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J Am Coll Cardiol, 2000; 35:277-283 © 2000 by the American College of Cardiology Foundation |
a Department of Cardiology, University of Vienna, Vienna, Austria
* Department of Clinical Chemistry, University of Vienna, Vienna, Austria
Institute for Medical Statistics, University of Vienna, Vienna, Austria
Department of Cardiology, University of Innsbruck, Innsbruck, Austria
Manuscript received February 2, 1999; revised manuscript received August 16, 1999, accepted October 18, 1999.
Reprint requests and correspondence: Dr. Thomas Neunteufl, Department of Cardiology, University of Vienna Medical School, Währinger Gürtel 18-20, A-1090 Vienna, Austria
TNEUNTEUFL{at}pop3.kard.akh-wien.ac.at
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Abstract |
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The aims of this study were to determine whether chronic or acute impairment of flow mediated vasodilation (FMD) in the brachial artery of smokers can be restored or preserved by the antioxidant vitamin E.
BACKGROUND
Transient impairment of endothelial function after heavy cigarette smoking and chronic endothelial dysfunction in smokers result at least in part from increased oxidative stress.
METHODS
We studied 22 healthy male smokers (mean ± SD, 23 ± 9 cigarettes per day) randomly assigned to receive either 600 IU vitamin E per day (n = 11, age 28 ± 6 years) or placebo (n = 11, age 27 ± 6 years) for four weeks and 11 age-matched healthy male nonsmokers. Flow mediated vasodilation and endothelium-independent, nitroglycerin-induced dilation were assessed in the brachial artery using high resolution ultrasound (7.5 MHz) at baseline and after therapy. Subjects stopped smoking 2 h before the ultrasound examinations. At the end of the treatment period, a third scan was obtained 20 min after smoking a cigarette (0.6 mg nicotine, 7 mg tare) to estimate transient impairment of FMD.
RESULTS
Flow mediated vasodilation at baseline was abnormal in the vitamin E (5.3 ± 3.8, p < 0.01) and in the placebo group (6.4 ± 3.5, p < 0.05) compared with nonsmoking controls (11.6 ± 4.7). Using a two-way repeated measures analysis of variance (ANOVA) to examine the effects of vitamin E on FMD, we found no effect for the grouping factor (p = 0.5834) in the ANOVA over time but a highly significant difference with respect to time (p = 0.0065). The interaction of the time factor and the grouping factor also proved to be significant (p = 0.0318). Flow mediated vasodilation values remained similar after treatment for four weeks in both groups but declined faster after smoking a cigarette in subjects taking placebo compared with those receiving vitamin E (p values from successive differences for the time/group factor: 0.0001/0.0017). The transient attenuation of FMD (calculated as the percent change in FMD) was related to the improvement of the antioxidant status, estimated as percent changes in thiobarbituric acid-reactive substances (r = –0.67, p = 0.0024). Nitroglycerin-induced dilation did not differ between study groups at baseline or after therapy.
CONCLUSIONS
These results demonstrate that oral supplementation of vitamin E can attenuate transient impairment of endothelial function after heavy smoking due to an improvement of the oxidative status but cannot restore chronic endothelial dysfunction within four weeks in healthy male smokers.
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Epidemiologic studies have found an inverse association between the frequency of coronary artery disease (CAD) and dietary intake of antioxidant vitamins (8,9). However, a possible cause and effect relation between the intake of antioxidants and a reduction in the complications of CAD has thus far only been shown for vitamin E (10).
With regard to the effects of antioxidant vitamins on endothelial function, controversial results have been obtained. Improvement of endothelial dysfunction in smokers was noticed in the forearm vasculature after intraarterial infusion of the watersoluble antioxidant vitamin C (11) and in the brachial artery after intravenous vitamin C infusion (12). The beneficial effect of vitamin C supplementation was associated with a decrease in thiobarbituric acid–reactive substances (TBARS) as an index of oxidative stress. In contrast, oral application of vitamin C for one month showed no beneficial effect on endothelial function in the forearm vessels of patients with hypercholesterolemia (13) but resulted in acute improvement of endothelium-dependent vasodilation in the brachial artery of patients with CAD when applied at a higher dose (14).
Recently, we demonstrated that the oral intake of vitamin E, one of the main lipid-soluble antioxidants in human plasma lipoproteins, in addition to lipid lowering therapy, restored vascular reactivity in the brachial artery of hypercholesterolemic men (15). In contrast, vitamin E alone failed to improve endothelial function in forearm circulation of patients with hypercholesterolemia (13). However, the effect of vitamin E supplementation on endothelial function in the brachial artery of smokers is unknown.
In this study, we hypothesized that vitamin E supplementation, through an antioxidant effect, may restore chronic or preserve acute impairment of endothelium-dependent vasodilation in the brachial artery.
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Methods |
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Assessment of vasodilation and blood flow. Endothelium-dependent FMD following reactive hyperemia and endothelium-independent nitroglycerin-induced dilation (NMD) were examined in the brachial artery according to the method described by Celermajer et al. (16). Using high resolution ultrasound (7.5 MHz linear array transducer), measurements of the right brachial artery were taken at rest after lying quietly for at least 10 min, after cuff deflation completing suprasystolic compression (250 mm Hg for 4.5 min) of the right upper arm and after sublingual application of 0.8 mg nitroglycerin. Scans of the brachial artery were taken proximal to the bifurcation of the radial and the ulnar artery above the antecubital fossa at end diastole, incident with the R wave on a continuously recorded electrogram by the same ultrasound operator. Diameter measurements were taken from one media-adventitia interface to the other for at least three times at baseline and every 30 s following reactive hyperemia and after administration of nitroglycerin. The maximum FMD and NMD diameters were calculated as the average of the three consecutive maximum diameter measurements following hyperemia and nitroglycerin, respectively. Vasodilation was then calculated as the percent change in diameter compared with baseline. The impairment of FMD after heavy smoking (delta FMD) was estimated as the percent change in FMD values compared with baseline.
In all subjects, blood flow was calculated at rest and within 15 s after cuff deflation by multiplying the velocity time integral of the flow signal by the heart rate and the vessel cross-sectional area (3.14 x D2/4). Reactive hyperemia was then calculated as percent change in blood flow during reactive hyperemia compared with baseline.
Ultrasound measurements were performed at baseline and after therapy for four weeks. Subjects stopped smoking 2 h before the examinations. At the end of the treatment period, a third scan was obtained 20 min after smoking a cigarette (0.6 mg nicotine, 7 mg tare) to estimate transient impairment of FMD.
The mean range of intraobserver difference for measurements of baseline diameter was 0.10 ± 0.07%, that of %FMD 2.4 ± 2.2% and that of %NMD 2.7 ± 1.9% as reported elsewhere (17).
Determination of lipids and lipoproteins. Cholesterol and triglycerides were measured enzymatically using assay kits from Boehringer Mannheim (Mannheim, Germany). High-density lipoprotein cholesterol was measured from the supernatant after precipitation with polyethylene glycol (Reagent A from Immuno A.G., Vienna, Austria). Low-density lipoprotein cholesterol (LDL-C) was calculated according to the Friedewald equation.
Alpha-tocopherol measurements in human plasma samples. The neutral lipid fraction (200 µl) including alpha-tocopherol, the most active form of vitamin E, was extracted in a basic extraction system consisting of 200 µl ethanol, 200 µl H2O and 200 µl hexane. The upper organic layer (20 µl) containing alpha-tocopherol was analyzed by high-performance liquid chromatography using an ExSil 100 20 x 0.46 cm silica column (mobile phase: hexane/1% ethanol, 1 ml/min) and fluorescence detection (Hitachi, E: 295 nm/Em: 390 nm) (18). Quantitation was performed by peak area comparison with external alpha-tocopherol standards of known concentrations. The alpha-tocopherol serum level was then normalized to LDL-C because there is evidence in the literature that the vitamin E content in LDL-C and not in the whole plasma correlates with lipid peroxidation (19,20). The increase of alpha-tocopherol/LDL-C (delta alpha-tocopherol/LDL-C) under therapy was calculated as the percent change in alpha-tocopherol/LDL-C.
Determination of malondialdehyde with thiobarbituric acid. The measurements of TBARS are based on the reaction of malondialdehyde, a secondary breakdown product of lipid hydroperoxides, with thiobarbituric acid (TBA). The assay was performed as described previously (21). The plasma sample was mixed with two volumes of cold 10% trichloroacetic acid for protein precipitation. Following centrifugation, the supernatant was mixed with an equal volume of 0.67% TBA in a boiling water bath for 10 min. After cooling, the absorbency was measured at 532 nm, and concentration of malondialdehyde was calculated from epsilon = 153 000 M–1 cm–1 (21). Measurements were performed in duplicate. The decrease of TBARS (delta TBARS) under therapy was calculated as the percent change in plasma TBARS levels.
Statistical analysis. Results are expressed as mean ± standard deviation (SD). Differences between groups were analyzed using one factor analysis of variance (ANOVA) followed by Scheffe’s test, when comparing all three study groups (including nonsmoking controls) and Student t test, when comparing the vitamin E with the placebo group only. To examine whether FMD can be restored or preserved by vitamin E, a two-way repeated measures ANOVA with the group factor vitamin E/placebo and the repeated factor time (three levels: baseline/after four weeks/after heavy cigarette smoking) was performed. Successive differences contrasts between neighboring points in time were calculated as supportive statistical analyses. Univariate analyses of the effects of delta alpha-tocopherol/LDL and delta TBARS on delta FMD as well as of cigarettes smoked per day on TBARS levels were performed with linear regression. Differences were considered significant at p < 0.05.
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Results |
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Comparing the two study groups consisting of male smokers with an age-matched control group of male nonsmokers, FMD was impaired in both the vitamin E (5.3 ± 3.8 vs. 11.6 ± 4.7, p < 0.01) and the placebo groups (6.4 ± 3.5 vs. 11.6 ± 4.7, p < 0.05).
By performing a two-way repeated measures ANOVA, we found no effect for the grouping factor (group factor p value = 0.5834) in the ANOVA over time. With respect to time, there was a highly significant difference (time factor p value = 0.0065), and the interaction of the time factor and the grouping factor also proved to be significant (p = 0.0318), thus confirming the beneficial effect of vitamin E supplementation (Fig. 1).
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Relationship between oxidative stress, smoking habits and FMD. Univariate analyses over both study groups revealed significant inverse correlations between the transient impairment of FMD after acute smoking (delta FMD) and the improvement of the antioxidant status (delta TBARS, r = –0.67, p = 0.0024; delta alpha-tocopherol/LDL, r = 0.51, p = 0.016; Fig. 3). Alpha-tocopherol serum levels or delta alpha-tocopherol were not related to the impairment of FMD. The number of cigarettes smoked per day correlated with TBARS at baseline (r = 0.49, p = 0.025). No significant correlation was found between pack-years and TBARS levels. The close correlation between delta FMD and delta TBARS (r = –0.71, p = 0.031) also remained following exclusion of the placebo group.
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Discussion |
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Effects of chronic smoking on endothelial dysfunction. Endothelial dysfunction has been observed in both peripheral and coronary arteries of long-term smokers (1,2). The precise mechanism of this impairment by chronic smoking has not yet been elucidated but may be related to oxidative stress. In support of this, cigarette smoke has been shown to contain large amounts of free radicals such as superoxide anion and hydroxyl radicals (11,22), agents known to degrade endothelium derived relaxing factor and to impair FMD (6). Superoxide anion, in turn, can rapidly react with nitric oxide to form peroxynitrite, a molecule with high cytotoxic potency (23,24). Moreover, it has been reported that cigarette smokers have higher rates of in vivo and in vitro lipid peroxidation (25). Thus, atherogenic effects of smoking may be mediated in part by free radical damage to lipids, in particular oxidative modification of LDL, which promotes foam cell formation (26,27), monocyte adhesion (28,29) and is cytotoxic to vascular cells (30).
Role of antioxidants in smokers. Epidemiologic studies have shown that plasma levels of antioxidant nutrients, such as vitamin C and E, are significantly lower in smokers compared with nonsmokers (31). In the Health Professionals’ follow-up study, a reduction in major coronary events among healthy male subjects taking 100 to 250 IU supplementation of vitamin E per day, with little further benefit at higher doses, was reported (8). In contrast, in a randomized therapeutic trial among Finnish smokers, no beneficial effect on cardiovascular events was observed for either vitamin E or beta-carotene intake (32). However, the dose of vitamin E applied in this study (50 mg per day) was below the protective range suggested in the Health Professionals’ follow-up study. Therefore, a cause-and-effect relation between vitamin E intake and a reduction in cardiovascular events has thus far been suggested only for vitamin E supplementation in patients with angiographically proven coronary atherosclerosis (10).
Effects of antioxidant vitamins on endothelial function in smokers.
Endothelium-dependent vasodilation in the forearm circulation of chronic smokers can be improved by acute intraarterial administration of vitamin C (11). Moreover, Motoyama et al. (12) demonstrated that an acute impairment of FMD in the brachial artery after heavy smoking is caused by an increase of oxidative stress within 10 min. This transient impairment of endothelial function lasts for 
90 min (4) and can be attenuated by intravenous infusion of vitamin C (12). In contrast, oral vitamin E supplementation did not improve endothelial function in the forearm circulation of normocholesterolemic chronic smokers (33). Our data suggest that the oral intake of vitamin E for four weeks is not able to reverse chronic endothelial dysfunction of smokers but does prevent the transient further impairment of endothelium-dependent vasodilation in the brachial artery after acute smoking. This acute effect is likely due to the antioxidant effect of vitamin E which is supported by its close correlation with
Interestingly, no improvement of chronic endothelial dysfunction was found, although the oxidative status was improved. These results are in accordance with previous observations in the forearm vasculature, in which vitamin E supplementation failed to improve endothelium-dependent vasodilation, although the susceptibility of LDL to oxidation could be reduced (34). However, the beneficial effect of vitamin E supplementation on the impairment of endothelial function after heavy smoking reported in this study is unlikely due to increased resistance of LDL-C to oxidation, as this process would last longer than the time elapsed between heavy smoking and the ultrasound examination (20). Thus, other mechanisms must be considered, such as the scavenging of superoxide or an improvement of the intracellular antioxidant status due to the accumulation of alpha-tocopherol in the vascular wall and in endothelial cells in particular (35).
A possible explanation for the discrepancy between the lack of effect on chronic endothelial dysfunction after vitamin E supplementation and its beneficial effect on transient endothelial dysfunction in our study may be the relatively low degree of oxidative stress while subjects refrained from smoking before the examination, compared with the burst of oxidative stress after heavy smoking. Recently, Heitzer et al. (33) revealed a beneficial effect of vitamin E supplementation on chronic endothelial dysfunction selectively in hypercholesterolemic smokers which was associated with a reduction of elevated autoantibody titers against oxidized LDL. Alternatively, chronic endothelial dysfunction caused by smoking may be due to other mechanisms not affected by the antioxidant or other properties of vitamin E. These mechanisms may include an increase in asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor (36), as well as more long-lasting endothelial cell damage, due to cytotoxic intermediates (23,24).
Study limitations. A possible drawback of this study may be that we did not measure TBARS after heavy smoking. However, an acute increase of TBARS after heavy smoking as well as an attenuation of this increase by an antioxidant has been demonstrated previously (12). In this study, TBARS levels and alpha-tocopherol serum levels normalized to LDL-C were measured to estimate the attenuation of oxidative stress under vitamin E supplementation. The fact that TBARS levels were not significantly different in both study groups (p = 0.06) is most likely due to the high SD of this parameter (19). However, differences in delta TBARS, alpha-tocopherol/LDL-C and delta alpha-tocopherol/LDL-C strongly suggest an improvement in the antioxidant status of subjects taking vitamin E. Finally, we cannot exclude that the duration of vitamin E treatment may have been insufficient. However, long term vitamin E supplementation for four months also failed to restore endothelial function in long-term smokers with a lipid profile similar to our study subjects (33).
Conclusions. This study demonstrates that oral vitamin E supplementation can attenuate the transient impairment of endothelial function after heavy smoking due to an improvement of the antioxidant status, but cannot restore chronic endothelial dysfunction within four weeks. These data are consistent with the concept that vitamin E may in part be beneficial due to direct tissue effects that are different from the prevention of the formation of oxidized LDL-C.
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