KEY WORDS: Oxidative stress, endurance, heat, malondialdehyde, palm vitamin E.

Generation of reactive oxygen species (ROS) increases during exercise in both animal (Davies et al., 1982; Gohil et al., 1986) and man (Dillard et al., 1978; Kanter et al., 1988). Under normal circumstances, however, ROS are neutralized by an elaborate endogenous antioxidant system, comprising of enzymatic and non-enzymatic antioxidants (Gohil et al., 1986; Urso and Clarkson, 2003). However, during increased oxygen utilization, as happens during strenuous exercise, the rate of ROS production may overwhelm the body's capacity to detoxify them (Sjodin et al., 1990; Urso and Clarkson, 2003), which can lead to increased oxidative stress and subsequent lipid peroxidation (Davies et al., 1982; Jenkins, 1988; Kanter et al., 1988). Free radical production reaches the highest level when exercise is exhaustive (Ji et al., 1998; Li et al., 1998; Sastre et al., 1992). In addition, strenuous physical activity is also associated with an increase in body temperature and elevated body temperature has also been shown to increase the rate of free radical production (Altan et al., 2003; Salo et al., 1991).

There is evidence that free radical production during exercise contributes to muscle fatigue (Barclay and Hansel, 1991; Novelli et al., 1990; Shindoh et al., 1990). Moreover, oxidative stress that results from antioxidant deficiency has been suggested to reduce endurance performance during exhaustive exercise (Coombes et al., 2002; Davies et al., 1982; Gohil et al., 1986). In this regards, high pre-race plasma vitamin E level has been associated with enhanced physical endurance in dogs (Piercy et al., 2001). But most human studies, however, have failed to demonstrate any performance enhancement associated with vitamin E supplementation (Lawrence et al., 1975; Rokitzki et al., 1994b; Sharman et al., 1971) except when exercise was performed at high altitude (Simon-Schnass and Pabst, 1988). Its role in enhancing endurance performance therefore remains debatable and unclear, particularly when exercising at high ambient temperature and humidity. Supplementation of vitamin E, on the other hand, has been consistently demonstrated to increase tissue resistance to exercise-induced lipid peroxidation (Dillard et al., 1978; Itoh et al., 2000; Kanter et al., 1993; Rokitzki et al., 1994a; Sumida et al., 1989). Moreover, vitamin E supplementation has also been shown to reduce the leakage of creatine kinase, a marker of exercise-induced muscle damage following exhaustive cycle exercise (Rokitzki et al., 1994a) and endurance running (Itoh et al., 2000). Whether these effects are also evident during exercise, in particular, when exercising in heat is unknown.

Vitamin E refers to the eight structural isomers of tocopherols and tocotrienols: alpha (α), beta (β), gamma (γ) and delta (δ) tocopherols and tocotrienols (Brigelius-Flohe and Traber, 1999; Kamal-Eldin and Appelqvist, 1996). However, most sports-related studies mentioned earlier have used alpha-tocopherols as the form of vitamin E. To our knowledge, to date, no scientific studies have examined free radical activity during exercise in a hot and humid environment, particularly, in heat-adapted recreational athletes, or examined the benefits of tocotrienol-rich palm vitamin E supplementation in these individuals when exercising in a hot and humid environment. Palm vitamin E contains natural tocotrienol-rich fractions dissolved in palm olein (Hood, 1996). Compared to α-tocopherol, α-tocotrienol is reportedly more effective in the inhibition of lipid peroxidation, being 40-60 times more potent than -tocopherol (Serbinova et al., 1991). In addition, tocotrienol has also been shown to possess hypocholesterolaemic, anti-thrombotic and anti-tumour effects, indicating that tocotrienol may serve as an effective agent in the prevention and/or treatment of cardiovascular disease and cancer (Theriault et al., 1999). The present study therefore investigates the effects of tocotrienol-rich palm vitamin E supplementation on exercise-induced lipid peroxidation and endurance performance in the heat in heat-adapted recreational athletes.

Eighteen, male heat-adapted recreational athletes participated in this study. Their age, height, weight, percent body fat and VO₂max were 24.9 ± 1.4 yrs, 1.69 ± 0.01 m, 59.6 ± 1.5 kg, 18.0 ± 0.9 % and 57.7 ± 1.5 ml·kg^-1·min^-1 respectively. An informed consent was obtained from each subject and the experimental procedures were approved by the Research and Ethics Committee of University Sains Malaysia.

After familiarization with treadmill running, the subjects performed two tests to determine the relationship between running speed and oxygen uptake and their maximum oxygen uptake (VO₂max). From these data, running speeds during warm-up at 50% VO₂max and endurance running performance at 70% VO₂max were established. All subjects were required to undertake a 60-minute trial run on a motorized treadmill at 70% VO₂max one week before the first experimental trial to familiarize with the experimental protocol.

A double blind, placebo-controlled, randomized crossover design was used to control within subject variability of all physiological measurements and the effect of order of the administration of the two supplements (tocotrienol-rich palm vitamin E and placebo). There was a washout period of 2 weeks between the two regimens. After a 6-week supplementation regimen with either tocotrienol-rich palm vitamin E (E) or placebo (P), endurance running performance tests were conducted at 31^oC and 70% relative humidity for both the groups. Each subject was required to run to exhaustion at 70% VO₂max on a motorized treadmill on two different occasions.

Palm vitamin E was supplemented in the form of capsules, provided by the Malaysian Palm Oil Board (MPOB). Each capsule contained 60 mg of palm vitamin E, a highly concentrated fraction of tocotrienols (33% alpha-tocopherol and 33% alpha-tocotrienol, 24% gamma-tocotrienol, 10% delta-tocotrienol) in 240 mg of superolein while each placebo capsule contained 300 mg of superolein. Participants were instructed not to take other forms of antioxidant supplements during the course of the study and to maintain their physical activity level throughout the supplementation period.

To minimize differences in resting muscle glycogen concentrations, subjects recorded their food intake for 3 days before the first experimental trial and were then instructed to follow the same diet before the second trial. They were also required to abstain from training the day before each trial and to observe a 10 h fast before their arrival to the laboratory. Upon arrival at the laboratory they were given a standardized breakfast, after which they were asked to empty their urinary bladder, and their nude body weight was recorded (Tanita, Japan). A rectal thermistor (Yellow Springs Instrument, USA) was inserted to a depth of 10 cm beyond the anal sphincter for the measurement of core temperature. In addition, skin thermistors (Yellow Springs Instrument, USA) were attached to the chest, biceps, thigh and calf for the measurement of mean skin temperature (Ramanathan, 1964). An indwelling cannula (Vasocan^®, B. Braun, Malaysia) was inserted into a forearm vein for repeated blood withdrawals. Patency of the cannula was maintained with heparinized saline (10 IU heparin sodium in 1 ml 0.9% NaCl, B. Braun, Malaysia).

After standing on the treadmill for 5 minutes, a resting blood sample was collected and CO₂ and O₂ concentrations in the expired air were measured (SensorMedics 2900, USA). Following a 5 minute warm-up run at 50% VO₂ max on the treadmill, the speed of the treadmill was increased to elicit 70% VO₂ max and the subjects were asked to run until exhaustion. Oxygen and CO₂ in the expired air samples were recorded immediately after warm-up, at 10 minutes into exercise and every 20 minutes thereafter until exhaustion. Venous blood (3 ml) was collected at rest, after warm-up, at 20-minute intervals during the trials, at exhaustion, and at 1 and 24 h post-exercise. Heart rate (Sport Tester PE 3000, Polar, Finland), ratings of perceived exertion (RPE), core and skin temperatures (Libra Medical ET 300R, USA), room temperature and relative humidity (whirling hygrometer, Brannan, England) were recorded every of 10 minutes throughout the exercise test. After completion of the run, the subjects towel-dried themselves and post-exercise nude body weight was measured.

Hematocrit was estimated in EDTA-treated blood on a microhematocrit centrifuge (Hettich-Haematokrit 20, Germany) and reader (Hawksley, England), and the percent change in plasma volume was calculated using the formula of van Beaumont et al., 1981. For the estimation of malondialdehyde and creatine kinase, 1 ml of EDTA-treated blood was centrifuged at 4000 rpm (Hettich-Rotina 46RS, Germany) and at 4^oC for 10 minutes, and the plasma from this sample was then divided into two equal portions and stored at -80^oC (Heto Ultra Freeze 3410, Denmark) for subsequent analysis of malondialdehyde and creatine kinase. Plasma malondialdehyde was determined as thiobarbituric acid reactive substances (TBARS) using high performance liquid chromatography (HPLC) (Nielsen et al., 1997). Plasma creatine kinase was analysed using a commercially available kit (Randox, U.K.) and determined colori-metrically (Hitachi Automatic Analyzer 912, Bohringer Mannheim, Germany). The remainder of the blood sample was allowed to clot and then centrifuged at 4,000 rpm for 10 minutes and 4^oC. The supernatant was divided into two portions and stored at -80^oC for the analysis of serum vitamin E and total antioxidant status. Serum total antioxidant status was analysed colorimetrically (Hitachi Automatic Analyzer 912, Bohringer Mannheim, Germany) using a reagent kit (Randox, U. Kingdom). Serum vitamin E was determined using HPLC (Agilent 1100 series, Agilent Technologies, Germany) after extraction with hexane. Briefly, 50 l of the sample was introduced through the injector into 2 normal phase Zorbax 5 micron (250 X 4.6 mm) columns connected in series. The mobile phase was 99.25:0.75 mixture (hexane: 2-propanol); which was set at a flow rate of 2 ml·min^-1. Hexane and 2-propanol mixture used in conjunction with Zorbax silica column has been found to be the optimal normal-phase system for the analysis of tocopherol and tocotrienol isomers (Tan and Brzuskiewicz, 1989). Isomers of vitamin E were detected with a fluorescence detector at EX (excitation wavelength) of 295 nm and EM (emission wavelength) of 325 nm. Standards for tocopherols/tocotrienols were purchased from Merck, Germany.

Statistical analysis
Two-way analysis of variance (ANOVA) with repeated measures was used to compare differences between groups. Bonferroni post-hoc test was used to determine significant mean differences. Comparison of the ratings of perceived exertion was done using Wilcoxon Signed-Rank test. The accepted level of significance was set at p < 0.05. Data are expressed as means ± SE.

Mean room temperature and relative humidity (RH) in the groups were well maintained at 30.9 ± 0.1° C, 70.1 ± 0.2% RH and 31.0 ± 0.1° C, 70.3 ± 0.3% RH respectively. Mean oxygen consumption during exercise was similar in both the groups (40.1 ± 0.4 ml·kg^-1·min^-1 and 40.6 ± 0.5 ml·kg^-1·min^-1 in the E-supplemented and placebo-supplemented groups respectively). These figures correspond to 70.1 ± 0.6% and 70.6 ± 0.7% of VO₂max, which was maintained during exercise in the E-supplemented and placebo-supplemented groups respectively. Running time of heat-adapted individuals to exhaustion was not significantly different between the E-supplemented and placebo-supplemented groups (81.1 ± 4.5 min vs. 76.9 ± 4.5 min respectively).

Serum vitamin E concentrations increased significantly (p < 0.001) after six weeks of tocotrienol-rich palm vitamin E supplementation (E-supplemented group; Figure 1A). In contrast, there were no significant differences in serum vitamin E before and after placebo supplementation. Serum vitamin E concentrations at exhaustion were higher when compared to their respective resting values in both groups but it was only statistically significant (p < 0.05) in the placebo-supplemented group. All differences calculated for serum vitamin E remained statistically significant even after the data were adjusted for plasma volume changes except between exhaustion and resting values in the placebo-supplemented group.

Resting serum total antioxidant status (TAS) in both groups was not different from their respective pre-supplementation values. However, serum TAS was significantly higher (p < 0.001) at exhaustion and at 1 h post-exercise when compared to their corresponding resting levels in both the groups (Figure 1B). Serum TAS at 24 h post-exercise was significantly (p < 0.05) lower than at exhaustion in the E-supplemented group. Similarly, in the placebo group serum TAS levels at exhaustion and 1 h after exercise were significantly higher than that at 24 h post-exercise (p < 0.001). In the E-supplemented group however, significant difference was only evident between the levels at exhaustion and 24 h after exercise (p<0.05) but not between 1 and 24 h post exercise. There were however no significant differences in serum TAS between the two groups.

Resting plasma malondialdehyde (MDA) levels were significantly lower (p < 0.05) after supplementation in the E-supplemented group when compared to the placebo-supplemented group (Figure 2A). Although at exhaustion, mean MDA level was lower in the E-supplemented group compared to the placebo-supplemented group, this difference, however, was not statistically significant (p = 0.090).

Creatine kinase (CK) levels at exhaustion, 1 h and 24 h post-exercise were significantly (p < 0.001) higher than their corresponding resting values in both groups (Figure 2B). Plasma CK levels, although were higher in the placebo-supplemented group during and after exercise, but when compared to the E-supplemented group, the differences were however not statistically significantly. All statistically significant differences for the plasma CK levels were maintained even after correction for changes in plasma volume.

Core temperature increased significantly (p < 0.001) over time from rest until exhaustion in both the groups (Figure 3A). However, there were no significant differences in core temperature between the two groups. Core temperature at exhaustion was 39.3 ± 0.1° C and 39.4 ± 0.1° C in the E-supplemented and placebo-supplemented groups respectively. RPE increased significantly from rest until exhaustion (p < 0.001) in both the groups (Figure 3B). However, RPE scores were similar throughout exercise in both groups. RPE at exhaustion was 18.7 ± 0.2 in both groups.

Plasma volume decreased over time during exercise in both groups (p < 0.001) (Table 1). Compared to the resting level, plasma volume in the E-supplemented groups was not different at 1 h post-exercise but was higher (p < 0.001) at 24 h post-exercise. Plasma volume in the placebo-supplemented group was not different at 1 h and 24 h post-exercise when compared to the resting level. The plasma volume changes during and after exercise were similar between E-supplemented and placebo-supplemented groups.

Although the subjects in this study were given a tocotrienol-rich palm vitamin E supplement, the concentrations of serum tocotrienol were very low despite a 6-week supplementation regimen. The reason for this remains unclear although similar findings have also been reported before (Mensink et al., 1999; Khor et al., 1995). The fate of supplemented tocotrienols and the relationship between its intestinal absorption, blood levels, and tissue distribution are still not fully understood (Watkins et al., 1999). It is possible tocotrienol is either metabolized immediately, or converted to alpha-tocopherol, or rapidly taken up by the adipose tissue and displacing alpha-tocopherol from these tissues. In this study, the supplementation of tocotrienol-rich palm vitamin E resulted in a significant increase in alpha-tocopherol but not in tocotrienol. Thus, serum vitamin E concentrations reported in this study are concentrations of alpha-tocopherol.

Pre-supplementation serum alpha-tocopherol levels (Figure 1A) indicate that the subjects in this study were not vitamin E-deficient. Supplementation of tocotrienol-rich palm vitamin E resulted in ~33% increase in serum alpha-tocopherol. Similar increases in serum alpha-tocopherol concentrations, after vitamin E supplementation have also been reported before (Dillard et al., 1978; Itoh et al., 2000; Rokitzki et al., 1994a; Sumida et al., 1989). Owing to its fat solubility alpha-tocopherol is easily retained in the body after absorption.

No significant difference was evident in the endurance running performance in heat following tocotrienol-rich palm vitamin E supplementation when compared to the placebo-supplemented group (81.1 ± 4.5 min vs 76.9 ± 4.5 min in the E-supplemented and placebo-supplemented groups respectively). Similar findings have also been reported before (Lawrence et al., 1975; Sharman et al., 1971; 1976), where vitamin E supplementation was also found not to improve endurance performance, although these studies were all done in thermoneutral environment and none were on heat-adapted individuals exercising in heat.

Apart from having no effect on endurance running performance, interestingly, tocotrienol-rich palm vitamin E supplementation also did not increase serum TAS (Figure 1B) and this despite a significant rise in serum alpha-tocopherol (Figure 1A) during the same period. The reason for this is not clearly apparent although it has also been reported before following supplementation of vitamins A and E (Jakeman and Maxwell, 1993; Schroder et al., 2000). One possible explanation for this observation is that the measurement of serum TAS reflects the overall antioxidant capacity of the serum, which includes both enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic antioxidants (vitamins A, C and E, glutathione, ubiquinones, α-lipoic acid, flavonoids and uric acid). Palm vitamin E supplementation to non-deficient individuals under resting conditions may be downgrading the synthesis and possibly the activity of some of these endogenous antioxidants, but with little net change in overall TAS activity. Clearly, more studies are needed to ascertain these possibilities. It nevertheless appears that significant elevation of serum vitamin E following supplementation in normal individuals with normal vitamin E levels does not necessarily correlate to overall serum TAS.

Although tocotrienol-rich palm vitamin E supplementation did not alter resting TAS, the concentration of MDA, a marker of lipid peroxidation, was nevertheless significantly lower (p < 0.05) in the E-supplemented group when compared to that in the placebo-supplemented group at rest (Figure 2A), suggesting an increased antioxidant activity at rest. Similar decrease in MDA at rest following supplementation with antioxidant vitamins has been reported before (Itoh et al., 2000; Kanter et al., 1993; Sumida et al., 1989). Interestingly, although tocotrienol-rich palm vitamin E supplementation decreased resting MDA levels, it however did not prevent exercise-induced increase in lipid peroxidation. The levels of plasma MDA at exhaustion increased in both groups (Figure 2A), although statistically significant increase was only evident in the E-supplemented group (p < 0.001) and not in the placebo-supplemented group (p = 0.081). Nevertheless, plasma MDA level in the E-supplemented group at exhaustion was slightly lower than that in the placebo-supplemented group, although the difference did not reach statistical significance (p = 0.099). The reason for this is also not apparent. Compared with the placebo-supplemented group, plasma MDA in the E-supplemented group was 11.1% and 6.7% lower at exhaustion and 1 h post-exercise respectively, suggesting that the positive antioxidant effect that was apparent at rest was carried over during exercise. When viewed together, these results indicate that tocotrienol-rich palm vitamin E supplementation only decreases lipid peroxidation at rest and, perhaps to a very small extent, during exercise in the heat. This evident benefit, albeit small, of tocotrienol-rich palm vitamin E supplementation on lipid peroxidation during exercise appears in contrast to some other studies, which found no beneficial effects of vitamin E supplementation on lipid peroxidation during exercise (Maxwell et al., 1993; Vasankari et al., 1997). The reason for this is unclear but it may be related to the type of vitamin E supplemented or due to different techniques used for the measurement of TBARS in plasma after exercise. However, more studies are needed to verify the benefit of vitamin E supplementation on lipid peroxidation during exercise, possibly measuring other markers of lipid peroxidation at the same time.

Increased oxidative stress is believed to be responsible for the slight muscle damage that follows exhaustive exercise and increases in plasma creatine kinase are considered to indicate muscle damage (McBride et al., 1998; Kanter et al., 1993). We estimated plasma creatine kinase activity to see if tocotrienol-rich palm vitamin E supplementation reduced muscle damage during exercise. Supplementation of tocotrienol-rich palm vitamin E, did not result in any significant changes in plasma creatine kinase (CK) activity from pre-supplementation values in both groups (Figure 2B) and plasma CK activity at exhaustion and post-exercise (1 and 24 hours) increased significantly (p < 0.001) from the resting values and somewhat equally in both groups (Figure 2B). The increase in plasma CK activity was particularly more marked after 24 hours. The delay is possibly due to the length of time it takes for creatine kinase released from damaged muscle fibres to enter the blood. These data suggest that muscle damage did occur during exercise in both trials, and although it was slightly lower in the E-supplemented group, supplementation of tocotrienol-rich palm vitamin E did not prevent muscle damage during strenuous exercise. This is in contrast to what has been observed before where vitamin E supplementation was found to prevent the elevation of enzyme leakage during exhaustive exercise in man and animal (McBride et al., 1998; Kanter et al., 1993). Subjects supplemented with antioxidant vitamins had a significantly lower creatine kinase value at 24 h post-race compared to non-supplemented subjects (Rokitzki et al., 1994a; 1994b). Vitamin E supplementation has also been shown to reduce the leakage of CK following 6 successive days of endurance running (Itoh et al., 2000) or after a 40-min run (Gillam et al., 1992). The reason for the difference in our findings and those mentioned above is unclear but it may be related to the duration of vitamin E supplementation and the hot and humid environment of exercise. In the study of Rokitzki et al., 1994a -tocopherol was supplemented for a period of 5-months, whereas tocotrienol-rich palm vitamin E was only supplemented for 6 weeks in this study.

Endurance performance is influenced by a number of other factors including changes in body core temperature and plasma volume during exercise. We also examined the effects of tocotrienol-rich palm vitamin E supplementation on these. During exercise, body core temperature increased significantly in both the groups reaching values of 39.3° and 39.4° C at exhaustion in the E-and placebo-supplemented groups respectively (Figure 3A), and the changes in body core temperature of the subjects were similar in both groups. The rise in body temperature during exercise is well established and the similar rise in both groups in this study is probably due to the same environmental temperature and relative humidity during the trials.

As was the case with body core temperature, there was also no significant difference in plasma volume changes during exercise in both the groups (Table 1). Similar significant reductions (p < 0.001) in plasma volume, from pre-exercise values, were evident in both groups. Reduction in plasma volume during exercise is a well-established occurrence and, has been attributed to redistribution of body fluids and loss of fluids through sweat and breathing during exercise. Of importance from this study are the findings that tocotrienol-rich palm vitamin E supplementation does not significantly affect changes in body core temperature and plasma volume before, during or after exhaustive exercise.

This is the first study carried out to investigate the effects of tocotrienol-rich palm vitamin E supplementation on lipid peroxidation and endurance running performance in the heat in heat-adapted individuals. It appears that 6 weeks of tocotrienol-rich palm vitamin E supplementation to heat-adapted recreational athletes with normal serum vitamin E levels does not improve endurance running performance. This is in agreement with a number of other studies although in most other studies α-tocopherol was the main source of vitamin E (Lawrence et al., 1975; Sharman et al., 1971; 1976). Despite the claims that tocotrienol is a more potent antioxidant than tocopherol (Serbivona et al., 1991), the present study showed that, as was the case with studies using alpha-tocopherol, endurance running performance was not enhanced after 6-weeks of tocotrienol-rich palm vitamin E supplementation. The reason for the lack of a positive effect of tocotrienol-rich palm vitamin E on endurance performance may be because the subjects in this study had adequate vitamin E stores and therefore additional supplementation did not bring any extra benefit to them. It is possible potential benefits may only be evident in vitamin E deficient individuals. The results of the present study however do indicate that supplementation with tocotrienol-rich palm vitamin E decreases the level of lipid peroxidation at rest.

Our special thanks are due to all the subjects who participated in this study. We wish to express our sincere appreciation to the Malaysian Palm Oil Board (MPOB) for providing the tocotrienol-rich palm vitamin E and placebo capsules and allowing us to perform the analysis of serum vitamin E at their Nutrition Laboratory. We also wish to extend our gratitude to B. Braun Medical Supplies (M) for providing the extension tubings, which were used for blood collection during the experimental trials. This research was financially supported by a short-term grant (304/PPSP/6131278) from Universiti Sains Malaysia.

Tocotrienol-rich palm vitamin E supplementation for 6 weeks:

• reduced lipid peroxidation at rest.
• did not enhance endurance running performance in the heat.
• did not prevent exercise-induced muscle damage as indicated by CK activity.