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Influence of dietary fat and vitamin E supplementation on α-tocopherol levels and fatty acid profiles in chicken muscle membranal fractions and on susceptibility to lipid peroxidation
PII:
s0309-1740(97)00010-7
Meat Science, Vol. 46, No. I, 9-22, 1997 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0309-1740197 $17.oo+o.ocl
ELSEVIER
Influence of Dietary Fat and Vitamin E Supplementation on cw-Tocopherol Levels and Fatty Acid Profiles in Chicken Muscle Membranal Fractions and on Susceptibility to Lipid Peroxidation C. Lauridsen,a* D. J. Buckleyb & P. A. Morrissey” UDepartment of Nutrition, University College Cork, Republic of Ireland bDepartment of Food Technology, University College Cork, Republic of Ireland (Received 3 November 1996; revised version received 3 January 1997; accepted 3 January 1997) ABSTRACT Broiler chickens were fed a basal feed supplemented with 10% tallow or olive oil and varying levels of vitamin E (20 and 200 mg vitamin E/kg feed). The concentration of IXtocopherol in the membranes of breast and thigh muscles was significantly influenced by the a-tocopherol level in the feed (p < 0.001). Deposition of vitamin E was not injuenced by the type of oil in the feed, except in the mitochondrial fraction of breast where the vitamin E concentration was higher in those fed olive oil than in those fed tallow (p <:0.05). Dietary oil injluenced the fatty acid composition of the muscle membranal fractions (p< 0.001). The oxidative stability of the membranal fractions tended to increase with increasing concentrations of u-tocopherol in the membranal fractions. In conclusion, the supplementation of vitamin E appeared to enhance the stabihty of muscle to oxidation. Thus, incorporation of a-tocopherol into the membranes via dietary manipulation helped in stabilizing the membrane-bound lipids. e 1997 Elsevier Science Ltd
INTRODUCTION Lipid oxidation is considered to be the major cause of quality deterioration in meat and meat products during refrigerated storage (Pearson et al., 1983). Lipid oxidation is primarily initiated in the highly unsaturated fatty acid components of membrane phospholipids (Gray & Pearson, 1987). Stabilization of membrane lipids by dietary supplementation with vitamin E has been shown to prevent the onset of lipid oxidation in chicken (Yamauchi et al., 1991) and pork muscle (Monahan et al., 1993). While vitamin E is widely distributed in tissues, it is primarily located within biological membranes, such as microsomes and mitochondria (Machlin, 1984). Localization of ar-tocopherol in the
membranes allows it to function very efficiently compared to other antioxidants. Diets for fast growing broilers are generally rich in polyunsaturated fatty acids and the degree of unsaturation in carcass fat is thereby increased. Furthermore, there is some *To whom correspondence should be addressed at: Department for Product Quality, Danish Institute of Animal Science, Research Centre Foulum, PO Box 39, DK-8830 Tjele, Denmark. 9
C. Lauridsen, D. J. Buckley, P. A. Morrissey
10
evidence that dietary oils can alter the fatty acid composition of mitochondrial and microsomal lipids (Tahin et al., 1981; Asghar et al., 1990). However, increasing the degree of unsaturation of the muscle membranes by dietary manipulation increases the peroxidizability of tissues and muscle foods (Igene et al., 1980; Pikul et al., 1984). Thus, there is a need to increase the antioxidant capacity of the tissues, and this is readily achieved by feeding a-tocopheryl acetate. Unfortunately, there is little quantitative information available on the effects of feeding different levels of vitamin E and different types of fat (varying in degree of unsaturation) on the concentration of a-tocopherol in the subcellular membranal fractions and their influence on the susceptibility to lipid peroxidation. The objective of the present study was to determine the effect of increasing the degree of monounsaturation of dietary oil and supplementation with cu-tocopherol on the deposition of a-tocopherol, the fatty acid composition, and the oxidative stability of membranal fractions of broiler dark and white meat.
MATERIALS
AND METHODS
Animals and diets
Sixty one-day old chicks were obtained from a commercial hatchery and housed in an environmentally controlled room. Chicks were randomly assigned to four groups and fed a basal diet (Table 1) supplemented with fat and a-tocopheryl acetate as follows: Group Group Group Group
1: 2: 3: 4:
Tallow, 10% + o-tocopheryl acetate, 20 mg/kg feed Tallow, 10% + a-tocopheryl acetate, 200mg/kg feed Olive oil, 10% + cr-tocopheryl acetate, 20mg/kg feed Olive oil, 10% + cr-tocopheryl acetate, 200mg/kg feed
Groups were housed separately in raised wire cages with six birds per cage in an environmentally-controlled room employing a 12 hr light/dark cycle. Feed and water was provided ad libitum. After 42 days, eight chicks from each group were selected at random and killed by cervical dislocation. Breast and thigh muscles were removed, vacuum packed and quickly frozen and stored at -20°C until analyzed. TABLE 1
Composition of Broiler Diets Ingredients
Ground maize Soybean meal Tallow/olive oil Limestone Dicalcium phosphate DL-Methionine NaCl Mineral mixa Vitamin mixb
% of diet
46.56 39.58 10 1 2 0.16 0.40 0.10 0.20
Chemical analysis
DM, g/kg feed Stoldt fat Crude protein (Nx6,25) Sugar Starch ME (MJ/kg DM)
glkg DM
906.5 137.3 259.6 57.9 347.9 15.15
“Containing per kg feed: MnS04, 4H20 333.3 mg; ZnSOz, 7H20 220.3mg; C6HJ07Fe, 3Hz0 450mg; CuS04, 5H20 35 mg; KI03 2mg; CoSO4 5HzO 1mg; Na2Se03 0.35mg. ‘Containing per kg feed: A/D3 6mg; K 2mg; Riboflavin 5mg; Ca panthotenate 15 mg; Niacin 30 mg; B12 0.015 mg; Folic acid 0.6mg; Pyridoxine 3.5 mg; Thiamine 2 mg; Choline chloride 12g; Biotin 0.2 mg; a-tocopheryl acetate 20 or 200 mg.
a-tocopherol levels and fatty acid projles in chicken
11
The chemical composition of the basal diets was determined by the AOAC. procedure (1984) except for crude fat, which was determined by the method described by Stoldt (1952). Isolation of mitochondrial
and microsomal fractions from muscles
Separation of muscle membranal fractions was carried out by a modification of the method by Asghar et al. (1990) as follows. A 50g sample of breast- and thigh muscle, respectively, of each chicken was homogenized with 500 ml of deaerated, nitrogen-saturated buffer solution (0.1 M KCl, 0.2% glycerol, 0.025M Tris-HCl) in a Warring Blender for 2min and centrifuged at 1OOOxgfor 10min in a Beckmann centrifuge. The supernatant was filtered through cheesecloth to remove material in the suspension and was then subjected to differential centrifugation to separate the mitochondrial and microsomal fractions. The mitochondrial extracts were sedimented at 15 OOOxg for 15 min. Calcium chloride was added to the sup rnatant to give a final concentration of 8mM for aggregating the microsomes. The mixture was centrifuged at 13 2OOxg for 30min to separate the microsomal fraction. The subcellular fractions were not purified further. Vitamin E analysis
Tocopherol was extracted from the feed after saponification (Bursting et al., 1994). Muscle ol-tocbpherol was extracted with hexane after saponification at 70°C x 30 min (Buttriss & Diplock, 1984) and determined by reverse-phase High Performance Liquid Chromatography (HPLC) as described by Sheehy et al. (1991). Fatty acid analysis
The fatty acid profiles of diets were determined by capillary gas chromatography after lipid extraction (Burton et al., 1985) and esterification (Christophersen & Glass, 1969). The fatty acid methyl esters were analysed by gas-liquid chromatography using a PyeUnicam PU 4500 gas chromatography equipped with a Machery-Nagel Fused Silica Capillary (25mx0.25mm i.d.) column. The oven temperature was held at 50°C for 3.5min, and increased by 3°C per min to a final temperature of 220°C which was maintained for 18min. Injector and detector temperatures were 250°C. A Shimadzy C-R3A integrator was used for calculation of peak areas. Fatty acid methyl esters were identified by comparison of retention times with those of authentic standards. The fatty acid profiles of samples were quantified following addition of an internal standard to each sample. Thioharbituric
acid-reactive substances
The lability of muscle membranal fractions to iron-stimulated lipid peroxidation was determined by a modification of the method of Kornbrust and Mavis (1980). Membranal fractions were incubated in 40mM tris-maleate buffer (pH 7.4) with 1 mM FeS04 in a total volume of 1 ml. At fixed time intervals aliquots were removed for the measurement of 2-thiobarbituric acid reacting substances (TBARS) bv the method of Buege and Aust (1978). Statistical
analysis
Statistical analyses were performed by means of analysis of variance using the general linear models (GLM) procedure of SAS (1995) with the concentration of vitamin E and
C. Lauridsen, D. J. Buckley, P. A. Morrissey
12
TABLE 2 Concentration of Vitamin E in Diets (added and analysed values) Added Diet
Analysed values
dl-cr-tocopherol acetate” (mglkg)
d-u-tocopherol
d-y-tocopherol
20 200 20 200
34 260 57 272
18 17 21 20
Tallow Tallow Olive oil Olive oil
“Provided by Hoffmann La Roche, a/s.
dietary fat and the interaction between the concentration of vitamin E and dietary fat as independent variables. Where significant variance ratios were detected, differences between treatment means were tested using the least significant difference (LSD.) procedure of SAS. Where data showed variance in-homogeneity, they were treated using the proc MIXED procedure of SAS package version 6.11 (SASR Institute, 1995) after the following model: Xv
=
(lli +
Eg, Eg
-N(O,$),i=
1,2 and j=
1,2
RESULTS Vitamin E in feeds The o-tocopherol concentration in the diets is shown in Table 2. The levels were higher in the diets containing olive oil compared to those containing tallow. The fatty acid profiles of feed samples are shown in Table 3. The diets containing tallow had 57.6% saturated, 32.0% monounsaturated, and 10.4% polyunsaturated fatty acids, whereas those with olive oil had 13.3% saturated, 70.4% monounsaturated, and 16.3% polyunsaturated fatty acids. Supplementation of vitamin E had no influence on the fatty acid composition of the feed. TABLE 3 Fatty Acid Composition (%) of Diets Containing Tallow or Olive Oil. Meanshsd
of 2 Analyses
Performed in Duplicate Fatty acid
14:o 16:O 16:l 18:O 18:l 18:2 18:3 Others
Tall0 w
Olive oil
Mean f sd
Mean f sd
4.6zkO.2 28.4 f 0.6 1.1 io.04 24.3 f 0.9 30.9 f 1.5 8.2 zt 0.4 1.3*0,04 1.21kO.8
0*3*0.1 12.2zko.2 0.8 i 0.04 0.6 f 0.2 69.6ztO.S 14.8hO.l 0.8 f 0.04 0.9 f 0.4
a-tocopherol levels andfatty acid profiles in chicken
13
Membranal fractions In general, the dietary treatments had no influence on the weight of the membranal fractions isolated from chicken breast @ > 0.05). However, a significant influence of dietary oil (p < 0.05) was observed on the weight of mitochondria of dark muscles, i.e. mitochondria of broilers fed tallow weighed 77 mg/g tissue versus 63 mg/g of tissue for broilers fed olive oil. Significant interactions between dietary oil and vitamin E on the mitochondrial (p < 0.05) and on the microsomal (p < 0.001) weight of dark muscles were found. No reasonable explanations are available for these observations. The average wet wt (mg/g of tissue) of mitochondrial fraction was 63.0 f 10.9mg and 69.8 & 17.0mg for breast and thigh muscles, respectively, and for microsomal fractions 70.6 f 22.3 and 78.3 f 24.1 for breast and thigh muscles, respectively. Table 4 shows the concentration of u-tocopherol in membranal fractions expressed on the basis of membrane weight, protein and total fat content. Supplementation with otocopherol had a significant influence @ < 0.001) on the concentration of a-tocopherol in the membranes of breast and thigh muscles. When a-tocopherol concentrations were expressed on a total lipid basis, the levels in the mitochondria and microsomes from breast muscle were lower than those observed for thigh muscle. These differences were most pronounced when the values were expressed on a wet wt basis. The membranal a-tocopherol concentrations were not influenced by the type of oil (JJ> O.OS), although significant interactions between vitamin E and oil were observed on the concentration of a-tocopherol per wet wt of microsomes (p < 0.01) as well as the concentration of cr-tocopherol per mg total fat in mitochondria @ < 0.01) and microsomes (p < 0.01) of dark muscles. Furthermore, a significant influence @ < 0.05) was observed on vitamin E expressed in relation to the content of protein in the microsomal fraction of breast muscle. The fatty acid composition of the muscle membranal fractions are shown in Tables 5 and 6. Dietary oil influenced the fatty acid composition of the muscle membranal fractions. In general, the concentration of saturated fatty acids (Ci4:e and C,& and Ci6:i was higher, and the concentration of monounsaturated (Cis:i) lower, in the mitochondrial fraction from broilers fed tallow compared to broilers fed olive oil. However, the content of C16:0in mitochondria of group 3 (olive oil + low a-tocopheryl acetate) was remarkably low, as was the content of C,s:i in the breast of group 4. The Cis:s content in mitochondria of groups fed tallow was slightly higher than in the olive oil fed groups. Overall, the relationship between saturated fatty acids and monounsaturated fatty acids of mitochondrial fractions was influenced by the dietary treatments, although the variation between groups was very high for groups fed olive oil. The data on the fatty acid profiles of the microsomes were unexpected when compared group-wise. The content of Ci6:, was higher, and the Cis:e lower, in groups fed tallow compared to groups fed olive oil. The content of Cis:i in microsom s of breast from broilers fed tallow was higher and the content of C20:4lower when compared to broilers fed olive oil. Oxidative stability of membrane-bound lipids Data on the rates of iron-induced lipid-peroxidation in the mitochondria and microsomes isolated from breast and thigh muscles from the four groups of broilers are shown in Figs 1 and 2, respectively. In general, the thiobarbituric acid-reactive substances (TBARS) were lower in the olive oil-fed groups than the tallow-fed group. Dietary o-tocopherol supplementation significantly (p < 0.001) increased the stability of membranal fractions to lipid peroxidation.
D: Olive + 200 mg Vit. E/kg feed
C: Olive + 20 mg Vit. E/kg feed
20.33 i 3.53 24.87& 14.12
30-87 f 17.78 46.66 f 19.80
4.94 rfr2-3’7 6.01 f 3.62
5.99 f 1.30
155zt68
302 z+z 53
Thigh
10.94k2.03 14.12*3.15
2.07 rf:0.53
1.94rtO.48
59st20
94i 105
Thigh 4,661 I.70
11.00 If: 12-05 11.45* 1.45 1.51 irO.61
i-29 f 044
35i25
7O~t26
Breast
85h33
32.87 f 5.57 37.66rt 17.2
7.04zto.91
5.23& 1.18
222 * 49
293 * 45
Thigh
224*661
27.54 f 744 13.94 28.05 LIZ
3.49f0.76
3.33 f 0.70
113r?c31
183k37
Breast
Breast
9.52 f 7.25
1l-49 4 5.74
I+36* 040
I-6110.72
3?1t 13
88zt33
4.90 f 1.50
Thigh
7.43 4 3.45
0,85 f 0.30
1.33 i 0.23
Microsomes
24rtr9
Mitochondria
Microsomes
57k 17
Mitochondriu
Microsomes
Breast
Mitochondria
TABLE 4 of cr-tocopherol in Membranal Fractions from Breast and Thigh Muscle of Broilers Fed Different Dietary Oils and a-tocopherol Levels. Means * sd of 8 Analyses Performed in Duplicate ____-.pg Vit. Ejmg fat kg Vit. E/mg wet wt protein Muscle pg Vit. E[g wet wt of membrane
B: Tallow + 200 mg Vit. E/kg feed
A: Tallow + 20 mg Vit. E/kg feed
~. Treatments
Concentration
c-14:0 C-16:0 C-16:1 C-18:0 C-18:1 C-18:2 C-18:3 c-20:0 c-20:4 c-205 C-22:6 SAFA MLJFA PUFA SAFA/MUFA
0.9*0.4 17-O& I.0 1.3*0.2 IO.3 f 0.9 40.3 f 2.9 17.8ztO.7 1.5* 1.5 0.1 f0.1 5.8zt4.6 1.2kO.5 3.7hO.3 28.2 41.6 30.0 0.68
Breast
Breast I.1 *0.4 16.4zt 1.2 1.6 f 0.4 9.9Ito.7 42.5 zt 3.4 16.7zt1.6 2.3 f I.3 0.1 kO.1 5.Oh2.1 0.9*0.3 3.7 f 1.o 27.5 43.9 28.6 0.63
1.6kO.5 14.5+ I,5 I.6 f 0.4 10.1 f I.2 42.8 f 4.2 20.2k I.9 2.0*0.5 0.1 kO.1 4.2zk4.3 0.4zto.4 2.5+ I.4 26.2 44.4 29.3 0.60
_____
Thigh 2.0*0.1 18.9& I.0 2.9kO.7 9.4 f 0.7 44.5*4-3 17.7zt3.1 1.0+0.3 0.3kO.5 1.8 * 1.4 0.6 f 0.4 I.1 +0.6 30.5 47.3 22.2 0.64
Group 2
0.1 kO.1 5.ozto.4 l.OkO.5 9.4 f 2.2 59.2 f 6.5 19.8 f 2.8 0.5 f 0.4 0.2ztO.l 1.4*0.4 0.2~tO.3 3.1*0.9 14.5 60.2 25.2 0.24
Breast
Thigh 1.2zkO.6 12.5h2.9 0.9 * 0.5 7.1 zt 1.3 53.4zt4.2 19.7* I.1 1.8+ 1.2 0.1 zto.1 2.3xt I.6 0.0 0.9+0.6 20.8 54.3 24.7 0.38
Group 4
0.9 f 0.6 19.1 f 2.5 0.9Ito.5 12.9zt2.3 35.41t2.1 2l.Ozt3.1 1.0*0.5 0.2 zt 0.2 5.7 f I.9 0.3 f 0.6 1.7f I.7 33.1 36.7 29.7 0.90
in Duplicate
Thigh
Performed Group 3
0.1 kO.1 5.7 f 0.9 0.6kO.3 8.7k I.9 64.1 zk4.8 16.3 f I.9 0.2*0.2 0.1 l 0.1 1.6+0.7 0.1 *0.3 2.6 f 0.8 14.5 64.7 20.8 0.22
Breast
TABLE 5 (%) of Mitochondria. Means f sd of 8 Analyses
Thigh
Group I
Fatty Acid Composition
c-14:0 C-16:0 C-16:1 C-l 8:0 C-18:1 C-18:2 C-18:3 c-20:0 C-20:4 C-20:5 C-22:6 SAFA MUFA POLY SAFA/MUFA
2.7zt2.1 16.6*3.3 1.7 f 1.o 9.1 f 1.4 44.7 * 5.9 17.7*2.9 2.3 f 1.4 0.4 It 0.4 2.8* 1.7 1.7f 1.6 1.71t 1.6 28.4 46.4 25.1 0.61
Breast
Breast 1.8*0.8 19.4zk 1.8 2.1 zkO.8 9.8+ 1.2 40.1 It 3.2 17.91t2.6 1.3*0.6 0.3 zt 0.3 4.3+ 1.4 1.6* 1.3 1.61t 1.3 31.3 42.2 26.5 0.74
3.052.3 15.4*0.8 1.8+0.4 10.4*0.5 38.4k6.9 20.2 f 2.0 4.5 f 3.3 0.5 zt 0.4 2.3* 1.2 2.4 f 1.2 2.3 zt 1.2 29.3 40.2 30.8 0.73
1.6ikO.6 17.ozt 1.4 2.4 zt 0.7 11.6zk 1.3 37.0 zk2.3 22.41t 1.3 1.2ztO.4 0.4ztO.2 3.2zk 1.2 2.7k 1.7 2.7zk 1.7 30.6 39.4 30.1 0.78
Thigh
Performed
1.6kO.7 19.2zk3.7 0.8 f 0.7 12.4* 1.6 34.1 f 3.4 18.4s 1.0 2.5 zt 0.9 0.7 f 0.6 6.3 f 3.4 2.2h2.3 2.2k 1.0 33.2 34.9 31.1 0.95
1.8* 1.3 15.Oh2.0 l.OkO.2 11.8* 1.7 39.3 E!Z 4.8 23.8* 1.8 1.0*0.3 0.6kO.3 3.8zt 1.6 1.6ztO.9 1.6ztO.9 29.2 40.3 30.6 0.72
Thigh 1.2kO.7 16.7* 1.4 0.9 f 0.2 14.9*2.4 33.8 f 5.0 25.2 III 1.9 1.1 It 1.0 0.3zkO.l 3.0f 1.4 0.9 + 0.8 2.2k 1.3 33.1 34.7 32.2 0.95
Group 4 Breast
in Duplicate
Thigh
Group 3
2.7& 1.2 18.650.3 0.8* 1.1 11.6* 1.3 34.7 f 2.1 15.4xto.4 3.8& 1.2 2.8 f 0.9 9.7 f 5.4 0 0 35.7 35.5 28.9 1.0
Breast
TABLE 6 Means k sd of 8 Analyses
Group 2
(%) of Microsomes.
Thigh
Group I
Fatty Acid Composition
wtocopherol levels and fatty acid profiles in chicken
17
Fractions from chicks fed the tallow-low E diet (Group 1) showed the highest rate of lipid peroxidation at 80 and 120 min for thigh muscles and at 120 min for breast muscles. The mitochondrial fractions from both leg and breast muscle were more stable to ironinduced lipid oxidation for all treatments than microsomal fractions. Furthermore, oxidative changes appeared to be more extensive in subcellular membranes isolated from thigh muscles compared to membranes from breast muscles. TBARS 61
s
60 80 Time (min) Olive/High
E
Olive/low E
Tallow/High
E
Tallow/low E
TBARS 8,
6 51 4
EC
_
C I
0
20
40
Olive/High
E
60 80 Time (min) Olive/)ow E
Tallow/High
100
E
Tallow/low
120
140
E
Fig. 1. Iron-induced lipid oxidation in mitochondria from breast (upper) and thigh (lower) muscle of broilers fed different dietary oils and cY-tocopherol levels. *
C. Lauridsen, D. .I. Buckley, P. A. Morrissey
18
DISCUSSION In order to meet the high energy demand of the fast growing broilers, fat has been widely supplemented in poultry feed. However, it has been shown that the utilization of the fat by broilers depends on the fatty acid composition of the oil as well as the quality of the fat. Polyunsaturated fatty acids are more effectively absorbed than the more saturated TBARS 12
86-
60
80 Time (min)
Olive/High E
Olive/low E
Tallow/High E
TallowJ low E
TBARS 14 I 12 10 8 6
2 0
0
20
40
60
80
100
120
140
Time (min) Olive/High E
Olive(,ow E
TallowLHigh E
Tallow I low E
Fig. 2. Iron-induced lipid oxidation in microsomes from breast (upper) and thigh (lower) muscle of broilers fed different dietary oils and cr-tocopherol levels. *
a-tocopherol levels and fatty acid projiles in chicken
19
ones, but PUFA are also more susceptible to oxidation than the saturated ones. An increase in the degree of unsaturation of carcass fat has been related to a decrease in the oxidative stability of poultry meat (Marion & Woodroof, 1963; Bartov et al., 1974; Sklan et al., 1983). In this experiment, olive oil was compared with tallow as a dietary fat source in broiler feeds. Olive oil is high in oleic acid, a monounsaturated fatty acid. Oxidative attack is believed to occur most frequently in fatty acids containing two or more double bonds, it was not expected that major differences would be observed between the dietary fat treatments on the susceptibility to oxidation of membranes. Our results demonstrate that supplementation with 200mg vitamin E to poultry feed significantly stabilized the membranal fractions to oxidation compared to groups fed only 20 mg vitamin E/kg feed. However, the difference between Groups 1 and 3 regarding the oxidative stability of membranal fractinns (Figs 1 and 2) could be related to the slightly higher content of vitamin E per mg fatty acids (Tables 4 and 5) in the group fed olive oil. Olive oil contains a higher natural content of a-tocopherol (as shown in Table 2) than tallow. Thus, it is likely that the content of vitamin E in membranes of Group 1 is consumed rapidly, which results in a more accelerated increase in TBARS numbers than in Group 3. One could say that the lag period, defined as the time prior to onset of maximal TBARS, is shorter due to the low concentrations of vitamin E. Hare1 and Kanner (1985) reported that the addition of low concentrations (1 to 10 PM) of a-tocopherol can inhibit in-vitro microsomal lipid oxidation initiated by hydrogenperoxide activated metmyoglobin, which means that relatively small differences of membranal vitamin E concentrations may cause different TBARS responses. In agreement with the results of Asghar et al. (1989), the subcellular fractions isolated from the thigh muscles seemed to be more susceptible to oxidation compared to membranes of breast muscles. Asghar et al. (1989) proposed that this effect is due to the differences in lipid content between the two tissues. There is evidence that more active muscles contain a greater quantity of phospholipids (Acosta et al., 1966) which are characterized by high levels of polyunsaturated fatty acids. However, Katz et al. (1966) showed that dark meat (leg) from chickens contains only about half as much phospholipids as white meat (breast). Previous studies with pigs (Monahan et al., 1993) and chickens (Asghar et al., 1989) showed that dietary a-tocopherol supplementation significantly increases the cr-tocopherol content of muscle membranes. In the present study, supplementation of the diet with a-tocopherol increased the a-tocopherol concentration in both mitochondria and microsomes, which is in contrast to Asghar et al. (1990) who found that mitochondria of white and dark meat as well as microsomes of white meat of cY-tocopherol-supplemented broilers did not accumulate vitamin E in any significant amount. Lin et al. (1989) proposed that the higher concentrations of a-tocopherol in thigh muscle compared with breast muscle may be associated with the more highly developed vascular system and higher lipid content of thigh muscle. It has also been demonstrated in beef that the accumulation of cY-tocopherol is muscle-dependent (Arnold et al., 1992, 1993). There are still no clear explanations why skeletal muscles differ in o-tocopherol concentration. Being a lipid soluble vitamin, o-tocopherol partitions into biomembranes, and its concentration increases with dietary supplementation. Thus, the higher lipid content in red muscles compared to white muscles is the likely explanation for the higher content of a-tocopherol in red muscle. Evarts and Bieri (1974) showed that the relative a-tocopherol content expressed as a percent of cY-tocopherol content divided by percent of total protein revealed that a-tocopherol distribution among various subcellular fractions is distinctly different from the protein distribution. As can be seen from Table 4, the content of ar-tocopherol from membranes of red meat is still higher than from white meat membranes even when the
20
C. Lauridsen, D. J. Buckley, P. A. Morrissey
content is corrected for the lipid content of the membrane. One reasonable factor could be the number and size of membranes, which differ between different muscles (Porter & Palade, 1957). More and smaller mitochondria may provide more membrane volume for potential retention of a-tocopherol as different fiber sizes could affect membrane volume. Another factor could be the greater capillary blood supply to red muscle fibers than to white fibers, which affects the a-tocopherol accumulations. Sosnicki et al. (1991) observed that the number of capillaries in turkey femoris were doubled compared with those of pectoralis muscle. Previous studies showed that the nature of dietary oils is reflected in the fatty acid composition of neutral lipids of membranes and, to a lesser degree, in fatty acid composition of the phospholipids (Asghar et al., 1990; Lin et al., 1989) In the present study we did not extract neutral and polar lipids separately, which may account for the reduced effects of dietary fatty acids. Based on Table 3 we expected more Ci6:e and CisO and less Cis:i, eventually C1sZ2,in membranes of tallow-groups compared to membranes of oliveoil fed groups. This was partly true for the mitochondrial fractions (Table 5), but the result was unexpected for the microsomal fraction (Table 6), e.g. the content of Cis:s was lower and the content of Cis:i higher in groups fed tallow compared to olive oil. This could probably be related to the feed, but differences in fatness of the chicken may influence the fatty acid composition of microsomes. The standard deviation was extremely high especially for fatty acids present in low concentrations. In conclusion, the relationship between saturated and monounsaturated fatty acid appeared to be influenced by dietary fatty acids to a higher degree in mitochondria than in microsomes. Studies have reported that dietary oil has no apparent influence on the deposition of CXtocopherol in the membranal fractions (Asghar et al., 1989; Yamauchi et al., 1991). However, in the study of Lin et al. (1990), the concentration of o-tocopherol in microsomes of dark meat was to some extent influenced by the nature of the dietary oil. In the present study we also found some influence of dietary vitamin E on the occurrence of single fatty acids in the membranal fractions, but no clear pattern was obtained. In conclusion, dietary cu-tocopherol acetate supplementation increased the antioxidant status of muscle. Muscle a-tocopherol levels appear to be a dominant factor in determining oxidative stability of muscle membranes rather than the fatty acid composition.
ACKNOWLEDGEMENTS The authors are grateful to the Commission of the European Communities for the financial support of this work. For providing analysis of vitamin E in diets Dr Ssren Krogh Jensen is gratefully acknowledged. REFERENCES Acosta, S. O., Marion, W. W. and Forsythe, R. H. (1966) Total lipids and phosphohpids in turkey tissues. Poultry Science 45, 169-184. AOAC (1984) Ojficial methods of analysis 14th edn: Association of Official Analytical Chemists, Washington D.C. Arnold, R. N., Arp, S. C., Scheller, K. K., Williams, S. N. and Schaefer, D. M. (1992) Tissue equilibration and subcellular distribution of vitamin E relative to myoglobin and lipid oxidation in displayed beef. Journal of Animal Science 71, 105-l 18. Arnold, R. N., Arp, S. C., Scheller, K. K., Williams, S. N. and Schaefer, D. M. (1993) Dietary cr-tocopheryl acetate enhances beef quality in Holstein and beef breed steers. Journal of Food Science 58, 28-33.
a-tocopherol levels and fatty acid profiles in chicken
21
Asghar, A., Lin, C. F., Gray, J. I., Buckley, D. J., Booren, A. M., Crakel, R. L. and Flegal, C. J. (1989) Influence of oxidised dietary oil and antioxidant supplementation on membrane-bound lipid stability in broiler meat. British Poultry Science 30, 815-823. Asghar, A., Lin, C. F., Buckley, D. J., Booren, A. M. and Flegal, C. J. (1990) Effect of dietary oils and a-tocopherol supplementation on membranal lipid oxidation in broiler meat. Journal of Food Science 55, 46-50 + 118. Bartov, I., Bornstein, S. and Lipstein, B. (1974) Effect of calorie to protein ratio on the degree of fatness in broilers fed on practical diets. British Poultry Science 15, 107-l 17. Buege, J. A. and Aust, S. D. (1978) Microsomal lipid peroxidation. Methods in Enzymology 105, 131-138. Burton, G. W., Webb, A. and Ingold, K. U. (1985) A mild, rapid, and efficient method of lipid extraction for use in determining vitamin E/lipid ratios. Lipids 20, 29-39. Buttriss, J. L. and Diplock, A. T. (1984) High-performance liquid chromatography methods for vitamin E in tissues. Methods in Enzymology 105, 13 l-l 38. Borsting, C. F., Engberg, R. M., Jakobsen, K., Jensen, S. K. and Andersen, J. 0. (1994) Inclusion of oxidized fish oil in mink diets. 1. The influence on nutrient digestibility and fatty-acid accumulation in tissues. Journal of Animal Physiology and Animal Nutrition 72, 132-145. Christophersen, S. W. and Glass, R. L. (1969) Preparation of milk fat methyl esters by alcholysis in an essentially nonalcoholic solution. Journal of Dairy Science 52, 1289-l 290. Evarts, R. P. and Bieri, J. G. (1974) Ratios of polyunsaturated fatty acids to alpha-tocopherol in tissues of rats fed corn or soybean oils. Lipids 9, 86&864. Gray, J. I. and Pearson, A. M. (1987) Rancidity and warmed-over flavor. Advances in Meat Research 3, 221-269.
Harel, S. and Kanner, J. (1985) Muscle membranal lipid peroxidation initiated by hydrogen peroxide-activated metmyoglobin. Journal of Agricultural and Food Chemistry 33, 1188-I 192. Igene, J. O., Pearson, A. M., Dugan, L. R. Jr. and Price, J. F. (1980) Role of triglycerides and phospholipids in development of rancidity in model meat systems during frozen storage. Food Chemistry 5, 263-276.
Katz, M. A., Dugan, L. R. Jr. and Dawson, L. E. (1966) Fatty acids in neutral lipids and phospholipids from chicken tissues. Journal of Food Science 31, 7 17-720. Kornbrust, D. J. and Mavis, R. D. (1980) Relative susceptibility of microsomes from lung, heart liver, kidney, brain and testes to lipid peroxidation: Correlation with vitamin E content, Lipid.7 15, 3 1S-322. Lin, C. F., Gray, J. I., Asghar, A., Buckley, D. J., Booren, A. M. and Flegal, C. J. (1989) Effects of dietary oils and cr-tocopherol supplementation on lipid composition and stability of broiler meat. Journal of Food Science 54, 1457-1460+ 1484. Machlin, L. J. (1984) Vitamin E. In Handbook of Vitamins. Nutritional, Biochemical, and Clinical Aspects. ed. L. J. Machlin, pp. 99-145. Marcel Dekker, New York. Marion, J. E. and Woodroof, J. G. (1963) The fatty acid composition of breast thigh, and skin tissues of chicken broilers as influenced by dietary fats. Poultry Science 42, 120221211. Monahan, F. J., Gray, J. I., Asghar, A., Haug, A., Shi, B., Buckley, D. J. and Morrissey, P. A. (1993) Effect of dietary lipid and vitamin E supplementation on free radical production and lipid oxidation in porcine muscle microsomal fractions. Food Chemistry 46, 1-6. Pearson, A. M., Gray, J. I., Wolzak, A. M. and Horenstein, N. A. (1983) Safety implications of oxidized lipids in muscle foods. Food Technology 37, 121-I 29. Pikul, J., Lesczynski, D. E., Bechtel, P. J. and Kummerow, F. A. (1984) Effects of frozen storage and cooking on lipid oxidation in chicken meat. Journal of Food Science 49, 838-843. Porter, K. R. and Palade, G. E. (1957) Studies on the endoplasic reticulum III. Its form and distribution in striated muscle cells. Journal of Biophysical and Biochemical Cytology 3, 269-299. SAS (1995) In Users guide: Statistics. SASR Institute Inc., Cary, NC 27513, USA. Sheehy, P. J. A., Morrissey, P. A. and Flynn, A. (1991) Influence of dietary cr-tocopherol on tocopherol concentrations in chick tissues. British Poultry Science 32, 391-397. Sklan, D., Tenne, E. and Budowski, P. (1983) The effect of dietary fat and tocopherol on lipolysis and oxidation in turkey meat stored at different temperatures. Poultry Science 62, 2017-202 I. Sosnicki, A. A., Cassens, R. G., Vimini, R. J. and Greaser, M. L. (1991) Distribution of capillaries in normal and ischemic turky skeletal muscle. Poultry Science 70, 343-348.
22
C. Lauridsen, D. J. Buckley, P. A. Morrissey
Stoldt, W. (1952) Vorslag zur Vereinheitlichung
der Fettbestimmung
in Lebensmitteln.
Fette, Seifen,
Anstrichmittel54, 206-20’7.
Tahin, Q. S., Blum, M. and Carafoli, E. (1981) The fatty acid composition of subcellular membranes of rat liver, heart, and brain: Diet-induced modifications. European Journal of Biochemistry 122, S-13.
Yamauchi, K., Murata, M., Ohashi, T., Katayama, H., Pearson, A. M., Okada, T. and Yamakura, T. (1991) Effect of dietary cr-tocopherol supplementation on the molar ratio of polyunsaturated fatty acids/a-tocopherol in broiler skeletal muscles and subcellular membranes and its relationship to oxidative stability. Nippon Shokuhin Kogyo Gakkaishi 38, 545-552.
s0309-1740(97)00010-7
Meat Science, Vol. 46, No. I, 9-22, 1997 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0309-1740197 $17.oo+o.ocl
ELSEVIER
Influence of Dietary Fat and Vitamin E Supplementation on cw-Tocopherol Levels and Fatty Acid Profiles in Chicken Muscle Membranal Fractions and on Susceptibility to Lipid Peroxidation C. Lauridsen,a* D. J. Buckleyb & P. A. Morrissey” UDepartment of Nutrition, University College Cork, Republic of Ireland bDepartment of Food Technology, University College Cork, Republic of Ireland (Received 3 November 1996; revised version received 3 January 1997; accepted 3 January 1997) ABSTRACT Broiler chickens were fed a basal feed supplemented with 10% tallow or olive oil and varying levels of vitamin E (20 and 200 mg vitamin E/kg feed). The concentration of IXtocopherol in the membranes of breast and thigh muscles was significantly influenced by the a-tocopherol level in the feed (p < 0.001). Deposition of vitamin E was not injuenced by the type of oil in the feed, except in the mitochondrial fraction of breast where the vitamin E concentration was higher in those fed olive oil than in those fed tallow (p <:0.05). Dietary oil injluenced the fatty acid composition of the muscle membranal fractions (p< 0.001). The oxidative stability of the membranal fractions tended to increase with increasing concentrations of u-tocopherol in the membranal fractions. In conclusion, the supplementation of vitamin E appeared to enhance the stabihty of muscle to oxidation. Thus, incorporation of a-tocopherol into the membranes via dietary manipulation helped in stabilizing the membrane-bound lipids. e 1997 Elsevier Science Ltd
INTRODUCTION Lipid oxidation is considered to be the major cause of quality deterioration in meat and meat products during refrigerated storage (Pearson et al., 1983). Lipid oxidation is primarily initiated in the highly unsaturated fatty acid components of membrane phospholipids (Gray & Pearson, 1987). Stabilization of membrane lipids by dietary supplementation with vitamin E has been shown to prevent the onset of lipid oxidation in chicken (Yamauchi et al., 1991) and pork muscle (Monahan et al., 1993). While vitamin E is widely distributed in tissues, it is primarily located within biological membranes, such as microsomes and mitochondria (Machlin, 1984). Localization of ar-tocopherol in the
membranes allows it to function very efficiently compared to other antioxidants. Diets for fast growing broilers are generally rich in polyunsaturated fatty acids and the degree of unsaturation in carcass fat is thereby increased. Furthermore, there is some *To whom correspondence should be addressed at: Department for Product Quality, Danish Institute of Animal Science, Research Centre Foulum, PO Box 39, DK-8830 Tjele, Denmark. 9
C. Lauridsen, D. J. Buckley, P. A. Morrissey
10
evidence that dietary oils can alter the fatty acid composition of mitochondrial and microsomal lipids (Tahin et al., 1981; Asghar et al., 1990). However, increasing the degree of unsaturation of the muscle membranes by dietary manipulation increases the peroxidizability of tissues and muscle foods (Igene et al., 1980; Pikul et al., 1984). Thus, there is a need to increase the antioxidant capacity of the tissues, and this is readily achieved by feeding a-tocopheryl acetate. Unfortunately, there is little quantitative information available on the effects of feeding different levels of vitamin E and different types of fat (varying in degree of unsaturation) on the concentration of a-tocopherol in the subcellular membranal fractions and their influence on the susceptibility to lipid peroxidation. The objective of the present study was to determine the effect of increasing the degree of monounsaturation of dietary oil and supplementation with cu-tocopherol on the deposition of a-tocopherol, the fatty acid composition, and the oxidative stability of membranal fractions of broiler dark and white meat.
MATERIALS
AND METHODS
Animals and diets
Sixty one-day old chicks were obtained from a commercial hatchery and housed in an environmentally controlled room. Chicks were randomly assigned to four groups and fed a basal diet (Table 1) supplemented with fat and a-tocopheryl acetate as follows: Group Group Group Group
1: 2: 3: 4:
Tallow, 10% + o-tocopheryl acetate, 20 mg/kg feed Tallow, 10% + a-tocopheryl acetate, 200mg/kg feed Olive oil, 10% + cr-tocopheryl acetate, 20mg/kg feed Olive oil, 10% + cr-tocopheryl acetate, 200mg/kg feed
Groups were housed separately in raised wire cages with six birds per cage in an environmentally-controlled room employing a 12 hr light/dark cycle. Feed and water was provided ad libitum. After 42 days, eight chicks from each group were selected at random and killed by cervical dislocation. Breast and thigh muscles were removed, vacuum packed and quickly frozen and stored at -20°C until analyzed. TABLE 1
Composition of Broiler Diets Ingredients
Ground maize Soybean meal Tallow/olive oil Limestone Dicalcium phosphate DL-Methionine NaCl Mineral mixa Vitamin mixb
% of diet
46.56 39.58 10 1 2 0.16 0.40 0.10 0.20
Chemical analysis
DM, g/kg feed Stoldt fat Crude protein (Nx6,25) Sugar Starch ME (MJ/kg DM)
glkg DM
906.5 137.3 259.6 57.9 347.9 15.15
“Containing per kg feed: MnS04, 4H20 333.3 mg; ZnSOz, 7H20 220.3mg; C6HJ07Fe, 3Hz0 450mg; CuS04, 5H20 35 mg; KI03 2mg; CoSO4 5HzO 1mg; Na2Se03 0.35mg. ‘Containing per kg feed: A/D3 6mg; K 2mg; Riboflavin 5mg; Ca panthotenate 15 mg; Niacin 30 mg; B12 0.015 mg; Folic acid 0.6mg; Pyridoxine 3.5 mg; Thiamine 2 mg; Choline chloride 12g; Biotin 0.2 mg; a-tocopheryl acetate 20 or 200 mg.
a-tocopherol levels and fatty acid projles in chicken
11
The chemical composition of the basal diets was determined by the AOAC. procedure (1984) except for crude fat, which was determined by the method described by Stoldt (1952). Isolation of mitochondrial
and microsomal fractions from muscles
Separation of muscle membranal fractions was carried out by a modification of the method by Asghar et al. (1990) as follows. A 50g sample of breast- and thigh muscle, respectively, of each chicken was homogenized with 500 ml of deaerated, nitrogen-saturated buffer solution (0.1 M KCl, 0.2% glycerol, 0.025M Tris-HCl) in a Warring Blender for 2min and centrifuged at 1OOOxgfor 10min in a Beckmann centrifuge. The supernatant was filtered through cheesecloth to remove material in the suspension and was then subjected to differential centrifugation to separate the mitochondrial and microsomal fractions. The mitochondrial extracts were sedimented at 15 OOOxg for 15 min. Calcium chloride was added to the sup rnatant to give a final concentration of 8mM for aggregating the microsomes. The mixture was centrifuged at 13 2OOxg for 30min to separate the microsomal fraction. The subcellular fractions were not purified further. Vitamin E analysis
Tocopherol was extracted from the feed after saponification (Bursting et al., 1994). Muscle ol-tocbpherol was extracted with hexane after saponification at 70°C x 30 min (Buttriss & Diplock, 1984) and determined by reverse-phase High Performance Liquid Chromatography (HPLC) as described by Sheehy et al. (1991). Fatty acid analysis
The fatty acid profiles of diets were determined by capillary gas chromatography after lipid extraction (Burton et al., 1985) and esterification (Christophersen & Glass, 1969). The fatty acid methyl esters were analysed by gas-liquid chromatography using a PyeUnicam PU 4500 gas chromatography equipped with a Machery-Nagel Fused Silica Capillary (25mx0.25mm i.d.) column. The oven temperature was held at 50°C for 3.5min, and increased by 3°C per min to a final temperature of 220°C which was maintained for 18min. Injector and detector temperatures were 250°C. A Shimadzy C-R3A integrator was used for calculation of peak areas. Fatty acid methyl esters were identified by comparison of retention times with those of authentic standards. The fatty acid profiles of samples were quantified following addition of an internal standard to each sample. Thioharbituric
acid-reactive substances
The lability of muscle membranal fractions to iron-stimulated lipid peroxidation was determined by a modification of the method of Kornbrust and Mavis (1980). Membranal fractions were incubated in 40mM tris-maleate buffer (pH 7.4) with 1 mM FeS04 in a total volume of 1 ml. At fixed time intervals aliquots were removed for the measurement of 2-thiobarbituric acid reacting substances (TBARS) bv the method of Buege and Aust (1978). Statistical
analysis
Statistical analyses were performed by means of analysis of variance using the general linear models (GLM) procedure of SAS (1995) with the concentration of vitamin E and
C. Lauridsen, D. J. Buckley, P. A. Morrissey
12
TABLE 2 Concentration of Vitamin E in Diets (added and analysed values) Added Diet
Analysed values
dl-cr-tocopherol acetate” (mglkg)
d-u-tocopherol
d-y-tocopherol
20 200 20 200
34 260 57 272
18 17 21 20
Tallow Tallow Olive oil Olive oil
“Provided by Hoffmann La Roche, a/s.
dietary fat and the interaction between the concentration of vitamin E and dietary fat as independent variables. Where significant variance ratios were detected, differences between treatment means were tested using the least significant difference (LSD.) procedure of SAS. Where data showed variance in-homogeneity, they were treated using the proc MIXED procedure of SAS package version 6.11 (SASR Institute, 1995) after the following model: Xv
=
(lli +
Eg, Eg
-N(O,$),i=
1,2 and j=
1,2
RESULTS Vitamin E in feeds The o-tocopherol concentration in the diets is shown in Table 2. The levels were higher in the diets containing olive oil compared to those containing tallow. The fatty acid profiles of feed samples are shown in Table 3. The diets containing tallow had 57.6% saturated, 32.0% monounsaturated, and 10.4% polyunsaturated fatty acids, whereas those with olive oil had 13.3% saturated, 70.4% monounsaturated, and 16.3% polyunsaturated fatty acids. Supplementation of vitamin E had no influence on the fatty acid composition of the feed. TABLE 3 Fatty Acid Composition (%) of Diets Containing Tallow or Olive Oil. Meanshsd
of 2 Analyses
Performed in Duplicate Fatty acid
14:o 16:O 16:l 18:O 18:l 18:2 18:3 Others
Tall0 w
Olive oil
Mean f sd
Mean f sd
4.6zkO.2 28.4 f 0.6 1.1 io.04 24.3 f 0.9 30.9 f 1.5 8.2 zt 0.4 1.3*0,04 1.21kO.8
0*3*0.1 12.2zko.2 0.8 i 0.04 0.6 f 0.2 69.6ztO.S 14.8hO.l 0.8 f 0.04 0.9 f 0.4
a-tocopherol levels andfatty acid profiles in chicken
13
Membranal fractions In general, the dietary treatments had no influence on the weight of the membranal fractions isolated from chicken breast @ > 0.05). However, a significant influence of dietary oil (p < 0.05) was observed on the weight of mitochondria of dark muscles, i.e. mitochondria of broilers fed tallow weighed 77 mg/g tissue versus 63 mg/g of tissue for broilers fed olive oil. Significant interactions between dietary oil and vitamin E on the mitochondrial (p < 0.05) and on the microsomal (p < 0.001) weight of dark muscles were found. No reasonable explanations are available for these observations. The average wet wt (mg/g of tissue) of mitochondrial fraction was 63.0 f 10.9mg and 69.8 & 17.0mg for breast and thigh muscles, respectively, and for microsomal fractions 70.6 f 22.3 and 78.3 f 24.1 for breast and thigh muscles, respectively. Table 4 shows the concentration of u-tocopherol in membranal fractions expressed on the basis of membrane weight, protein and total fat content. Supplementation with otocopherol had a significant influence @ < 0.001) on the concentration of a-tocopherol in the membranes of breast and thigh muscles. When a-tocopherol concentrations were expressed on a total lipid basis, the levels in the mitochondria and microsomes from breast muscle were lower than those observed for thigh muscle. These differences were most pronounced when the values were expressed on a wet wt basis. The membranal a-tocopherol concentrations were not influenced by the type of oil (JJ> O.OS), although significant interactions between vitamin E and oil were observed on the concentration of a-tocopherol per wet wt of microsomes (p < 0.01) as well as the concentration of cr-tocopherol per mg total fat in mitochondria @ < 0.01) and microsomes (p < 0.01) of dark muscles. Furthermore, a significant influence @ < 0.05) was observed on vitamin E expressed in relation to the content of protein in the microsomal fraction of breast muscle. The fatty acid composition of the muscle membranal fractions are shown in Tables 5 and 6. Dietary oil influenced the fatty acid composition of the muscle membranal fractions. In general, the concentration of saturated fatty acids (Ci4:e and C,& and Ci6:i was higher, and the concentration of monounsaturated (Cis:i) lower, in the mitochondrial fraction from broilers fed tallow compared to broilers fed olive oil. However, the content of C16:0in mitochondria of group 3 (olive oil + low a-tocopheryl acetate) was remarkably low, as was the content of C,s:i in the breast of group 4. The Cis:s content in mitochondria of groups fed tallow was slightly higher than in the olive oil fed groups. Overall, the relationship between saturated fatty acids and monounsaturated fatty acids of mitochondrial fractions was influenced by the dietary treatments, although the variation between groups was very high for groups fed olive oil. The data on the fatty acid profiles of the microsomes were unexpected when compared group-wise. The content of Ci6:, was higher, and the Cis:e lower, in groups fed tallow compared to groups fed olive oil. The content of Cis:i in microsom s of breast from broilers fed tallow was higher and the content of C20:4lower when compared to broilers fed olive oil. Oxidative stability of membrane-bound lipids Data on the rates of iron-induced lipid-peroxidation in the mitochondria and microsomes isolated from breast and thigh muscles from the four groups of broilers are shown in Figs 1 and 2, respectively. In general, the thiobarbituric acid-reactive substances (TBARS) were lower in the olive oil-fed groups than the tallow-fed group. Dietary o-tocopherol supplementation significantly (p < 0.001) increased the stability of membranal fractions to lipid peroxidation.
D: Olive + 200 mg Vit. E/kg feed
C: Olive + 20 mg Vit. E/kg feed
20.33 i 3.53 24.87& 14.12
30-87 f 17.78 46.66 f 19.80
4.94 rfr2-3’7 6.01 f 3.62
5.99 f 1.30
155zt68
302 z+z 53
Thigh
10.94k2.03 14.12*3.15
2.07 rf:0.53
1.94rtO.48
59st20
94i 105
Thigh 4,661 I.70
11.00 If: 12-05 11.45* 1.45 1.51 irO.61
i-29 f 044
35i25
7O~t26
Breast
85h33
32.87 f 5.57 37.66rt 17.2
7.04zto.91
5.23& 1.18
222 * 49
293 * 45
Thigh
224*661
27.54 f 744 13.94 28.05 LIZ
3.49f0.76
3.33 f 0.70
113r?c31
183k37
Breast
Breast
9.52 f 7.25
1l-49 4 5.74
I+36* 040
I-6110.72
3?1t 13
88zt33
4.90 f 1.50
Thigh
7.43 4 3.45
0,85 f 0.30
1.33 i 0.23
Microsomes
24rtr9
Mitochondria
Microsomes
57k 17
Mitochondriu
Microsomes
Breast
Mitochondria
TABLE 4 of cr-tocopherol in Membranal Fractions from Breast and Thigh Muscle of Broilers Fed Different Dietary Oils and a-tocopherol Levels. Means * sd of 8 Analyses Performed in Duplicate ____-.pg Vit. Ejmg fat kg Vit. E/mg wet wt protein Muscle pg Vit. E[g wet wt of membrane
B: Tallow + 200 mg Vit. E/kg feed
A: Tallow + 20 mg Vit. E/kg feed
~. Treatments
Concentration
c-14:0 C-16:0 C-16:1 C-18:0 C-18:1 C-18:2 C-18:3 c-20:0 c-20:4 c-205 C-22:6 SAFA MLJFA PUFA SAFA/MUFA
0.9*0.4 17-O& I.0 1.3*0.2 IO.3 f 0.9 40.3 f 2.9 17.8ztO.7 1.5* 1.5 0.1 f0.1 5.8zt4.6 1.2kO.5 3.7hO.3 28.2 41.6 30.0 0.68
Breast
Breast I.1 *0.4 16.4zt 1.2 1.6 f 0.4 9.9Ito.7 42.5 zt 3.4 16.7zt1.6 2.3 f I.3 0.1 kO.1 5.Oh2.1 0.9*0.3 3.7 f 1.o 27.5 43.9 28.6 0.63
1.6kO.5 14.5+ I,5 I.6 f 0.4 10.1 f I.2 42.8 f 4.2 20.2k I.9 2.0*0.5 0.1 kO.1 4.2zk4.3 0.4zto.4 2.5+ I.4 26.2 44.4 29.3 0.60
_____
Thigh 2.0*0.1 18.9& I.0 2.9kO.7 9.4 f 0.7 44.5*4-3 17.7zt3.1 1.0+0.3 0.3kO.5 1.8 * 1.4 0.6 f 0.4 I.1 +0.6 30.5 47.3 22.2 0.64
Group 2
0.1 kO.1 5.ozto.4 l.OkO.5 9.4 f 2.2 59.2 f 6.5 19.8 f 2.8 0.5 f 0.4 0.2ztO.l 1.4*0.4 0.2~tO.3 3.1*0.9 14.5 60.2 25.2 0.24
Breast
Thigh 1.2zkO.6 12.5h2.9 0.9 * 0.5 7.1 zt 1.3 53.4zt4.2 19.7* I.1 1.8+ 1.2 0.1 zto.1 2.3xt I.6 0.0 0.9+0.6 20.8 54.3 24.7 0.38
Group 4
0.9 f 0.6 19.1 f 2.5 0.9Ito.5 12.9zt2.3 35.41t2.1 2l.Ozt3.1 1.0*0.5 0.2 zt 0.2 5.7 f I.9 0.3 f 0.6 1.7f I.7 33.1 36.7 29.7 0.90
in Duplicate
Thigh
Performed Group 3
0.1 kO.1 5.7 f 0.9 0.6kO.3 8.7k I.9 64.1 zk4.8 16.3 f I.9 0.2*0.2 0.1 l 0.1 1.6+0.7 0.1 *0.3 2.6 f 0.8 14.5 64.7 20.8 0.22
Breast
TABLE 5 (%) of Mitochondria. Means f sd of 8 Analyses
Thigh
Group I
Fatty Acid Composition
c-14:0 C-16:0 C-16:1 C-l 8:0 C-18:1 C-18:2 C-18:3 c-20:0 C-20:4 C-20:5 C-22:6 SAFA MUFA POLY SAFA/MUFA
2.7zt2.1 16.6*3.3 1.7 f 1.o 9.1 f 1.4 44.7 * 5.9 17.7*2.9 2.3 f 1.4 0.4 It 0.4 2.8* 1.7 1.7f 1.6 1.71t 1.6 28.4 46.4 25.1 0.61
Breast
Breast 1.8*0.8 19.4zk 1.8 2.1 zkO.8 9.8+ 1.2 40.1 It 3.2 17.91t2.6 1.3*0.6 0.3 zt 0.3 4.3+ 1.4 1.6* 1.3 1.61t 1.3 31.3 42.2 26.5 0.74
3.052.3 15.4*0.8 1.8+0.4 10.4*0.5 38.4k6.9 20.2 f 2.0 4.5 f 3.3 0.5 zt 0.4 2.3* 1.2 2.4 f 1.2 2.3 zt 1.2 29.3 40.2 30.8 0.73
1.6ikO.6 17.ozt 1.4 2.4 zt 0.7 11.6zk 1.3 37.0 zk2.3 22.41t 1.3 1.2ztO.4 0.4ztO.2 3.2zk 1.2 2.7k 1.7 2.7zk 1.7 30.6 39.4 30.1 0.78
Thigh
Performed
1.6kO.7 19.2zk3.7 0.8 f 0.7 12.4* 1.6 34.1 f 3.4 18.4s 1.0 2.5 zt 0.9 0.7 f 0.6 6.3 f 3.4 2.2h2.3 2.2k 1.0 33.2 34.9 31.1 0.95
1.8* 1.3 15.Oh2.0 l.OkO.2 11.8* 1.7 39.3 E!Z 4.8 23.8* 1.8 1.0*0.3 0.6kO.3 3.8zt 1.6 1.6ztO.9 1.6ztO.9 29.2 40.3 30.6 0.72
Thigh 1.2kO.7 16.7* 1.4 0.9 f 0.2 14.9*2.4 33.8 f 5.0 25.2 III 1.9 1.1 It 1.0 0.3zkO.l 3.0f 1.4 0.9 + 0.8 2.2k 1.3 33.1 34.7 32.2 0.95
Group 4 Breast
in Duplicate
Thigh
Group 3
2.7& 1.2 18.650.3 0.8* 1.1 11.6* 1.3 34.7 f 2.1 15.4xto.4 3.8& 1.2 2.8 f 0.9 9.7 f 5.4 0 0 35.7 35.5 28.9 1.0
Breast
TABLE 6 Means k sd of 8 Analyses
Group 2
(%) of Microsomes.
Thigh
Group I
Fatty Acid Composition
wtocopherol levels and fatty acid profiles in chicken
17
Fractions from chicks fed the tallow-low E diet (Group 1) showed the highest rate of lipid peroxidation at 80 and 120 min for thigh muscles and at 120 min for breast muscles. The mitochondrial fractions from both leg and breast muscle were more stable to ironinduced lipid oxidation for all treatments than microsomal fractions. Furthermore, oxidative changes appeared to be more extensive in subcellular membranes isolated from thigh muscles compared to membranes from breast muscles. TBARS 61
s
60 80 Time (min) Olive/High
E
Olive/low E
Tallow/High
E
Tallow/low E
TBARS 8,
6 51 4
EC
_
C I
0
20
40
Olive/High
E
60 80 Time (min) Olive/)ow E
Tallow/High
100
E
Tallow/low
120
140
E
Fig. 1. Iron-induced lipid oxidation in mitochondria from breast (upper) and thigh (lower) muscle of broilers fed different dietary oils and cY-tocopherol levels. *
C. Lauridsen, D. .I. Buckley, P. A. Morrissey
18
DISCUSSION In order to meet the high energy demand of the fast growing broilers, fat has been widely supplemented in poultry feed. However, it has been shown that the utilization of the fat by broilers depends on the fatty acid composition of the oil as well as the quality of the fat. Polyunsaturated fatty acids are more effectively absorbed than the more saturated TBARS 12
86-
60
80 Time (min)
Olive/High E
Olive/low E
Tallow/High E
TallowJ low E
TBARS 14 I 12 10 8 6
2 0
0
20
40
60
80
100
120
140
Time (min) Olive/High E
Olive(,ow E
TallowLHigh E
Tallow I low E
Fig. 2. Iron-induced lipid oxidation in microsomes from breast (upper) and thigh (lower) muscle of broilers fed different dietary oils and cr-tocopherol levels. *
a-tocopherol levels and fatty acid projiles in chicken
19
ones, but PUFA are also more susceptible to oxidation than the saturated ones. An increase in the degree of unsaturation of carcass fat has been related to a decrease in the oxidative stability of poultry meat (Marion & Woodroof, 1963; Bartov et al., 1974; Sklan et al., 1983). In this experiment, olive oil was compared with tallow as a dietary fat source in broiler feeds. Olive oil is high in oleic acid, a monounsaturated fatty acid. Oxidative attack is believed to occur most frequently in fatty acids containing two or more double bonds, it was not expected that major differences would be observed between the dietary fat treatments on the susceptibility to oxidation of membranes. Our results demonstrate that supplementation with 200mg vitamin E to poultry feed significantly stabilized the membranal fractions to oxidation compared to groups fed only 20 mg vitamin E/kg feed. However, the difference between Groups 1 and 3 regarding the oxidative stability of membranal fractinns (Figs 1 and 2) could be related to the slightly higher content of vitamin E per mg fatty acids (Tables 4 and 5) in the group fed olive oil. Olive oil contains a higher natural content of a-tocopherol (as shown in Table 2) than tallow. Thus, it is likely that the content of vitamin E in membranes of Group 1 is consumed rapidly, which results in a more accelerated increase in TBARS numbers than in Group 3. One could say that the lag period, defined as the time prior to onset of maximal TBARS, is shorter due to the low concentrations of vitamin E. Hare1 and Kanner (1985) reported that the addition of low concentrations (1 to 10 PM) of a-tocopherol can inhibit in-vitro microsomal lipid oxidation initiated by hydrogenperoxide activated metmyoglobin, which means that relatively small differences of membranal vitamin E concentrations may cause different TBARS responses. In agreement with the results of Asghar et al. (1989), the subcellular fractions isolated from the thigh muscles seemed to be more susceptible to oxidation compared to membranes of breast muscles. Asghar et al. (1989) proposed that this effect is due to the differences in lipid content between the two tissues. There is evidence that more active muscles contain a greater quantity of phospholipids (Acosta et al., 1966) which are characterized by high levels of polyunsaturated fatty acids. However, Katz et al. (1966) showed that dark meat (leg) from chickens contains only about half as much phospholipids as white meat (breast). Previous studies with pigs (Monahan et al., 1993) and chickens (Asghar et al., 1989) showed that dietary a-tocopherol supplementation significantly increases the cr-tocopherol content of muscle membranes. In the present study, supplementation of the diet with a-tocopherol increased the a-tocopherol concentration in both mitochondria and microsomes, which is in contrast to Asghar et al. (1990) who found that mitochondria of white and dark meat as well as microsomes of white meat of cY-tocopherol-supplemented broilers did not accumulate vitamin E in any significant amount. Lin et al. (1989) proposed that the higher concentrations of a-tocopherol in thigh muscle compared with breast muscle may be associated with the more highly developed vascular system and higher lipid content of thigh muscle. It has also been demonstrated in beef that the accumulation of cY-tocopherol is muscle-dependent (Arnold et al., 1992, 1993). There are still no clear explanations why skeletal muscles differ in o-tocopherol concentration. Being a lipid soluble vitamin, o-tocopherol partitions into biomembranes, and its concentration increases with dietary supplementation. Thus, the higher lipid content in red muscles compared to white muscles is the likely explanation for the higher content of a-tocopherol in red muscle. Evarts and Bieri (1974) showed that the relative a-tocopherol content expressed as a percent of cY-tocopherol content divided by percent of total protein revealed that a-tocopherol distribution among various subcellular fractions is distinctly different from the protein distribution. As can be seen from Table 4, the content of ar-tocopherol from membranes of red meat is still higher than from white meat membranes even when the
20
C. Lauridsen, D. J. Buckley, P. A. Morrissey
content is corrected for the lipid content of the membrane. One reasonable factor could be the number and size of membranes, which differ between different muscles (Porter & Palade, 1957). More and smaller mitochondria may provide more membrane volume for potential retention of a-tocopherol as different fiber sizes could affect membrane volume. Another factor could be the greater capillary blood supply to red muscle fibers than to white fibers, which affects the a-tocopherol accumulations. Sosnicki et al. (1991) observed that the number of capillaries in turkey femoris were doubled compared with those of pectoralis muscle. Previous studies showed that the nature of dietary oils is reflected in the fatty acid composition of neutral lipids of membranes and, to a lesser degree, in fatty acid composition of the phospholipids (Asghar et al., 1990; Lin et al., 1989) In the present study we did not extract neutral and polar lipids separately, which may account for the reduced effects of dietary fatty acids. Based on Table 3 we expected more Ci6:e and CisO and less Cis:i, eventually C1sZ2,in membranes of tallow-groups compared to membranes of oliveoil fed groups. This was partly true for the mitochondrial fractions (Table 5), but the result was unexpected for the microsomal fraction (Table 6), e.g. the content of Cis:s was lower and the content of Cis:i higher in groups fed tallow compared to olive oil. This could probably be related to the feed, but differences in fatness of the chicken may influence the fatty acid composition of microsomes. The standard deviation was extremely high especially for fatty acids present in low concentrations. In conclusion, the relationship between saturated and monounsaturated fatty acid appeared to be influenced by dietary fatty acids to a higher degree in mitochondria than in microsomes. Studies have reported that dietary oil has no apparent influence on the deposition of CXtocopherol in the membranal fractions (Asghar et al., 1989; Yamauchi et al., 1991). However, in the study of Lin et al. (1990), the concentration of o-tocopherol in microsomes of dark meat was to some extent influenced by the nature of the dietary oil. In the present study we also found some influence of dietary vitamin E on the occurrence of single fatty acids in the membranal fractions, but no clear pattern was obtained. In conclusion, dietary cu-tocopherol acetate supplementation increased the antioxidant status of muscle. Muscle a-tocopherol levels appear to be a dominant factor in determining oxidative stability of muscle membranes rather than the fatty acid composition.
ACKNOWLEDGEMENTS The authors are grateful to the Commission of the European Communities for the financial support of this work. For providing analysis of vitamin E in diets Dr Ssren Krogh Jensen is gratefully acknowledged. REFERENCES Acosta, S. O., Marion, W. W. and Forsythe, R. H. (1966) Total lipids and phosphohpids in turkey tissues. Poultry Science 45, 169-184. AOAC (1984) Ojficial methods of analysis 14th edn: Association of Official Analytical Chemists, Washington D.C. Arnold, R. N., Arp, S. C., Scheller, K. K., Williams, S. N. and Schaefer, D. M. (1992) Tissue equilibration and subcellular distribution of vitamin E relative to myoglobin and lipid oxidation in displayed beef. Journal of Animal Science 71, 105-l 18. Arnold, R. N., Arp, S. C., Scheller, K. K., Williams, S. N. and Schaefer, D. M. (1993) Dietary cr-tocopheryl acetate enhances beef quality in Holstein and beef breed steers. Journal of Food Science 58, 28-33.
a-tocopherol levels and fatty acid profiles in chicken
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