39**
2.53
0.34
7c-
0.20**
0.59
0.03
9c-
4.78**
1.85
0.31
11c-
0.30**
0.03
0.02
13c-
0.01
0.12**
0.07
9c-17:1
1.11**
0.47
0.04
Total 18:1t
4.25**
2.67
0.24
Total 18:1c
41.3**
28.4
0.86
0.14** 38.0**
0.09 27.4
0.01 0.81
11c-
1.91**
0.46
0.08
12c-
0.12**
0.09
0.01
13c-
0.58**
0.06
0.04
14c-
0.04**
0.00
0.01
15c-
0.16**
0.08
0.01
16c-
0.09*
0.07
0.00
17-
0.01
6c-8c9c/10c-
0.18
0.19
9c-20:1
0.11*
0.10
0.01
11c-20:1
0.31**
0.21
0.02
15c-24:1
0.01**
0.06
0.01
9c-14:1/14:0
0.34**
0.03
0.02
9c-16:1/16:0
0.17**
0.07
0.01
9c-17:1/17:0
0.93**
0.31
0.06
9c-18:1/18:0 9c11t-18:2/11t-18:1
3.85** 0.55**
1.00 0.23
0.19 0.04
SEM standard error of the mean
Relative % of total FAMEs
*P < 0.05, beef differs from muskox, **P < 0.01, beef differs from muskox
2.50
**
2.00
Beef
1.50
Muskox 1.00
**
0.50 0.00
**
6-8t .
** **
9t
** **
10t 11t 12t 13,14t 15t trans- 18:1 isomers
**
16t
Fig. 3 Muskox and beef subcutaneous adipose tissue trans-18:1 composition. Bars and vertical lines represent mean ± SEM, n = 16 for each species. For each isomer, bars with asterisks are significantly different (P < 0.01)
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the predominant trans-18:1 isomer irrespective of the grain type fed, whether corn [35], barley [36], a barley/oat mixture [37], or a mixture to several ingredients [38]. On the other hand, feeding a lower concentrate to forage ratio increases both 11t- and 10t-18:1, but 11t-18:1 remains the major trans-18:1 isomer [39–42]. Comparable changes in 10t-18:1 levels in milk fat have also been found when dairy cows are fed differing concentrate to forage ratios [43–46]. A recent study shows that the shift toward 10t-18:1 production can be eliminated by feeding a grass/hay basal diet supplemented with linseed oil when the forage to concentrate ratio is 64:36 [47]. The ionophore monensin has also been shown recently to increase 10t-18:1 in milk fat when dairy cows are fed a concentrate containing supplementary sunflower seed oil [48]. High levels of 10t-18:1 appear to be related to changes (and instability) in the rumen environment and altered bacterial populations when highly fermentable (i.e., pH reducing) diets are fed [36, 49, 50]. CLA The complete analysis of the CLA isomers in beef and muskox required a combination of GC and Ag+-HPLC, reference standard no. UC-59M from Nu Chek Prep Inc., and isomerization of individual CLA standards using iodine [20]. Peaks were identified by comparison to previously established elution orders by GC and Ag+-HPLC [20, 34, 51, 52]. The 7t,9c-18:2 isomer was observed as a distinct shoulder on the 9c,11t-18:2 peak in the GC chromatogram but by Ag+-HPLC these isomers were clearly resolved (Fig. 4). On the other hand, 9c,11t- and 9t,11c-18:2 were resolved by GC but co-eluted with using Ag+-HPLC (Fig. 4). The relatively high content of 21:0 observed in muskox lipids eluted in the region of the minor CLA isomers in the GC chromatogram and the presence of these isomers was confirmed using Ag+-HPLC (Fig. 4) [20, 34]. The adipose tissue of beef had significantly more total CLA than muskox (Table 5). The most abundant CLA isomer in both beef and muskox was 9c, 11t-18:2, which was significantly higher in beef than in muskox as a percent of total FAME (Fig. 5), but both had ~67% 9c,11t-CLA in total CLA. There were, however, significant differences in the remaining CLA isomers. Among the minor CLA isomers present, beef had significantly greater amounts of amounts of 7t,9c- and 9t,11c-18:2 (P < 0.01; Fig. 5) and contained trace amounts of 10t,12c-18:2 (data not presented). The CLA profile of beef fat in this study was similar to that reported by others in beef [40, 53] and dairy cows [44, 54] fed concentrates. Muskox adipose tissue had significantly greater amounts of 11t,13c-18:2 (P < 0.01; Fig. 5) and 11t,13t-18:2 (Fig. 4, Ag+–HPLC). For details regarding the identification of the CLA isomers see reference by CruzHernandez et al. [20]. The minor trans, trans-CLA isomers
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confirmed by comparison with iodine isomerized products of linoleic and linolenic acids [58].
9c,11t9c,11t-
Ag+-HPLC
GC
7t,9c-
48
11t,13c-
PUFA
11c,13t10c,12t-
12,14 c/t
8t,10c
21:0
trans,trans 12,14 11,13 10,12 9,11
11t,13t9t,11t/10t,12c-
11t,13c-
9t,11c-
7t,9c-
21:0 / 10c,12t-
Beef
Muskox
49
min
22
26
30
34
min
Fig. 4 Partial GC and Ag+-HPLC chromatograms of the CLA region of adipose tissue FAME from wild muskox and barley fed beef. Details of GC temperature program A and silver-ion HPLC separation are given in the M&M sections
(
