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Effect of Chronic Oral Supplementation With Vitamins on the Endothelial Function in Chronic Smokers

Posted on: Sunday, 5 December 2004, 03:00 CST

Cigarette smoking has been associated with endothelial dysfunction including impaired endothelium-dependent flow-mediated dilation (FMD). In cigarette smokers, increased oxygenderived free radicals have been suspected of being one of the major causes of endothelial dysfunction, owing possibly to the inactivation of nitric oxide by free radicals. Vitamins C and E are widely used antioxidant vitamins, which have also been reported to effectively improve the endothelial function in several conditions. To test the effect of moderate-term oral antioxidant vitamin supplementation on the endothelial function in smokers, the authors evaluated the combined effect of vitamins C and E, administered in normal dosages, on FMD in young male smokers. A prospective interventional study was performed. In 15 healthy male subjects (mean age, 24.4 2.5 years old). They studied FMD in the brachial artery by using high- resolution ultrasound. The vascular effects of moderate-term oral supplementation with vitamin C (1.0 g/day) and vitamin E (500 mg/ day) were determined during reactive hyperemia, which causes endothelium-dependent FMD. They performed a vascular function study 3 times including before vitamin supplement, after 25 days of vitamin supplement, and 4 weeks after the cessation of the vitamin supplement. The flow-mediated dilator response measurements were repeated twice a day before vitamin supplements, and the repeatability obtained from these measurements was found acceptable (variability of FMD <2%). The oral antioxidant vitamin supplement significantly restored FMD (3.8 2.2% vs 5.9 2.5%; p<0.05), however, this effect disappeared 4 weeks after the vitamin supplementations ended. The combined usual dosage of vitamins C and E supplements was found to improve the endothelial function in chronic smokers.

Introduction

In cigarette smokers, vascular endothelial function has been examined in several studies and has generally been found to be impaired to various extents.1-3 Normal endothelium contributes to the physiological function of the vessel wall and the peripheral circulation. It regulates the local vasomotor tone and maintains a nonthrombogenic surface of the vascular wall. Normal endothelium also prevents atherosclerosis by controlling proliferation of vascular adhesion of neutrophils, monocytes, and lymphocytes. Thus, in the presence of abnormal endothelial function, atherosclerosis is prone to progress. For these reasons, abnormal endothelial function in cigarette smokers is considered to be one of the major pathological causes of coronary atherosclerosis.4 Since circulating reactive oxygen intermediates derived from cigarette smoke are important factors5 in impairing endothelial function, it seems reasonable to administer antioxidant compounds in order to improve an abnormal endothelial function and so prevent pathological vasoconstriction, thrombus formation, and leukocyte adhesion in smokers.

Antioxidant vitamins, such as vitamins C and E, can be safely administered to humans as potent antioxidant materials. The intravenous administration of vitamin C has been reported to improve endothelium-dependent dilation6,7 in smokers, and the oral intake of vitamin C also modulates leukocyte adhesiveness to the endothelium in smokers.8 Vitamin E is a lipid-soluble antioxidant that augments the effect of water-soluble antioxidant vitamin C in vivo.9 However, the combined effect of vitamins C and E on endothelial dysfunction in chronic smokers has not been closely investigated. In addition, the clinical effect of vitamin E as demonstrated in multicenter, randomized clinical trials remains controversial.10-13

In this study, we selected young male smokers to test the effects of the usual dosage of oral antioxidant vitamins C and E on vascular endothelial function. In particular, we studied the effect of the combined oral supplementation with vitamin C (1.0 g/day) and vitamin E (500 mg/ day) on brachial artery flow-mediated dilation (FMD) by using high-resolution ultrasound.

Methods

Study Population

The study population included 15 healthy male volunteers aged from 20 to 30 years (24.4 2.5 years) who had a history of 2 or more pack-years (1 pack-year defined as smoking 20 cigarettes per day for 1 year or equivalent periods). All subjects were normotensive, nondiabetic, and nonhypercholesterolemic, and none had a family history of premature vascular disease. Further exclusion criteria were the current use of any antioxidants or vasoactive medications. All subjects were aware of the investigative nature of the study and gave their informed consent. The research protocol received the approval of the ethics committee in this institution. All subjects were asked to continue smoking as usual throughout the study.

Brachial Endothelial Vasomotor Function Study

A brachial endothelial vasomotor function study was performed according to previously reported methods.14-16 All studies were performed around 10:00 in the morning after overnight fasting in a temperature-controlled quiet room. In addition, all subjects were asked to refrain from smoking until the end of the brachial endothelial vasomotor function study in the morning on the day of testing; other than this, subjects were allowed to continue smoking throughout the study. The brachial artery diameter and flow velocity were imaged by using a 7.5 MHz linear-array transducer ultrasound system (Hewlett Packard, SONOS 1500, Andover, MA). The flow volume was calculated by multiplying the velocity-time integral of the Doppler flow signal (corrected for angle) by the heart rate and the vessel cross-sectional area. Hyperemia was induced by a cuff (Hokanson SC-10, Seattle, WA) that was inflated on the most proximal portion of the forearm to occlusive pressure (40 mm Hg higher than systolic blood pressure) for 5 minutes. Ultrasound images of the brachial artery were obtained at baseline and 60 s after deflation. After a 20minute rest period (to allow the arterial diameter to return to the baseline size), brachial images were again obtained before and 4 minutes after the sublingual administration of nitroglycerin (0.3 mg). In each phase, ultrasound images of 3 cardiac cycles were digitized, and the vessel diameter was measured and averaged by personnel blinded to the image sequence and antivitamin treatment phase using a quantitative coronary angiography analysis computer (Kontron Electronic, Cardio 500, Munich, Germany) containing a digitizing board. The percent diameter changes in the brachial artery from the baseline in response to hyperemia was calculated and defined as FMD. Thus, to calculate FMD, the percent diameter changes were determined by the following formula: ([diameter after reactive hyperemia - baseline diameter] / baseline diameter) 100. The percent diameter changes to nitroglycerin was also calculated similar to FMD and defined as the endothelium-independent nitroglycerin-induced dilation (NTG-D). The relative increase in the blood flow during reactive hyperemia was calculated as the maximum flow recorded in the first 15s after cuff deflation divided by the flow at the baseline scan. In order to assess the repeatability of FMD, 15 subjects underwent brachial artery endothelial function study twice a day before administration of the antioxidant supplement.

Oral Antioxidant Vitamin Supplement Protocol

All subjects were administered 1.0 g of vitamin C and 500 mg of vitamin E (dl-alpha tocopherol nicotinate) as an antioxidant supplement for 25 days. We performed brachial endothelial function studies 3 times in each subject before administering the antioxidant supplement (the first study), 25 days after administering the oral antioxidant vitamin supplement (the second study), and 4 weeks after the subjects stopped taking the vitamins (the third study). The antioxidant vitamins were administered up until the start of the second study.

Biochemical Analysis

The plasma concentrations of vitamin C, alpha-tocopherol, cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride were measured in the morning of the first, second, and third brachial endothelial function study. The plasma concentration of vitamin C and alpha-tocopherol was determined by high- performance liquid chromatography. Low-density lipoprotein (LDL) cholesterol was calculated by using Friedewald's formula.17

Statistical Analysis

All data are presented as the means SD, and all data were examined by using repeated-measure ANOVA. The Pearson product- moment correlation coefficient was calculated to investigate the relationship between the magnitude of augmentation in FMD and the vitamin concentration or the amount of pack-years of cigarette smoking. A post hoc analysis was performed using the Scheffe method. The repeatability of FMD measurements was assessed by the method reported by Bland et al.18 Differences were considered significant if p < 0.05.

Results

Patient Characteristics

Clinical characteristics of the study groups are presented in Table I. All subjects included in this study were men who had a history of 5.1 3.0 pack-years cigarette smoking. There was no difference in the blood pressure, heart rate, and lipid profile in the 3 studies. The vitamin C concentration was not different in the 3 studies. Vitamin E concentrations, which are a reflection of the a\lpha-tocopherol levels, were significantly higher in the second study than in the first and third studies. The brachial artery vessel size was also not significantly different in the 3 studies.

Effects of Oral Antioxidant Vitamins on FMD and NTG-D

The results of a vascular function study for 15 male smokers are summarized in Table II and Figure 1. The flow-mediated dilator response measurements were repeated twice a day during the first study, and the repeatability obtained from these measurements was found to be acceptable (variability of FMD < 2%, Figure 2). As a result, FMD was significantly higher in the second study (5.9 2.5%, on vitamins), than in both the first study (3.8 2.2%, baseline) and the third study (4.6 2.0%, after cessation of vitamins). The FMD in both the first study and the third study was abnormal when compared with that of a healthy nonsmoker in our previous report.14 NTG-D was not different in the 3 studies. In addition, the degree of reactive hyperemia did not differ in the 3 studies; no side effects were observed after the combined administration of vitamins C and E. We analyzed the relation between the magnitude of augmentation in FMD and the vitamin concentration or the amount of pack-years of cigarette smoking, but we could not find any significant relation among these parameters.

Discussion

This study demonstrated the endothelium-dependent dilation to be augmented after vitamin administration while the endothelial function in the brachial artery also improved after the combined supplementation with vitamins C and E in young male smokers. In this study, we observed that the 25-day oral administration of antioxidant vitamins at normal dosages, which can be controlled on a daily basis at outpatient clinics, improved the endothelial function significantly in current smokers. Such vitamin administration, however, had no effect on NTG-D and the degree of hyperemia (Table II). This beneficial effect of the antioxidant vitamins on the vascular function did not continue for 4 weeks after the administration of the vitamins was stopped. These findings suggest that this combined vitamin supplement directly affects the endothelium.

Table I. Clinical characteristics of 15 male smokers and plasma concentrations of vitamins C and E (alpha-tocopherol) in the present study.

Since few studies have been conducted on the chronic effect of vitamins in smokers,19-21 the findings in this study are important, and our study shows a beneficial effect of the regular normal dosage of oral antioxidant vitamins on the endothelial function in smokers. Antioxidant vitamins have been shown to improve endothelial function in several conditions. However, the beneficial effect of antioxidant vitamins on vascular function has not yet been studied in detail regarding current smokers. In smokers, the oxygen-derived free radical is thought to be a crucial determinant of endothelial dysfunction by inactivating nitric oxide. Motoyama et al7 reported that the intravenous administration of vitamin C acutely improved FMD in chronic smokers, and Zhang et al22 also reported that vitamin C acutely improved microcirculation in chronic smokers. Besides the effect on smokers, vitamin C and vitamin E have also recently been tried as an antioxidant in diabetes,23 hypertension,24 and hypercholesterolemia25 and in patients with known coronary heart disease.26 Most investigators have reported a consistent positive beneficial effect on the endothelial function; however, it has yet to be clarified whether this antioxidant may also be a clinically useful medication in our daily outpatient clinic. We showed that the normal dosage of antioxidant therapy was effective for current smokers in this study. As a result, it is thus possible to establish an antioxidant therapy protocol for smokers that we can easily administer on an outpatient basis.

Figure 1. Effects of oral antioxidant vitamins on flow-mediated dilation in each subject (n = 15). Mean SD. *p < 0.05 vs the first and third studies.

Figure 2. Relationship between the differences in the measurements of flow-mediated dilation and the mean of those measurements; the assessment of repeatability.

We used antioxidant therapy as a combination of water-soluble antioxidant and lipid-soluble antioxidants, vitamins C and E, and these 2 different types of antioxidant vitamins demonstrated their antioxidant effects synergistically. Earlier reports have shown vitamin C to improve the endothelial function in several circumstances,24,26-28 but few reports have shown the chronic effect of vitamin C as an antioxidant. Recently, Neunteufl et al20 reported that chronic supplementation with vitamin E (600 IU) failed to improve endothelial function in smokers despite a significant reduction of oxidative stress. In our study, chronic oral supplementation with combined vitamins was observed to effectively improve endothelial function in smokers. From these observations, we speculate that such combination therapy appears to be an effective antioxidant treatment for smokers.

When the mechanism of endothelial dysfunction in smokers is considered, smokers are suggested to be a more sensitive population to antioxidant therapy, even though our patients were younger than the hypercholesterolemic patients studied in the earlier reports. Gilligan et al29 reported the effect of chronic oral antioxidant vitamin on endothelium-dependent vasodilation in hypercholesterolemic patients. In their study, they could not find any positive effect on endothelial function. The subject population was middle-aged with high plasma cholesterol concentrations, and thus it is reasonable to conclude that endothelial function was impaired in these individuals. In spite of the significant antioxidant effect on the LDL-cholesterol oxidation test and the significant increase in vitamin levels, no effect was observed regarding endothelial function. Since oxidative stress is an important factor in impairment of endothelial function in hypercholesterolemic patients, we speculate that a beneficial effect of the combination of water-soluble and lipid-soluble vitamins can thus be achieved by the same oral supplementation method used in this study. We hypothesize that the combined supplementation with vitamins C and E thus plays an important role in the induction of an antioxidant effect in humans since vitamin C turnover is rapid and vitamin E can possibly delay this.

In our observations, the plasma vitamin C concentration was similar throughout the study, while plasma vitamin E concentration was significantly higher at the second test when the subjects were receiving antioxidant medication. Since we would like to avoid any acute effects of vitamin C, we decided to use a daily dosage of 1.0 g of vitamin C, and vitamin C was discontinued on the day when the second study was started. As a result, the vitamin C level after treatment (the second study) was not different from the baseline level (the first study) in our study. In a clinical setting, the acute administration of vitamin C has been reported to improve endothelial function in several conditions such as diabetes,27 hypercholesterolemia,28 and smoking,5 and in patients with known cardiovascular disease.26 In previous reports, vitamin C was acutely given either intraarterially or orally. There have been no positive reports regarding chronic antioxidant therapy using vitamin C. In contrast, vitamin C improved endothelium-dependent vasodilation when 2.0 g of vitamin C was given orally to the populations reported above. In this regimen, the plasma vitamin C concentration was 3 times higher than the baseline from 2 to 5 hours after administration.

Regarding the vitamin E dosage, we basically used a normal dosage and thus expected a stable plasma concentration of alpha tocopherol. Most investigators use 400-800 IU of vitamin E and 500-1,000 mg of vitamin C. The intrinsic alpha tocopherol concentration is almost the same during the day and it is known that there is no circadian variation of its concentration.30 Tocopherol is created from tocopherol nicotinate through hydrolysis in the intestine and it is then transported to the plasma and lymph systems. If 500 mg of vitamin E is given orally, tocopherol and tocopherol nicotinate appear in the blood 3 hours after they are absorbed from the intestine. As a result, the plasma tocopherol concentration is highest at 10 hours after administration. The tocopherol half-time is as long as 39 hours.31 In our regimen, we prescribed vitamin E for 25 days. The alpha-tocopherol levels were thus considered to be at plateau levels in all subjects in this study.

Recently, endothelial dysfunction in coronary arteries32 has been shown to be associated with an untoward outcome such as cardiac events, and FMD in the brachial artery has also been reported to be related to the endothelial function of coronary arteries.16,33-35 It is thus reasonable to consider an impaired FMD to be an adverse effect of smoking on coronary arteries. Aggressive intervention with several strategies to improve endothelial function could thus prevent pathological vasoconstriction and thrombus formation in coronary circulation and thereby decrease the incidence of coronary events. In this regard, antioxidant vitamins are considered to be one promising medication to ameliorate endothelial function in several conditions in which oxidative stress is closely linked to pathogenesis.

As to study limitations, in this study, non-smokers were not included as controls. And we did not study whether or not a similar beneficial effect could be obtained in nonsmokers. However, it might be less valuable in clinical implication on controls because no endothelial dysfunction and no side effects were observed after the combined administration of vitamins C and E in this study. Lastly, to further confirm our observation, the present study should be duplicated as a ran\domized, placebo-controlled design with measurements of oxidative stress to see if differential effects of vitamins C and E exist.

In conclusion, the chronic combined dietary supplementation with vitamin C (1.0 g/day) and vitamin E (500 mg/day) was found to improve a parameter representative of endothelial function and is therefore considered to have a potentially preventive effect against the progression of atherosclerosis in smokers.

Acknowledgment

We wish to express special gratitude to Helen Webb, PhD, for English editorial assistance.

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Bonpei Takase, MD, Hirokuni Etsuda, MD, Yoshihiro Matsushima, MD, Makoto Ayaori, MD, Hiroyuki Kusano, MD, Akira Hamabe, MD, Akimi Uehata, MD,* Fumitaka Ohsuzu, MD, Masayuki Ishihara, PhD, and Akira Kurita, MD, Saitama and Tokyo, Japan

Angiology 55:653-660, 2004

From the National Defense Medical College and *Self Defense Force Central Hospital, Saitama and Tokyo, Japan

Correspondence: Bonpei Takase, MD, FACC, National Defense Medical College, Research Institute, Division of Biomedical Engineering, 3- 2 Namiki Tokorozawa, Saitama, Japan, 359-8513

E-mail: duil577@db3.so-net.ne.jp

2004 Westminster Publications, Inc, 708 Glen Cove Avenue, Glen Head, NY 11545, USA

Copyright Westminster Publications, Inc. Nov/Dec 2004

Source: Angiology

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