Improved oxidative stability of veal lipids and cholesterol through dietary vitamin E supplementation

Meat Science35(1993) 1-15

Improved Oxidative Stability of Veal Lipids and Cholesterol through Dietary Vitamin E Supplementation N i c k i J. E n g e s e t h , J. I a n G r a y , * A l d e n M. B o o r e n & Ali A s g h a r Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824, USA (Received 23 December 1991; accepted 25 May 1992)

A BSTRA CT The influence of dietary vitamin E supplementation on the ct-tocopherol content of muscle membranes and on the resultant oxidative stability of veal was investigated. Daily supplementation of veal calves with 500 mg vitamin E in the form of a-tocopherol acetate for 12 weeks after birth increased muscle and membranal ot-tocopherol concentrations approximately 6-fold over those of control animals. Oxidative stability of mitochondrial and microsomal lipids was enhanced by dietary supplementation as indicated by the results of an oxidative assay using hydrogen peroxideactivated metmyoglobin as the catalyst of oxidation. Muscle lipid and cholesterol stability was also improved by supplementation.

INTRODUCTION Lipid oxidation is a major deteriorative reaction in meats during storage. It is responsible for a wide variety of undesirable reactions such as loss of fresh meat colour and flavour (Benedict et al., 1975; Pearson et al., 1983), oxidation product reactions with proteins, with concomitant losses of protein functionality and nutritional value (Matsushita, 1975; Gardner, 1979), and possible deleterious biological effects such as cardiovascular disease (Addis, 1986; Yagi, 1 9 8 8 ) a n d cancer (Pearson et al., 1983; Addis, 1986). Products of cholesterol oxidation, in particular, have been reported to produce a variety of adverse biological effects such as * To whom correspondence should be addressed. 1

Meat Science 0309-1740/93/$06.00© 1993 Elsevier SciencePublishers Ltd, England. Printed in Great Britain

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N.J. Engeseth, J. I. Gray, A. M. Booren, A. Asghar

inhibition of cholesterol biosynthesis (Kandutsch & Chen, 1973), atherogenesis (Taylor et al., 1979; Imai et al., 1980; Cox et al., 1988), cytotoxicity (Peng et al., 1979; Sevanian & Peterson, 1986), mutagenesis (Ansari et al., 1982; Sevanian & Peterson, 1986) and carcinogenesis (Bischoff, 1957; Black & Lo, 1971). There have been many reports on the protective effect of dietary vitamin E on lipid stability in various muscle foods. For example, supplementation of veal calves with vitamin E resulted in improved oxidative stability of rendered fat (Ellis et al., 1974), even in animals whose diets had been supplemented with high levels of linoleic acid (approximately 14%, as encapsulated safflower oil). Shorland et al. (1981) observed a protective effect of vitamin E supplementation on lipid stability in longissimus dorsi tissues. However, this effect was not consistent from tissue to tissue. Faustman et al. (1989a,b) theorized that the improved lipid and colour stability of muscle from Holstein steers fed diets supplemented with 370 IU vitamin E/day was the result of incorporation of vitamin E into membranes. However, the ot-tocopherol content of the membranes was not determined. Several recent studies have focused on the effects of vitamin E supplementation on the incorporation of a-tocopherol into membranes and the resultant oxidative stability of membranes and muscles in different species. Monahan et al. (1989) showed incorporation of ot-tocopherol into mitochondria and microsomes as a consequence of supplementing pig diets with 200 mg a-tocopherol acetate/kg feed and a resultant improvement in lipid stability. Asghar et al. (1989) reported increased concentrations of a-tocopherol in porcine tissues with increasing dietary levels of vitamin E, and a resultant improvement in muscle and membranal lipid stability. Similar trends were found in poultry (Lin et al., 1989; Asghar et al., 1990). While no such studies have been carried out with veal, Ellis et al. (1974) pointed out that species differences in response to vitamin E supplementation do exist. The objective of the present study was to investigate the effects of dietary vitamin E on the oxidative stability of veal muscle lipids including cholesterol. MATERIALS AND METHODS

Samples Eight male Holstein calves were housed at the Michigan State University (MSU) dairy barn and randomly divided into two groups. Control animals (n = 4) were fed whole milk (2.82 × 10-3 mg ot-tocopherol/ml)

Improved oxidative stability of veal lipids and cholesterol

3

twice a day. Supplemented animals (n -- 4) received, in addition, 500 mg of ot-tocopherol acetate powder (BASF Corp., Wyandotte, MI), added directly to the milk. These feeding regimens were continued until the time of slaughter at twelve weeks. At two-week intervals, plasma was collected for a-tocopherol analyses. At slaughter, approximately 50 g of liver, heart, lung, kidney and perinephric adipose tissue were removed from each animal, immediately frozen in liquid nitrogen, packaged, and held at -80°C until analysed for a-tocopherol. Leg muscle (semitendinosus, semimembranosus, adductor and biceps femoris) samples were also removed, vacuum packaged in polyethylene-laminated nylon pouches (Koch, Kansas City, MO) and frozen at -80°C for future analysis. These pouches (3.5 mil) had a water vapor transmission rate of 0.041 ml/(m2 day mm Hg) and an oxygen transmission rate of 0.124 ml/(m2 day mm Hg) at 22.7°C, 50% relative humidity.

Determination of ~-tocopherol Plasma

Sample plasma (2 ml) was added to 2 ml 200 proof ethanol (absolute, USP), 0.3 ml saturated ascorbic acid (3.4 g in 10 ml deionised water) and internal standard (0.1 ml of 100 /~g/ml dl-a-tocopherol acetate, Sigma Chemical Co., St Louis, MO). After vortexing, the samples were extracted twice with 6 ml hexane containing butylated hydroxytoluene (BHT) (0.05% (w/v)). The extracts were combined and evaporated under nitrogen to dryness. Methanol (200 ~1) was added and the tubes were vortexed prior to transfer to 0.5 ml microfuge tubes and freezing for future HPLC analysis.

Muscle and other tissues Ground tissues were homogenised with an equal weight of nitrogensaturated deionised water containing 5% ascorbic acid. Homogenates (2 g) were accurately weighed and 1-5 ml 6 M urea and 100 tzl a-tocopherol acetate (0.2 mg/ml) were added. After vortexing (2 min), 1 ml 0.1 M sodium dodecyl sulphate (SDS) solution was added and the mixture vortexed again (1 min). Pyrogallol (4 ml 1% ethanol solution) was added and the mixture again vortexed (1 min). Samples were extracted three times with petroleum ether. The combined extracts were dried under nitrogen and redissolved in 200 ~1 ethanol. Membranes Mitochondria and microsomes were assayed for a-tocopherol in a manner similar to muscle, using a buffered membrane solution (volume

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N.J. Engeseth, J. I. Gray, A. M. Booren, A. Asghar

determined to achieve approximately 0.5 g membranes) to which 0.5 ml 0.5% ascorbic acid and 1 ml 6 M urea and 100 /xl a-tocopherol acetate (0.2 mg/ml) were added; tubes were capped and vortexed. The next step was SDS addition and the procedure was followed as for muscle. Adipose tissue Perinephric adipose tissue (0.5 g) was weighed accurately and 0.5 ml 2% pyrogallol in ethanol and 100/zl 25% ascorbic acid solution (dissolved in water, then made to volume in ethanol) were added. The tubes were flushed with nitrogen, capped and placed in a water bath at 75°C for 5 min. After the addition of 0.5 ml 10 N KOH the tubes were capped and heated for 30 min at 75°C with periodic shaking. The tubes were cooled and 1 ml ethanol and 1 ml water were added prior to extraction with 8 ml petroleum ether (3 times). The extracts were combined and washed with 8 ml aqueous sodium chloride solution (1%). The petroleum ether layer was evaporated under nitrogen and 200/zl of ethanol were added. Milk Composite milk samples were obtained from the MSU dairy barn at periodic intervals during the feeding study. Milk (1 ml) was added to 2 ml 1% pyrogallol in methanol and 0.5 ml 50% KOH solution. The samples were saponified for 30 min in a 70°C water bath. Two ml water were added and the tubes were allowed to cool to room temperature. Alpha-tocopherol was extracted with two 3 ml aliquots of hexane. The hexane extracts were combined, 100/zl a-tocopherol acetate (0.2 mg/ml) added, and the extracts evaporated to dryness under nitrogen. Samples were redissolved in 400/zl ethanol. H P L C analysis o f a-tocopherol A Waters high performance liquid chromatograph (Millipore Corporation, Waters Chromatography Division, Milford, MA) equipped with a 5/z, 4.6 × 150 mm Ultrasphere-ODS Column (Beckman Instruments Inc. Fullerton, CA) and a fixed wavelength detector (Waters, 440 absorbance detector) set at 280 nm was used. Peak areas were integrated using a Hewlett-Packard 3380A integrator (Hewlett-Packard Co., Avondale, PA). Standard areas were prepared using dl-a-tocopherol (Sigma Chemical Co., St Louis, MO).

Fatty acid analysis Fatty acid composition of muscle, mitochondria, and microsomes was determined by extracting total lipids by a dry-column procedure (Marmer & Maxwell, 1981). The neutral lipids and phospholipids were

Improved oxidative stability of veal lipids and cholesterol

5

fractionated by silica gel thin-layer chromatography (Pikul et al., 1984). Neutral lipid and phospholipid bands were scraped from the plates and eluted with 9:1 methylene chloride:methanol, dried and derivatised by the method of Maxwell & Marmer (1983). The fatty acid methyl esters were analysed by gas chromatography using a Hewlett Packard 5890A system, equipped with a 30 m, narrow-bore 0.25 mm i.d. DB-225 capillary column (0.25 g film, J & W Scientific/Anspec, Ann Arbor, MI). The gas chromatograph was programmed from 175°C (10 min) to 200°C at 1.5°C/rain and held for 40 min, with an injection temperature of 275°C and detector temperature of 300°C.

Preparation of samples for oxidative stability studies Steaks (approximately 1 in thick) from the semitendinosus muscle of each animal were cooked by broiling in a conventional oven to an internal temperature of 170°F (measured using a thermocouple: Omega Thermocouple Thermometer, Model 660, Omega Engineering, Inc., Stamford, CT). Mitochondria and microsomes were isolated from muscle of each animal by a centrifugation procedure similar to that reported by Kanner & Harel (1985). Two hundred grams of ground muscle were blended with 4 volumes of 0-12 M KC1, 5 mM histidine buffer (pH 7.3). After centrifugation for 10 min at 600 x g, the supernatant was centrifuged at 1000 x g (10 min). The pH of the supernatant was then readjusted to 7.3 and centrifuged at l0 000 x g for 15 min to precipitate the mitochondrial fraction, which was frozen under nitrogen. Microsomes were isolated from the supernatant by centrifuging at 100 000 x g for 1.25 h at 4°C using a Beckman L7 ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA). The microsomes were further purified by resuspending in 0.6 M KC1, 5 mM histidine buffer (pH 7.3) and centrifuging at 100 000 x g for one hour.

Oxidative stability studies The distillation method of Tarladgis et al. (1960), as modified by Crackel et al. (1988), was used to determine thiobarbituric acid (TBA-reactive substances, TBARS) in raw and cooked meats at 0, 2, and 4 days of storage at 4°C. Cholesterol oxide determinations were carried out on total lipid extracts (dry column procedure of Marmer & Maxwell, 1981) from 5 g of muscle. Extracts were dried by rotary evaporation and redissolved in 4 ml hexane:ethyl acetate (9:1). The extract was applied to a silica gel column, prepared and eluted as described by Park & Addis (1985).

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N.J. Engeseth, J. I. Gray, A. M. Booren, A. Asghar

The acetone fraction containing the cholesterol oxides was collected, rotary evaporated, redissolved in 4 ml ethyl acetate and frozen until derivatisation. Samples were derivatised by evaporating the ethyl acetate under nitrogen and adding 100 /zl pyridine and 50 /xl N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) silylating reagent (Pierce Chemical Co., Rockford, IL). The samples were wrapped in aluminium foil and held at room temperature for 30 min. GLC analysis of the silylated compounds was carried out on a Hewlett-Packard HP 5890A gas chromatograph using a 15 M × 0.25 mm i.d. polydimethylsiloxane column (Supelco, Bellefonte, PA), with helium as a carrier gas (1-2 ml/min) and a flame ionisation detector (300°C). The oven temperature was programmed from 180°C to 230°C at 10°C/min and then to 242°C at 0.2°C/min. This final temperature was held for 30 min. A split ratio of 16 was used. Areas were obtained using a Hewlett-Packard 3392A integrator. The oxidative stability of mitochondrial and microsomal lipids was determined by the method of Kanner & Harel (1985), using metmyoglobin/hydrogen peroxide as the catalyst. Protein determinations were carried out as described by Lowry et al. (1951) in order to adjust membrane solutions to equal protein concentrations prior to assay. The stability was expressed as concentration of malonaldehyde/g protein of membrane solution. Statistical treatment of data

Statistical analyses were carried out using MSTAT-C (Microcomputer Statistical Program, Michigan State University, East Lansing, MI). Student's t-tests were done to determine significant differences between control and supplemented plasma tocopherol, tissue tocopherol, fatty acid composition and membranal lipid stability. Also, for TBARS of raw and cooked meats over 4 days' storage at 4°C, a completely randomised factorial design was developed with factor C (raw versus cooked) being a split plot on factor A (control versus supplemented) and factor B (day of storage). Statistical significance was declared at P < 0.05. RESULTS AND DISCUSSION Concentrations of a-tocopherol in plasma and other tissues

Concentrations of ot-tocopherol in the plasma from supplemented animals were significantly higher (P < 0-05) than those in plasma from control animals during the entire period, with the exception of the last

Improved oxidative stability of veal lipids and cholesterol

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TABLE 1 Plasma a - T o c o p h e r o l Concentrations 0zg/ml o f Veal Calves Fed Control and Vitamin E-Supplemented Diets a

Age (days)

Control

0-14 b 15-28 c 29~2 d 43-56 e 57-70 f 71-84 g 85-98 h

1 + 0-4 3 + 0.6 3 + 1.2 2 + 0.4 1 _+ 0.4 3 + 0.8 4 + 2.0

Supplemented 14 13 11 10 11 14 8

+ 1.9 + 1.2 + 1.5 + 1.9 _+ 2.4 + 3.4 + 2.2

a Value represents mean of samples from four animals + standard error. b Control different from supplemented (P < 0.01). c Control different from supplemented (P < 0.001). d Control different from supplemented (P < 0.01). e Control different from supplemented (P < 0.01). f Control different from supplemented (P < 0.01). g Control different from supplemented (P < 0.05). h Control not significantly different from supplemented at P < 0.05.

collection (Table 1). Slaughtering of the eight calves occurred on two different days, approximately 2V2 weeks apart. The last collection of plasma thus represents only five animals because they were in the second group to be slaughtered. Therefore, the variability in the smaller group led to a lack of significance. The results generally agree with those of Asghar et al. (1991a), who reported that supplementation of swine with vitamin E increased plasma a-tocopherol concentrations relative to those of control pigs for the entire period. Monahan et al. (1989) also observed significantly higher concentrations (P < 0.01) in plasma from the supplemented animals. Plasma a-tocopherol concentrations were not reported in previous veal studies. Tissues from supplemented animals had higher a-tocopherol concentrations than those from control animals (P < 0.05); however, adipose tissue concentrations, which were determined in fewer samples (control n = 2; supplemented n = 3) had greater variability (supplemented greater than control at P < 0.01) (Table 2). Concentrations of a-tocopherol for organs decreased in the order: liver > kidney > lung > heart. Adipose tissue of the control animals was intermediate between liver and kidney, but had the highest level in the supplemented animals.

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N.J. Engeseth, J. L Gray, A. M. Booren, A. Asghar

TABLE 2

Mean a-Tocopherol Concentrations (/zg/g tissue, wet basis) in Tissues from Control and Vitamin E-Supplemented Veal Calves a Tissue

Control

Supplemented

Liver b Heart C Lung d Kidney e Adipose f (perinephric)

6 + 1.7 0 + 0.0 0 + 0.1 2 + 0.4 3 + 0.9

19 + 3.64 5 + 0.5 11 + 0-8 16 + 0-9 44 + 12.8

Value represents mean of samples from four animals + standard error (except adipose, where n -- 3). bControl different from supplemented (P < 0.05). c Control different from supplemented (P < 0.01). d Control different from supplemented (P < 0.01). Control different from supplemented (P < 0.01). f Control not significantly different from supplemented at (P < 0.05). e

The observation that a-tocopherol increased in tissues with vitamin E supplementation is consistent with other veal reports. Shorland et al. (1981) collected omental and perinephric tissues from control and supplemented (500 I U vitamin E/day) veal calves fed corn oil or coconut oil as milk fat replacers. Perinephric adipose tissue a-tocopherol concentrations increased with supplementation in the group receiving coconut oil but not in those receiving corn oil as a milk fat replacer. Tissue concentrations in control and supplemented tissue were 19.1 and 43.7/zg/g, respectively. Perinephric adipose tissue ot-tocopherol concentrations in the present study were 3.0 and 44.5/zg/g tissue for control and supplemented animals, respectively. Ellis et al. (1974) reported approximately 3-fold higher a-tocopherol concentrations in perinephric adipose tissue from animals supplemented with vitamin E as opposed to commercial veal. Concentrations of a-tocopherol in muscle, mitochondria, and microsomes o f supplemented animals were approximately 5-8 times greater than those in their counterparts from the control animals (Table 3). Shorland et al. (1981) also noted increased ot-tocopherol (upon vitamin E supplementation) in L. dorsi muscle o f veal calves receiving corn oil as a milk fat replacer, but not in those muscles from calves receiving coconut oil. Concentrations o f c~-tocopherol in the semitendinosus muscle o f the present study were lower in the control animals than those of Shorland et al. (1981) in L. dorsi (1.0 versus 3.4/zg/g, respectively). However, muscle

Improved oxidative stability of veal lipids and cholesterol

9

TABLE 3 M e a n a - T o c o p h e r o l C o n c e n t r a t i o n s in Muscles a n d Membranes of Control and Vitamin E-Supplemented Veal Calves a

Control Muscle b

1 + 0-2

Supplemented 6.1 + 0.8

(~g/g) Mitochondria c

(~g/g protein) Microsomesa (p~g/gprotein)

73 + 38

584 + 103

139 + 86

707 + 162

a Value represents m e a n o f four samples + s t a n d a r d

error. bControl different from supplemented(P < 0.01). cControl different from supplemented (P < 0.05). dControl different from supplemented(P < 0.01). a-tocopherol concentrations in supplemented animals were similar (6.1 versus 5-2 /xg/g). The increase in muscular a-tocopherol concentration in the present study was slightly more dramatic, perhaps due to a longer feeding time of 12 weeks as opposed to 8 weeks by Shorland et al. (1981). Asghar et al. (1991b) demonstrated that the deposition of a-tocopherol in L. dorsi muscle and subceilular membranes of pigs was dependent upon the concentration of vitamin E in the feed. Muscle from pigs supplemented with 200 IU vitamin E/kg feed had an a-tocopherol concentration of 4.7 ~g/g tissue, while that from control pigs (10 IU vitamin E/kg feed) was 0.5 t~g/g tissue. Monahan et al. (1989) also reported L. dorsi a-tocopherol concentrations in pigs supplemented with 200 mg of a-tocopherol acetate/kg feed to be 20.6 ~g/g protein (control concentration was 6.9 /zg/g protein). These values expressed on a tissue weight basis are somewhat similar to those obtained in this study.

Fatty acid composition of veal muscle In general, vitamin E supplementation had no significant effect on fatty acid composition of the neutral and phospholipids of veal. The neutral lipid fraction of muscles from the supplemented animals had less 20:3w6 and 20:4w6 than those from the control animals (data not shown). In the phospholipid fraction, muscles from the supplemented animals had lower 14:0, 16:0, and 18:0 contents. Shorland et al. (1981) reported higher C18:0 in the neutral lipid fraction with reduction in

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N.J. Engeseth, J. L Gray, A. M. Booren, A. .4sghar

C12:0, C14:0 and C16:1w7. They also found a higher level of C18:0 in the phospholipid fraction, which is the opposite of the trend in the present study. It was not expected that dietary supplementation with vitamin E would affect the fatty acid composition significantly. Other researchers have demonstrated no effect of vitamin E supplementation on fatty acid composition of pigs (Monahan et al., pers. comm.) or broilers (Lin et al., 1989). Thus, it would appear that fatty acid composition would not be a significant factor in determining the oxidative stability of muscle lipids from the control animals versus those supplemented with vitamin E. Oxidative stability of veal

Veal steaks from the vitamin E supplemented animals were more stable than those from the control calves (Table 4). Dietary supplementation with vitamin E resulted in significantly lower (P < 0.05) TBARS values in both raw and cooked steaks held at 4°C for 4 days. Initial TBARS values (day 0) for muscles from the control animals were much higher than expected. This can be attributed, in part, to the fact that the samples were held in frozen storage for approximately 6 months prior to analysis. TBARS values of muscles from control animals increased over time of refrigerated storage, while those of muscles from the vitamin E supplemented group did not change to any extent over the same period. Vitamin E supplementation had a protective effect on the oxidative stability of muscle lipids upon frozen and refrigerated storage. This is in agreement with the findings of Lin et al. (1989), who improved the stability of both white and dark meat of broilers fed vitamin E (100 IU/kg feed), and those of M o n a h a n et al. (1989), who improved the TABLE 4 TBARS Values (mg malonaldehyde/kg meat) for Raw and Cooked Veal Steak from Control and Vitamin E-SupplementedAnimals, Held at 4°C for 4 daysa'b'c Raw

Day0 Day2 D~ 4

Cooked

Control

Supplemented

Control

Supplemented

3.8±1.0 6.3±0.6 7.6±0.7

0.3±0-0 0-4±0.0 0.4±0.1

6.2±0.9 9.7±0.7 12.4±1.0

0.4±0.1 1.9±0.5 5.0±0.5

QValue represents mean of four samples + standard error. bControl different from supplemented (P < 0.001). c Time/treatment interaction not significantat P < 0.05.

Improved oxidative stability o f veal lipids and cholesterol

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TABLE 5 Cholesterol Oxide Concentrations (p.g/g sample) in C o o k e d Veal Held at 4°C for 4 days a,b,"

Oxide

/3-epoxide cr-epoxide 7-/3-OH triol 7-keto 25-OH Total

Day 0

Day 4

Control

Vitamin E d

Control

Vitamin E

1.1 0-1 2.7 ND 5.5 ND 9-4

6-4 0.5 1.5 ND 0.2 ND 8.6

4.4 0-3 4.0 ND 7-2 0.9 16.8

1.1 0.2 1.8 ND 2.7 ND 5.8

a Mean of four samples. b N D = not detected (detection limit: 1 ng). c tr = trace amount. d Vitamin E = supplemented veal.

stability of raw and cooked pork by supplementing with 200 E/kg feed. Asghar et al. (1991b) also found that TBARS values of raw pork supplemented with 100 and 200 IU vitamin E/kg feed were lower than control samples. Those muscles from the 200 IU supplemented group were more stable than those from the 100 IU group. Cooked muscle TBARS values were greater than those of raw muscle. This is expected as cooking disrupts the muscle membranes, thus exposing lipid substrates to oxidative catalysts (Rhee, 1988). Cholesterol oxide development was determined in raw (data not shown) and cooked (Table 5) veal muscles after 0 and 4 days' storage at 4°C. Vitamin E supplementation was effective in controlling the development of cholesterol oxides in both raw and cooked muscles during storage. Day 0 values were not clearly reduced by supplementation. As indicated previously, these samples were frozen for several months prior to analysis. Thus, day 0 values for cholesterol oxides were greater than expected. One problem with the cholesterol oxide values from the supplemented group is that one animal in the group had levels of cholesterol oxides much greater than the others. Because of the small sample size (n -- 4), the group means were high. This points to the need for larger sample sizes. Control of cholesterol oxidation by vitamin E supplementation, however, was obvious upon storage for 4 days of both raw and cooked samples. Vitamin E supplementation resulted in a reduction of cholesterol oxide levels by approximately 65% after 4 days' storage at

12

N.J. Engeseth, J. 1. Gray, A. M. Booren, A. Asghar 80 .-~ 70 o

o. 60 O)

• CONTROL o SUPPLEMENTED

E so

Q Z

"6

4O

E 3o

~ 2o <

~

10 ¢ !

OI

3'0

6'0

9'0

140

140

1110

Time (rain)

Fig. 1. Metmyoglobin/hydrogen peroxide-catalysed lipid oxidation (as measured by TBARS) in mitochondria from control and vitamin E-supplemented veal calves.

4°C. As found with other indices of oxidation, the oxide development was greater in cooked samples. This is consistent with previous findings and is explained by the harsh conditions of cooking which leads to the disruption of the membranes and subsequent exposure of lipid substances to oxidative catalysts (Rhee, 1988). One would expect this to apply to cholesterol as well, since most of the cholesterol in muscle is located in the membranes (Dugan, 1987; Hoelscher et al., 1988). Cholesterol oxide development was found to increase with increasing rancidity (indicated by TBARS values). Mitochondrial and microsomal membranes of the control animals oxidised to a greater extent than those from calves fed the vitamin E supplement (Figs 1 and 2, respectively). However, these differences were not significant at the P < 0.05 level due to great variation among .c 40 0

p 3s

O.

30

• CONTROL

T ~

E 2s

<

15 ~

10. 5~ -1-

30 I,,-

i

60

i

90

i

120

i

150

i

180

Time (rnin)

Fig. 2. Metmyoglobin/hydrogen peroxide-catalysed lipid oxidation (as measured by TBARS) in microsomes from control and vitamin E-supplemented veal calves.

Improved oxidative stability of veal lipids and cholesterol

13

membranes from different animals and the relatively small number (n -- 4) of animals in each group. The protective effect of vitamin E on membranal lipid oxidation in this study was also found in pork membranes (Asghar et al., 1991b; Monahan et al., 1989) and in poultry dark muscle microsomes (Asghar et al., 1990). In the present study, oxidation was greater in control mitochondrial membranes than microsomal membranes. This was also reported by Asghar et al. (1991b) for pork membranal lipid oxidation, using the same initiators as the present study. However, Monahan et al. (1989) reported similar rates of oxidation for pork mitochondrial and microsomal lipids in iron-induced oxidation studies. The fact that Monahan et al. (1989) reported higher a-tocopherol concentrations in microsomes than mitochondria and Asghar et al. (1990a) reported higher ot-tocopherol in mitochondria than microsomes implies that other factors, besides o~-tocopherol concentrations, played a role in the observed rates of membranal lipid oxidation. Values for TBARS (reported as malonaldehyde/mg protein) production obtained in this study were much greater than those reported in other studies. For instance, Harel & Kanner (1985) reported malonaldehyde production after 120 min of incubation under similar reaction conditions in dark chicken microsomes to be approximately 17 nmol/mg protein. Incubation of veal muscle microsomes in this study for 120 min resulted in a level of approximately 22 nmol/mg protein (mitochondria > 30 nmol/mg protein). Asghar et al, (1989) reported malonaldehyde levels at 180 min in pork mitochondria to be slightly less than 35 nmol/mg protein. This would be reasonable in comparison to those presented in this study for microsomes. Mitochondrial TBARS development was much higher due to two animals whose membranes were tested in duplicate at different times. The TBARS values of these two animals were not thrown out as outliers because they fell within 2 standard deviations of the mean. In summary, vitamin E supplementation of veal calves (500 mg a-tocopherol acetate, from birth to 12 weeks) resulted in increased incorporation of c~-tocopherol into muscle membranes, and improved oxidative lipid stability of the membranes and muscle. This indicates the potential for a supplementation program in food-producing animals in order to produce higher-quality meat and meat products and to protect the consumer from the potentially deleterious biological effects of lipid oxidation products.

ACKNOWLEDGEMENTS This study was supported in part by the Michigan Agricultural Experiment Station and by the National Live Stock and Meat Board.

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N.J. Engeseth, J. I. Gray, A. M. Booren, A. Asghar REFERENCES

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