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Antioxidant and biological activities of palm oil vitamin E


Elena A. Serbinova and Lester Packer

Abstract

The present study assessed the antioxidant properties of a -tocopherol, a -tocotrienol, and palm oil vitamin E, which contained 45% tocopherols and 55% tocotrienols. When vitamin E-deficient rats were fed either a -tocopherol- or a -tocotrienol-enriched diets, a -tocotrienol accumulated in the hearts and liver more slowly than a -tocopherol. The rate of lipid peroxidation induced in vitro in heart homogenate from rats supplemented with a -tocotrienol was approximately two-thirds as high as that from rats with an equivalent concentration of a -tocopherol. Thus palm oil vitamin E may be more efficient than a -tocopherol alone in protecting the heart against injury from ischaemia and reperfusion. In addition, supplementation with a -tocopherol or a -tocotrienol protects skeletal muscles against exercise-induced increases in protein oxidation Thus palm oil vitamin E protects biological systems against both lipid and protein oxidation.

 

Distribution and antioxidant activity of a -tocopherol and a -tocotrienol in different tissues

The pathogenesis of many diseases can involve free radical-mediated lipid peroxidation in biological membranes. Vitamin E is the major chain-breaking antioxidant in membranes; although it is present in extremely low concentrations, it is very efficient in inhibiting the development of conditions such as heart disease, cancer, cataracts, neuropathies, and myopathies [1]. Vitamin E is a generic name for tocopherol and tocotrienol derivatives that have vitamin E activity. Therefore it is important to establish which of these forms is the most effective antioxidant under defined conditions.

It is now well appreciated that different isomers of tocopherols (a , , g , d ) are not equally absorbed by the intestine or transported by lipoproteins. Nor are they equally sequestered by certain tissues [2]. The a -tocopherol-binding protein appears to be present in hepatocytes (and possibly other cell types too) [3]. Once returned to the liver by chylomicron remnants, a -tocopherol is selectively bound to the cellular binding protein for resecretion in nascent hepatic lipoproteins, whereas -, g -, and d -tocopherols rapidly disappear [3]. Tocotrienols are initially transported like any lipid-soluble compound, most likely incorporated along with triglycerides in the core of the triglyceride-rich chylomicron [4]. An essential lack of tocotrienols has been found in lowdensity lipoproteins (LDL) and high-density lipoproteins (HDL), in contrast to a -tocopherol, which points to striking differences in their transport and underscores the likelihood that tocotrienols and tocopherols associate with different lipid moieties during transport in plasma [4].

We studied the distribution of pure a -tocopherol and a -tocotrienol in rat tissues after feeding a diet containing 3 g of either of these forms of vitamin E per kilogram of diet to vitamin E-deficient animals (with 0.05-0.1 nmol of vitamin E per milligram of protein in their tissues) for eight weeks. No detectable amounts of tocotrienols were found in brain, kidneys, or skin. In the liver, a -tocotrienol was observed after two weeks of supplementation and reached its maximum level of 1.1 nmol per milligram of protein after five weeks (fig. 1). In the heart, the level was 0.1 nmol per milligram of protein after two weeks and reached a maximum of 0.9 nmol per milligram after seven weeks (fig. 2). The maximum level of a -tocotrienol accumulated in the heart was about half to one-third as high as that of a -tocopherol, and its accumulation was slower.

FIG. 1. Alpha -tocotrienol in liver and heart homogenates of three rats fed 3 g of pure a -tocotrienol per kilogram of diet-measured by HPLC

FIG. 2. Accumulation of a -tocopherol and a -tocotrienol in rat hearts

Tocotrienols have greater physiological efficiency in inhibiting growth than tocopherols. Proliferation of human and mouse tumour cells (sarcoma 180, Ehrlich carcinoma, IMC carcinoma) was suppressed after exposure to tocotrienols for 72 hours in vitro; tocopherol showed no significant effect [5-7].

Different forms of vitamin E exhibit different degrees of antioxidant protection in membranes. In vitro studies suggest that a -tocotrienol has 40-60 times as much antioxidant activity as a -tocopherol against lipid peroxidation induced by Fe2+ + ascorbate and Fe2+ + NADPH in rat liver microsomal membranes. In addition, a -tocotrienol protects cytochrome P-450 against oxidative damage 6.5 times as well as a -tocopherol [8].

Feeding vitamin E-deficient rats either pure a -tocopherol or a -tocotrienol increased the resistance of the tissues to lipid peroxidation in vitro. The susceptibility of tissue homogenates from both heart and liver to lipid peroxidation induced by Fe(II) + ascorbate decreased as tissues became more loaded with either substance over time. When the concentrations in the heart tissue were approximately equal (0.5 nmol per milligram of protein)-on day 10 of the experiment for x-tocopherol and day 21 for x-tocotrienol-the rate of lipid peroxidation was 62.5% as high in the tissue homogenate of the former and 40% as high in that of the latter as in that of the vitamin E-deficient controls (fig. 3). Hence we suggest that x-tocotrienol could be a more potent antioxidant than x-tocopherol not only in vitro but also in vivo.

FIG. 3. Susceptibility of rat heart tissue with approximately 0.5 nmol of a-tocopherol or a -tocotrienol per milligram of protein to in vitro lipid peroxidation induced by Fe(II) + ascorbate

 

Protection provided by palm oil vitamin E

Protection against ischaemia-reperfusion injury in vivo

It is well known that reactive oxygen species can oxidize lipids and proteins, and many studies have been conducted to investigate their role in the injury of the heart. In particular, heart ischaemia-reperfusion, anthracycline, and iron studies have shown that reactive oxygen species contribute to myocardial injury [9,10], which suggests that administration of antioxidants may lessen oxidative damage. One highly potent antioxidant is vitamin E [11].

We predict that tocotrienols may have specific health benefits for protecting the heart, as well as the brain, against ischaemia-reperfusion injury. In a previous investigation we studied the protection afforded by palm oil vitamin E (POE), a mixture of 55% tocotrienols and 45% tocopherols, in Langendorff perfused rat hearts subjected to 40 minutes of global ischaemia [12]. After 40 minutes of ischaemia and 20 minutes of reperfusion, the mechanical recovery rates of hearts from the POE-supplemented group (7 g per kilogram of diet) was 90%, compared with 80% for hearts from rats fed a diet supplemented with x-tocopherol (10,000 IU per kilogram) and 24% for hearts from rats kept on a normal diet (30 IU of a -tocopherol per kilogram).

The concentration of endogenous vitamin E in rat heart homogenates was 0.4-0.6 nmol per milligram of protein. After six weeks of dietary supplementation with POE, the concentration of endogenous a -tocopherol tripled. The concentrations of a -tocotrienol and a -tocopherol in hearts were 1.2+0.2 and 1.8+0.3 nmol per milligram of protein respectively; there were no significant differences after 60 minutes of perfusion. However, 40 minutes of ischaemia followed by 20 minutes of reperfusion caused a pronounced decrease in vitamin E levels. Only 41% of tocopherol and 21% of tocotrienol remained after ischaemia and reperfusion in the POE-supplemented group, while the amount of vitamin E in hearts from the control group was less than 4%.

Our results indicated that POE is more efficient than tocopherol in protecting the isolated Langendorff heart against ischaemia-reperfusion injury as measured by its mechanical recovery. Palm oil vitamin E completely suppressed LDH enzyme leakage from ischaemic hearts, prevented decreases in adenosine triphosphate (ATP) and creatine phosphate levels, and inhibited the formation of endogenous lipid peroxidation products [12].

In the present investigation we used two different approaches to follow the development of oxidative stress in post-ischaemia-reperfused hearts: the assay of carbonyls indicating oxidative modifications of proteins, and electron spin resonance (ESR) dimethyl proline oxide (DMPO) spin trapping, indicating the generation of short-lived free radicals. In hearts from control animals the level of oxidatively modified proteins increased remarkably during reperfusion, but in hearts from animals fed the palm oil-supplemented diet it did not differ from the values without reperfusion (table 1). We found, in accord with other data [13], a pronounced transient increase in steady-state concentrations of DMPO-spin adduct formation (of hydroxyl radicals) in the early reperfusion period in the control hearts. This burst of generation of oxygen radicals in the course of reperfusion was fully prevented in the hearts from POE-fed animals (fig. 4).

These results do not afford direct quantitative comparison of the protective effects of a -tocotrienol with that of a -tocopherol since both were present in the POE concentrate in proportion 55:45. However, there is indirect evidence of higher a -tocotrienol efficiency. Comparison of consumption in the course of ischaemia and reperfusion showed the loss of a -tocotrienol to be significantly more pronounced than that of a -tocopherol. In other words, a -tocotrienol was more active in radical scavenging and was preferentially consumed in the course of ischaemia-reperfusion-induced oxidative stress [12].

TABLE 1. Effect of palm oil vitamin E on the carbonyl content of proteins extracted from isolated perfused rat hearts (N = 3)

 

Carbonyl content (nmol/mg protein)

Control group

Supplemented groupa

60-min perfusion

2.20 0.25

2.27 0.25

10-min perfusion, 40-min ischaemia

2.41 0.30

2.20 0.30

10-min perfusion, 40-min ischaemia, 20-min reperfusion

4.50 0.25

2.00 0.20*

a. Animals were kept on a diet containing 7 g of palm oil vitamin E (POE) per kilogram of diet for 40 days.
*Significantly different from control group (p < .05).

FIG. 4. Effect of palm oil vitamin E (POE) on the time course of dimethyl proline oxide spin adducts in rat heart perfusate after 40 minutes of global ischaemia

In a rat resorption-gestation test, D-a -tocopherol exhibited the highest biopotency (100%), whereas D-a -tocotrienol manifested only 30% of this activity [14]. The significance of these estimates for health benefits is not clear, since vitamin E is considered physiologically the most important lipid-soluble chain-breaking antioxidant of membranes. We noted above that other physiological activities of a -tocotrienol, such as antitumour activity and inhibition of cholesterol biosynthesis, were reported to be much higher than those of a-tocopherol. Thus a -tocotrienol may have higher physiological activity under conditions of oxidative stress. In particular, our data indicate that a POE mixture containing both substances may be more efficient than a-tocopherol alone in protecting the heart against oxidative stress induced by ischaemia and reperfusion.

Protection in skeletal muscles before and after exercise

The possible mechanisms of free radical generation during exercise have recently been reviewed [15, 16]. Exercise may trigger an increase of free radical production by an increase in O2 generation in the mitochondrial electron transport chain or by an increase in metal-catalysed free radical production due to mechanical and morphological damage to muscles. In addition, higher levels of catecholamines, which are elevated many-fold during exercise, can potentially generate free radicals through the process of auto-oxidation [16].

It has been shown, using ESR techniques, that exercise can induce free radical production in muscles and liver [17]. Usually, when oxidative stress is of a large enough magnitude to overwhelm the antioxidant defense systems, oxidative damage will occur. Several studies on exercise-induced lipid peroxidation have been reported in the literature [18], but only recently has exercise-induced protein oxidation in muscle due to exercise has been reported [19]. In that study, muscle but not liver was significantly affected by extended exercise, as higher levels of protein oxidation were observed in muscles after endurance training [19].

We found that 60 to 90 minutes of exercise to exhaustion caused a marked depletion of vitamin E content in gastrocnemius and white quadriceps muscle of normally fed animals (table 2) but not in red quadriceps [20]. Similar observations were made in previous studies [21], in which endurance exercise depleted vitamin E levels in muscles as well as in the livers of animals.

However, in the present study, in the red quadriceps muscle vitamin E, ubiquinol, and ubiquinone levels were not altered or even slightly increased in exercised animals compared with resting controls. This may be because red quadriceps muscle uses mainly aerobic respiration, and its fibres are the first recruited in exercise and the most fatigue-resistant. Because of the continuous oxygen flux in this muscle, it may have many other protective antioxidant systems besides those measured that protect it adequately even during exhaustive exercise. Indeed, of the three muscles, the red quadriceps had the highest level of vitamin E, ubiquinol, and ubiquinone in the normally fed animals.

On the other hand, the white quadriceps muscle, which is heavily reliant on anaerobic metabolism, had the lowest levels of these membrane antioxidants, with especially low levels of ubiquinol and ubiquinone. This is expected, since white quadriceps muscle is not recruited except in severe or extended exercise and thus may have lower antioxidant protection mechanisms. The gastrocnemius muscle, which has a mixture of red (70%-80%) and white fibres had intermediate levels of vitamin E in the resting normally fed animals, as well as showing a decline in vitamin E and ubiquinol concentrations intermediate between those of red quadriceps and white quadriceps [20].

TABLE 2. Effects of various diets on the content of lipophilic antioxidants and protein carbonyls in the gastrocnemius muscle of control rats and rats subjected to a single bout of endurance exercise to exhaustion (N= 4)

  Content (nmol/g tissue) Change (%)
Control Exercised
Normal diet
Antioxidants
Vitamin E

15.74 1.08

12.90 0.94++

-18.1

ubiquinol

7.93 3.32

6.72 1.65

-15.3

ubiquinone

34.46 7.28

35.53 11.8

+3.1

Carbonyls

2.14 0.23

2.51 0.11

+17.28

Tocopherol-supplemented diet

Antioxidants
vitamin E

83.05 24.5

51.34 9.95+

-38.2

ubiquinone

21.9 7.94

32.71 16.86

+49.3

Carbonyls

1.45 0.06***

1.57 0.13***

+8.27

POE-supplemented diet (tocopherol + tocotrienol)

Antioxidants
vitamin E

22.45 5.92

26.67 7.95

+ 18.7

ubiquinone

31.5 + 12.13

35.47 11.46

+ 12.6

Carbonyls

1.73 0.13(*)

1.83 0.15(**)

+5.78

Different from control: +p < .05. ++p < .01
Different from normal diet: *p<.1. **p<.05. ***p<.001.

Another interesting observation that emerged from this study is that in gastrocnemius and white quadriceps muscles, where exercise caused a decrease of vitamin E and ubiquinol in animals fed a normal diet (30 IU of vitamin E [as D-a -tocopherol] per kilogram of diet), there was a concomitant increase of protein carbonyls. In red quadriceps muscle, where no decrease in these membrane components occurred, there was also minimal change in carbonyl content in exercised animals versus controls.

Probably the most important finding of this work was that, in all cases where animals were fed a high vitamin E diet (10,000 IU of a -tocopherol or 7 g of POE per kilogram), in control as well as in exercised animals, reductions in protein carbonyl concentrations were seen both at rest and at exhaustion,compared with animals fed a normal diet. In fact, the protein carbonyl levels in the muscles of exhausted supplemented animals were always lower than those of resting nonsupplemented animals. Since our procedure for extracting proteins for the carbonyl assays calls for the use of 0.1 % digitonin in phosphate buffer, it is possible that a good portion of the proteins extracted by this procedure are sarcolemma membrane proteins, which may be most affected by changes in the availability of vitamin E, ubiquinol, and ubiquinone in membranes. Indeed, in all muscles in which vitamin E levels were higher than in normally fed animals, the concomitant value of protein oxidation was lower [20].

In summary, an increase in protein oxidation in skeletal muscle after a single bout of exercise, which is correlated with an exercise-induced decrease in lipophilic antioxidants, can be prevented by supplementation with a -tocopherol or palm oil vitamin E (tocopherol + tocotrienol).

 

Acknowledgements

This research was supported by grant CA 47597 from the US National Institutes of Health, and by the Palm Oil Research Institute of Malaysia (PORIM). Mr. A. Gapor (PORIM) provided the tocotrienols.

 

References

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