Malaysian Journal of Medical Sciences School of Medical Sciences, Universiti Sains Malaysia ISSN: 1394-195X Vol. 9, Num. 2, 2002, pp. 34-42

Malaysian Journal of Medical Sciences, Vol. 9, No. 2, July 2002, pp. 34-42

Role Of Vitamin E On Oxidative Stress In Smokers

Nafeeza Mohd. Ismail, Asma Harun**, Ahmad Asmadi Yusof, Zaiton Z*, and Alini Marzuki**,

Depts. of Pharmacology, *Physiology, Faculty of Medicine, **Dept. of Pharmacy, Faculty of Allied Health Sciences, Universit1 Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
Correspondence: Assoc. Prof. Dr. Nafeeza Mohd. Ismail, M.B.B.S., Ph.D. Dept. of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.
e-mail: nafeeza@medic.ukm.my

Submitted-22.5.2001
Revised-21.8.2001
Accepted-16.11.2001

Code Number: mj02015

Cigarette smoke contains numerous oxygen free radicals that when inhaled, overwhelm antioxidant defenses and produce a condition of oxidative stress. This study investigated whether or not supplementation with vitamin E can affect the state of oxidative stress in healthy smokers. In this randomised double blind trial, 32 smokers received 200 mg of vitamin E or placebo daily for 8 weeks. All smokers in the vitamin E group completed the trial whilst only nine in the placebo group completed the trial. Plasma vitamin E concentrations increased significantly [P<0.02] in the vitamin E group. The release of malondialdehyde [MDA] from erythrocytes was not significantly different between the two groups at baseline and was clearly reduced [P<0.01] after 8 weeks of vitamin E supplementation. Vitamin E increased erythrocyte superoxide dismutase activity [P<0.02] and decreased gluthathione peroxidase activity [P<0.02]. No changes were detected in plasma MDA. We conclude that daily supplementation with 200 mg of vitamin E for 8 weeks improved the oxidative stress state in smokers.

Key words : Vitamin E, smokers, oxidative stress, antioxidants

INTRODUCTION

Cigarette smoke has an abundance of oxidants that lead to a tremendous production of free radicals. There is overwhelming evidence from epidemiological and experimental studies showing the detrimental effects of free radicals on health. The body has the ability to produce endogenous antioxidants such as superoxide dismutase, catalase and glutathione peroxidase.

Under normal circumstances, there is a balance between these endogenous antioxidants and the production of free radicals in the body. In the instance where there are excessive free radicals, the available tissue antioxidants may become depleted, leading to oxidative damage as in the case of cigarette smoke. The most logical course to defeat smoking is the implementation of smoking cessation programmes, but given the poor success rates of these programmes, there is compelling evidence for a beneficial role of exogenous antioxidants to minimise smoking-related oxidative damage.

Compounds, which possess antioxidant properties, have the potential to decrease oxidative stress and thus may protect against smoking-induced pathology. Compounds that have been investigated are lipoic acid, taurine, ubiquinone, selenium, garlic, ginkgo biloba and polyphenols (1 - 6). Other compounds, which have been widely tried for their antioxidant properties, are vitamins such as alpha-tocopherol (7,8), and ascorbic acid (9, 10). Vitamin C doses of between 500 mg and 2000 mg have produced controversial results (9, 10). Despite these antioxidative and anti atherogenic effects, the clinical results of vitamin E supplementation in human subjects varied (7, 8). The Cambridge Heart Antioxidant Study using alpha-tocopherol showed that a higher dose of 800 iu daily did not have additional advantage over a dose of 400 iu daily (7).

Of the tocopherols, alpha-tocopherol exhibits the greatest antioxidant potential in vivo. Although there is no clear evidence that the antioxidant capacity of tocotrienols is superior to tocopherols, tocotrienols have been found to be more potent antioxidants (11). Therefore, the objective of this study was to assess the effect of a tocotrienol-rich mixture on the status of oxidative stress in otherwise healthy smokers.

Methods

1) Study subjects

a) Recruitment of study subjects

Subjects were volunteers who responded to an advertisement to participate in this study. Thirty two volunteers who satisfied all the criteria for inclusion were included in the study.

b) Inclusion criteria

The criteria for inclusion were males, aged between 25 - 45 years, who are current smokers and had been smoking 20 cigarettes/day for at least 5 years.

c) Baseline investigations

All subjects underwent a clinical examination. Blood samples (10 ml) were drawn after 12 hour of fasting for lipid profile, blood sugar, liver and renal function tests. Lung function test was performed and FEV₁, FVC and FEV₁/FVC were determined. Chest X-ray and an electrocardiogram [ECG) were done.

d) Health status

All volunteers did not have a history of asthma or other respiratory illnesses. All volunteers had normal biochemical parameters, lung function test, chest X-ray and ECG.

e) Drug and additional vitamin E restriction

During the study, drugs such as aspirin and allopurinol as well as additional intake of vitamin C or vitamin E were not permitted.

2) Study design

a) Ethical clearance

This study was approved by the Ethics committee of the Faculty of Medicine, Universiti Kebangsaan Malaysia.

b) Randomisation

The volunteers were randomised into two experimental groups of 16 subjects.

c) Vitamin E dose

The volunteers were given either 200 mg soft gel capsules of vitamin E containing approximately 30 % tocopherol [TF] and 70 % tocotrienol [TT] or placebo.

d) Placebo

The placebo was visually identical to the vitamin E soft gels and contained palm olein of negligible vitamin E content.

e) Treatment and duration

The period of treatment for both groups were 8 weeks. The groups consisted of all males, with a mean age of 31.6 ± 8.9 years. All subjects signed an informed consent form, which was approved by the Universiti Kebangsaan Malaysia.

3) Chemical analyses

The release of MDA from erythrocytes in vitro was used as a functional measure of vitamin E status in the subjects. The plasma vitamin E levels were determined using HPLC. The indicators measured to reflect the status of oxidative stress were plasma MDA, antioxidant enzymes such as superoxide dismutase and glutathione peroxidase. All parameters were measured at the beginning and at the end of the treatment period.

a) Measurement of MDA release from erythrocyte

The determination of MDA release from erythrocytes in vitro was done according to a method described previously (12). Isolated erythrocytes were washed twice with phosphate buffered saline at a pH of 7.4. Two aliquots of 0.2 ml erythrocytes were placed in two separate test tubes. One aliquot was placed in a test tube (test tube 1) containing 3.8 ml phosphate buffered saline and the second aliquot of packed RBCs was suspended in another test tube (test tube 2) containing 3.8 ml phosphate buffered saline to which sodium azide had been added. Both suspensions were vortexed for 15 seconds.

One ml of the erythrocyte suspension was taken from test tubes 1 and 2 and mixed with 1.0 ml of 3 % hydrogen peroxide and 0.75 % hydrogen peroxide, respectively. These samples were prepared for duplicate incubation. All test tubes were vortexed for 10 seconds prior to incubation at 37×C in a shaking water bath. After an hour, 1 ml of trichloroacetic acid (TCA) in sodium arsenite was added to all tubes. Thiobarbituric acid (1 ml) was added to 2 ml of supernatant that was removed from each tube following centrifugation (3000 x g for 10 minutes). These specimens were then boiled for 10 minutes in a water bath, cooled to room temperature and absorbance at 535 determined using a spectrophotometer. The concentration of MDA in the samples was obtained from a standard absorption curve for MDA.

b) Determination of plasma vitamin E levels

The sample preparation and the analysis of vitamin E by HPLC according were performed to a method described by Lang et al. (13). The Gilson HPLC system was used with a fluorescent detector. Vitamin E was detected at an excitation wavelength of 294 nm and an emmision wavelength of 330 nm. The concentration of vitamin E was determined using a standard curve.

c) Determination of plasma MDA

The plasma content of MDA was determined using a method described by Ledwozyw et al. (14) with some modifications. A plasma sample of 0.5 ml was acidified with 2.5 ml of 1.22 M trichloroacetic acid /0.6 M HCl and left to stand at room temperature for 15 min after which 1.5 ml of 0.67% thiobarbituric acid/0.05 M NaOH was added. The samples were incubated in a 100^oC water bath for 30 min. Subsequently it was left to cool at room temperature before the addition of 4 ml of n-butanol. After thorough mixing, the mixture was centrifuged for 10 min. at 1500 x g. The absorbency of the upper phase was read at 535 nm. Various amounts of MDA standard, freshly prepared by acidification of 1,1,3,3-tetraethoxypropane were subjected to the identical test procedure as the basis for constructing a standard curve for thiobarbituric acid reactivity as MDA equivalent.

d) Analysis of superoxide dismutase

The measurement of this enzyme was done in erythrocytes according to previously described methods (15). This method was based on the ability of superoxide dismutase (SOD) to inhibit the reduction of nitro blue tetrazolium whereby 1 unit of SOD was taken as a 50% inhibition of reduction of nitro blue tetrazolium. A mixture that consisted of a substrate solution (L-methionine, Triton X-100 and nitro blue tetrazolium) and 20 ml sample of lysed erythrocytes or PBS (control) were vortexed. A riboflavine mixture (riboflavine, 0.1 ml of EDTA in PBS, pH 7.8) was then added. The tubes were then placed in a box lined with aluminium paper and lighted with a flourescent lamp (20 W). After 7 minutes, the absorbance of the mixture at 560 nm was determined using a spectrophotometer (Shimadzu UV-160 A) against a blank without the sample.

e) Analysis of glutathione peroxidase

The method used was that of Lawrence & Burk (16). A 0.1 ml sample of lysed erythrocytes and a buffered solution 1 mM NaN₃, 1mM EDTA, 0.2 mM NADPH, 50 units of glutathione reductase (GSSG) and 1mM GSH, 50mM PBS, (pH 7) was incubated at room temperature for 5 minutes, then 0.1 ml 0.25 mM hydrogen peroxide was added. The absorbance of the mixture at 340 nm was determined using a spectrophotometer (Shimadzu UV-160 A) against a blank without the sample.

4) Determination of lung function

Spirometric measurements were made using a Cosmed Pony Graphic 3.5 spirometer with subjects in a sitting position. After practice blows, recordings were repeated until three satisfactory tracings were obtained. Analysis was based on the average of these three readings. The measurement that was considered in this study was the percent ratio of FEV₁ and FVC.

5) Statistical Analysis

All values are expressed as mean ± S.E.M. Wilcoxon's nonparametric signed rank test for paired observations was used for comparison of all parameters before and after supplementation with vitamin E. A p value <0.05 was considered significant.

RESULTS

Plasma Vitamin E levels

There was no significant difference in total plasma vitamin E levels between the two groups at baseline (fig. 1). The plasma levels increased significantly (p<0.02) fig. 1 or fig 2 between the two groups after 8 weeks of supplementation with vitamin E. There was no difference in plasma vitamin E levels in the placebo group.

Effects of Vitamin E Supplementation on Erythrocyte Antioxidant Defence

There was no significant difference in the extent of erythrocyte susceptibility to peroxidation between the two groups at baseline (fig. 2). Vitamin E supplementation reduced erythrocyte release of MDA compared to the respective baseline levels (p<0.01]. Erythrocytes from the placebo group showed a greater susceptibility to hydrogen-induced lipid peroxidation than those from the vitamin E supplemented group (p<0.030). Before supplementation, erythrocyte GSHPx and SOD activities were comparable. After supplementation with vitamin E, the erythrocyte SOD activity was increased (p<0.02) and the erythrocyte GSHPx activity decreased (p<0.02) compared to the respective baseline levels.
(fig. 3) (fig. 4) (fig. 5)

Plasma MDA Levels

There was no difference in baseline levels of plasma MDA levels between the two groups. Vitamin E supplementation did not affect the plasma MDA levels.

Lung Function Test

There was no difference in FVC (fig. 6), FEV₁ (fig.7) and the percent ratio of FEV₁ and FVC (fig. 8) before or after supplementation with vitamin E between the two groups. There was also no difference in the FVC, FEV₁ and the percent ratio of FEV₁ and FVC after vitamin E supplementation compared to the baseline values.

DISCUSSION

Presently, it is widely known and accepted that free radical mediated lipid peroxidation has a crucial role in the pathogenesis of many disease processes such as atherosclerosis, diabetes mellitus, carcinogenesis, inflammation and many other conditions. Hence, the use of various antioxidant supplements to prevent or reduce damage to biological tissues are currently extensively investigated in various disease conditions. The amount of exogenous antioxidants required by individuals will be influenced by the oxidative stress status of the individual as this will affect the endogenous cellular antioxidant defence system. Antioxidant defences in the lung are provided by endogenous enzyme systems and by dietary antioxidants, particularly vitamin C and E (16 -18). Smokers have a higher index of oxidative stress resulting from inhalation of cigarette smoke that is known to contain oxidizing substances. Consequently, smokers have been shown to have a lower antioxidant status (19 - 24) that predisposes them to developing free radical mediated lung injury.

In this study, we assessed the antioxidant defences [in individuals with high oxidant stress] in response to supplementation with vitamin E as an antioxidant and compared this response to a placebo intake. It has previously been shown that erythrocytes from smokers are more susceptible to lipid peroxidation (25). This study demonstrated that vitamin E reduces the susceptibility of erythrocytes to lipid peroxidation. The end product of lipid peroxidation, MDA was measured by fluorometric determination. Although this assay is not specific only for MDA and measures other substance that can react with thiobarbituric acid and give rise to the colored end points detected, the modifying effect of vitamin E on the generation of TBA-reacting species implies that it is a valid indicator of peroxidation in vitro.

It has also been shown that erythrocyte activity is increased in smokers and this may reflect an increased spontaneous regeneration of superoxide in the cells (26). We found that vitamin E supplementation can further increase the SOD. The function of SOD is to metabolise the oxygen radical (O₂^-). Vitamin E is also a scavenger for superoxide. Therefore, in the presence of vitamin E, SOD is utilized less thus the pool of SOD is increased. On the other hand, the constant intake of vitamin E ensures its continued presence and increases the process of scavenging O₂^-, thereby generating hydrogen peroxide, the substrate for GSHPx. With increased production of its substrate, the utilization of GSHPx is more. There is a consequent fall in the reserves of GSHPx as the cellular utilization of GSHPx exceeds the production of this enzyme. Other studies have shown that there is a decline in glutathione (GSH) concentration with increasing erythrocyte vitamin E concentration and this may indicate an increased utilization of GSH in the reductive regeneration of the vitamin E its peroxyl radical (25). Glutathione depletion studies in mice demonstrate that the rate of decline of total GSH in tissues and blood reflects its rate of utilization (27).

There have been various reports on the intake of antioxidants and lung function (28 -30). Although this study demonstrates that vitamin E can improve the antioxidant status, there was no improvement of lung function. Britton et al. showed that a combined intake of vitamin C and E correlated positively with FEV₁ and FVC. It is possible that a higher dose of vitamin E is necessary to achieve an improvement in lung function, hence the improvement seen when vitamin E intake is combined with vitamin C (30). The fat-soluble vitamin E and water-soluble vitamin C are thought to act cooperatively in a system whereby vitamin E, which is mainly sequestered in cell membranes and other lipid structures, is maintained in a reduced state by interacting with water-soluble vitamin C.

The endogenous and exogenous factors influencing the cellular protection against free radicals are many and varied. Our study suggests that one such factor is the constant availability of vitamin E in the body. Although vitamin E improved the integrity of erythrocytes membrane shown by improved protection against lipid peroxidation in vitro, it also affected the regulation of the endogenous antioxidant enzymes. Until the biological relevance of this is made clear, the recommendation of vitamin E supplements against smoking induced lipid peroxidation should be done cautiously.

ACKNOWLEDGEMENT

The study was jointly supported by Renong Berhad and Bank of Commerce (Grant code number D/4/96).

REFERENCES

1. Franconi, F. Plasma and platelet taurine are reduced in subjects with insulin-dependent diabetes mellitus: Effects of taurine supplementation. Am. J. Clin. Nutr. 1995; 61, 1115-1119.
2. Weber, C. Antioxidative effect of dietary Coenzyme Q10 in human plasma. Internat. J. Vit. Nutr. Res. 1994; 624, 311-315.
3. Imai, J. Antioxidant and radical scavenging effects of garlic extract and its constituents. Planta Med. 1994; 60, 417-420.
4. Marcocci, L. Antioxidant action of ginkgo biloba extract Egb 761. Methods in Enzymology 1994; 234, 462-475.
5. Fuhrman, B. Consumption of red wine with meals reduces the susceptibility of human plasma and low density lipoprotein to lipid peroxidation. Am. J. Clin. Nutr. 1995; 61, 549-554.
6. Kagan, V. E. Dihydrolipoic acid, a universal antioxidant both in the membrane and in the aqueous phase. Biochemical 1992; 44, 1637 - 1649.
7. Stephans, N. G., Parsons, A., Schofield, P. M., Kelly, F., Cheeseman, K. and Mitchinson M.J. randomised controlled trial of vitamin E in patients with coronary heart disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 1996; 347, 781 - 786.
8. Stampfer, M., Hennekens, C., Manson, J. E., Colditz, G. A. and Rosner, B. Vitamin E consumption and the risk of coronary disease in women. N. Engl. J. Med. 1993; 328 (20), 1444 -1449.
9. Brown, K.M., Morrice, P.C., Duthie. Erythrocyte vitamin E and ascorbate concentrations in relation to erythrocyte peroxidation in smokers and non smokers: dose response to vitamin E supplementation. Am. J. Clin. Nutr. 1997; 65, 496 - 502.
10. Aghdassi, E., Royall, D. and Allard, J.P. Oxidative stress in smokers supplemented with vitamin C. Internat. J. Vit. Nutr. Res. 1999; 69, 1.
11. Suarna, C., Hood, R.L, Dean, R.T. and Stocker R. Comparative antioxidant activity of tocotrienols and other natural lipid soluble antioxidants in a homogeneous system, and in rat and human lipoproteins. Biochim. Biophys. Acta., 1993; 1166, 163 - 170.
12. Cynamon, H. A., Isenberg, J.N. and Nguyen, C.H. (1985) Erythrocyte malondialdehyde release in vitro: a functional measure of vitamin E status. Clinica. Chimica. Acta., 1993; 151, 169 - 176.
13. Lang, J.K., Gohil, K. and Packer, L. Simultaneous determination of tocopherol, ubiquinols and ubiquinones in blood, plasma, tissue homogenates and subcellular fractions. Analytical Biochemistry, 1986; 157, 106-116.
14. Ledwozyw, A., Michalak, J., Stepien, A. and Kadziolka, A. The relationship between plasma triacylglycerols, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clinica. Chimica. Acta. 1986; 155, 275-284.
15. Beyer, W.F. and Fridovich, I. Assaying for superoxide dismutase activity: some large consequences of minor changes in condition. Anal. Biochem. 1987; 161, 559 - 566.
16. Bast, A., Haenen G.R.M.M. and Doelman J.A. Oxidants and antioxidants: State of the art. Am J. Med. 1991; 91,2S-13S.
17. Cantin A. and Crstal R.G. Oxidants, antioxidants and the pathogenesis of emphysema. Eur. J. of Respir. Dis. 1985; 66, 7- 17.
18. Heffner J.E. and Repine J.E. Pulmonary strategies of antioxidant defense. Am. Rev. Respir. Dis. 1989; 140, 531-554.
19. Brooke M. and Grimshaw J.J. Vitamin E concentration of plasma and leukocytes as related to smoking habit, age, and sex of humans. Am. J. Clin. Nutr. 1968; 21,1254-1258.
20. Calder, J.H., Curtis, R.C. and Fore, H. Comparison of vitamin C in plasma and leukocytes of smokers and non-smokers. Lancet i, 1963; 556.
21. Schectman, G., Byrd, J.C., and Gruchow, H.W. The influence of smoking on vitamin C status in adults. Am J. Public Health 79, 1989; 158-162.
22. Kallner, A.B., Hartmann, D. and Hornig, D.H. On the requirements of ascorbic acid in man: steady state turnover and body pool in smokers. Am. J. Clin. Nutr. 1981; 34, 1347-1355.
23. Duthie, G.G., Authur, J.R., James, W.P. Effects of smoking and vitamin E on blood antioxidant status. Am. J. Clin. Nutr. 1991; 53, 1061S.
24. Jendryczko, A., Szpyrrka, G., Gruszczynski, J. and Kozowicz, M. Cigarette smoke exposure of school children: Effect of passive smoking and vitamin E supplementation on blood antioxidant status. Neoplasma, 1993; 40(3), 199-203.
25. Duthie, G.G., Authur, J.R., and Beattie, J.A.G. J. Cigarette smoke, antioxidants, lipid peroxidation, and coronary heart disease. Ann. NY Acad. Sci. 1993; 686, 120-129.
26. Scarpa, V., Viglino, P.Cotri, D. and Rigo, A. Generation of superoxide ion in human red blood cell lysates. J. Biol Chem. 1984; 259, 10657-10659.
27. Griffith, O.W. and Meister, A. Glutathione:interorgan translocation, turnover and metabolism. Proc Natl Acad Sci U.S.A. 1979; 76, 5606-5610.
28. Grievink, L, Jansen S.M.A, Veer, P.V. and Bruneureef, B. Acute effects of ozone on pulmonary function of cyclist receiving antioxidant supplements. Occup Environ Med. 1998; 55, 13-17.
29. Dow, L., Tracey, M., Villar, A., Coggon, D., Margetts, B.M., Campbell, M.J., and Holgate, S.T Does dietary intake of vitamin C & E influence lung function in older people? Am J Respir Crit Care Med. 1996; 154(5), 1401-1404.
30. Britton, J.I., Parord, K. Richards, A. Knox, A. and Wisniewski et al. Dietary antioxidant vitamin intake and lung function in the general population sample. Am J Respir Crit Care Med. 1995; 151, 1383-1387.

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