Abstract

Background:

Essential hypertension is characterized by endothelial dysfunction, arterial stiffness, and increased oxidative stress. We evaluated the effect of short-term combined treatment with the antioxidants vitamins C and E on endothelial function, arterial stiffness, and oxidative stress in untreated essential hypertensive patients.

Methods:

A randomized, double-blind, placebo-controlled, crossover study design was used to assign 30 male essential hypertensive patients to either vitamin C (1 g) and vitamin E (400 IU) or placebo for 8 weeks. Endothelium-dependent response was assessed as flow-mediated dilation (FMD) of the brachial artery. Arterial stiffness was assessed as central pulse wave velocity (PWV) and augmentation index (AIx). Plasma markers of oxidative stress and antioxidant status were measured.

Results:

After vitamin supplementation, FMD was significantly improved. Central PWV was significantly reduced, while AIx tended to decrease. Plasma vitamin levels and antioxidant capacity increased significantly. Levels of oxidative stress decreased. Changes in central PWV were related to changes in levels of oxidative stress.

Conclusions:

Combined treatment with vitamins C and E has beneficial effects on endothelium-dependent vasodilation and arterial stiffness in untreated, essential hypertensive patients. This effect is associated with changes in plasma markers of oxidative stress. Am J Hypertens 2007;20: 392–397 © 2007 American Journal of Hypertension, Ltd.

Essential hypertension is an important risk factor for cardiovascular disease.1 Most of the cardiovascular complications associated with hypertension are caused by alterations in vascular structure and function. In particular, hypertension is associated with endothelial dysfunction and increased arterial stiffness.2–4 Both endothelial dysfunction5 and arterial stiffness4 are increasingly being recognized as independent cardiovascular risk factors.

Oxidative stress, defined as an excessive amount of oxidative substances relative to endogenous antioxidant capacity, is known to be increased in conditions such as hypertension, resulting in reduced nitric oxide (NO) availability in these subjects.6,7 It is hypothesized that antioxidants provide vascular defense against oxidative stress by reducing free radicals and protecting NO from inactivation, thereby exerting beneficial effects on vascular function and structure.

Data are already available indicating that acute oral administration of vitamin C (2 g) can have beneficial effects on arterial stiffness measured as augmentation index (AIx) in healthy subjects.8 In hypertensive patients, intra-arterial administration of vitamin C improved endothelium-dependent vasodilation in the forearm microcirculation of hypertensive patients, primarily by restoring NO availability.6 However, it was noted that this beneficial effect in hypertensive patients was observed only at supra-physiologic doses of ascorbic acid.6,9 Thus, prolonged oral administration of ascorbic acid at 500 mg/d for 1 month did not improve endothelial function in essential hypertensive patients.10 Moreover, administration with vitamin E as the sole antioxidant did not improve arterial endothelial function in older adults.11

Different studies assessing the effect of antioxidant supplementation have combined vitamin C with vitamin E, a lipid soluble vitamin with a protective effect on lipid peroxidation.12,13 This combination is of special interest, as it is known that vitamin E can have pro-oxidant properties and appropriate concentrations of vitamin C are necessary for the regeneration of vitamin E.14 Because of synergism between vitamins C and E, it is conceivable that combined supplementation with both vitamins exerts a greater antioxidant effect.

At the present time no data are available concerning the possible effects of combined vitamins C and E on arterial stiffness and endothelial function in a population of untreated hypertensive patients. Therefore, the aim of this study was to evaluate the effect of short-term combined antioxidant treatment with vitamins C and E on these vascular parameters and oxidative stress in untreated essential hypertensive patients.

Methods

Study Population

The study included 30 never-treated, male, essential hypertensive patients (mean age, 50 years; range, 42 to 60 years). Patients were recruited among newly diagnosed cases in our outpatient clinic. Inclusion criteria were age between 40 and 60 years, sitting clinical blood pressure (BP; after 10 min of rest) values between 140/90 and 160/99 mm Hg confirmed on two separate occasions within 1 month, and in absence of target organ damage according to European Society of Hypertension–European Society of Cardiology (ESH-ESC) guidelines.1 Exclusion criteria were history of cardiovascular disease, diabetes mellitus, dyslipidemia, body mass index (BMI) more than 30 kg/m2, alcohol consumption (>50 g/d), smoking, currently performing vigorous physical exercise, or taking mineral or vitamin supplements, antihypertensive drugs, or statins.

Study Design

According to a randomized, double-blind, placebo-controlled, crossover study design, patients received combined vitamin C (1g) and vitamin E (400 IU) or placebo, once a day, for 8 weeks. After a wash-out period of 4 weeks, patients were assigned to the inverse treatment (Fig. 1). At baseline, after 8 weeks, and at the end of the study, vascular studies were performed twice, on 2 consecutive days. In between, subjects underwent 24-h BP monitoring (ABPM) to check for BP changes. Blood samples were collected for determination of lipid and glucose profile, plasma markers of oxidative stress, and antioxidant status. Patients were asked not to change their physical activity habits and dietary intake during the study. Dietary intake of foods and nutrients was assessed at baseline, after 8 weeks, and at the end of the study by a 3-day food record. Physical activity levels were assessed and controlled with a modified Baecke questionnaire.15 The investigation conformed to the principles outlined in the Declaration of Helsinki.16 All study participants gave written informed consent to the study.

Outline of study design. *Vascular studies, ABPM monitoring, blood samples for plasma markers of oxidative stress, and antioxidant status were performed.

Vascular Function

After an overnight fast, measurements were performed with the subjects in supine position in a quiet, air-conditioned room (22° to 24°C). Radial tonometry was performed by one trained operator (YP). Blood pressure was calculated as the mean value of three measurements taken at 3-min intervals by an automatic device on the dominant arm (OMRON-950 CP, OMRON Healthcare Europe, Hoofddorp, The Netherlands) to calibrate the measurements. A hand-held probe was placed on the radial artery from the wrist of the dominant arm and 10 to 15 subsequent images were recorded. Pulse wave analysis (PWA) (SphygmoCor, AtCor Medical, Sydney, Australia) was used to transform the radial pressure waveform into the aortic pressure waveform by using a validated transfer function.17 Three successive measurements were recorded. Augmented pressure (AP) was calculated as the difference between the first and the second systolic peak, and the AIx was calculated as the ratio between AP and pulse pressure (PP). In this study, because AIx correlated with heart rate, values were normalized at a heart rate of 75 beats/min. Central pulse wave velocity (CPWV) was assessed with the same device, recording waveforms at the femoral and carotid site sequentially. Surface distance between the two recording sites was measured. A simultaneously recorded electrocardiogram (ECG) was used as a reference to calculate wave transit time. An intraobserver reproducibility study in our laboratory showed a coefficient of variation of 14% for AIx and 13% for CPWV.

Endothelium-dependent response was assessed as dilation of the brachial artery to increased flow (flow mediated dilation, FMD), as previously described.18 Briefly, a B-mode scan of the right brachial artery was obtained in longitudinal section between 5 and 10 cm above the elbow using a 7.5-MHz linear array transducer, held by a stereotactic clamp to ensure a constant image. The B-mode images were triggered to the ECG signal to obtain only end-diastolic frames. Arterial flow velocity was obtained by pulsed Doppler signal at 70° to the vessel with the range gate (1.5 mm) in the center of the artery. A cuff was placed around the forearm just below the elbow and was inflated for 5 min at 250 mm Hg and then deflated to induce reactive hyperemia. Endothelium-independent dilation was obtained by administration of a low dose (25 μg) of sublingual glyceryl trinitrate (GTN).

Brachial artery diameter (BAD) measurements were performed after studying the acquired frames by the computerized edge detection system.19 Baseline vessel size was considered as the mean of measures obtained during the first minute. The FMD and response to GTN were calculated as the maximal percent increase in diameter above baseline. Doppler flow velocity was measured at baseline and within 15 sec after cuff release. Volume of blood flow was calculated by multiplying Doppler flow velocity (corrected for the angle) by heart rate and vessel cross-sectional area (πr2). Reactive hyperemia (RH) was the maximum percent increase in flow after cuff release as compared to baseline flow.

Blood Sampling and Biochemical Measurements

Venous blood was collected in lithium–heparin or EDTA tubes and immediately placed on ice. Plasma was immediately centrifuged and stored at −70°C until assayed. Total serum cholesterol, triglycerides, HDL-cholesterol, and plasma glucose were assessed by enzymatic methods (Roche, Diagnostic, Mannheim, Germany). The LDL-cholesterol was calculated with Friedewald's equation. Oxidative stress was evaluated through measurement of plasma malondialdehyde (MDA) by spectrophotometric assay20 and plasma lipoperoxides (LOOH) with a colorimetric method, as previously described.18 Antioxidant capacity was measured as plasma total antioxidant capability by measuring ferric-reducing antioxidant power (FRAP; spectrophotometric assay).21

For detection of vitamin C, tubes were incubated for 10 min at 2° to 8°C and then centrifuged at 10,000 rpm for 10 min. For vitamin E, blood samples were centrifuged for 5 min at 2000 rpm. Then plasma was stored at −20°C and protected from light until analyzed. High performance liquid chromatography (HPLC) was used to determine plasma vitamin C (HPLC-Analytik, Immundiagnostik AG, Bensheim, Germany) and vitamin E (Chromsystems, Instruments & Chemicals GmbH, Munchen, Germany).22

Statistical Analysis

Data are expressed as means ± SD, unless otherwise stated, and were analyzed as change from baseline by using Student t tests for paired data or nonparametric test if necessary. Differences were considered statistically significant when P < .05. Univariate (simple correlation) and multivariate (multiple regression model) analyses were used to examine associations between variables. All statistical procedures were performed using the Statview program (Abacus Concepts, Inc., SAS Institute, Cary, NC). Vascular responses (AIx, CPVW, FMD, response to GTN) were calculated as the mean value of the measures obtained in the 2 following days.

Results

During the study, dietary and physical activity habits and body weight were not modified. Clinical characteristics were similar in each phase of the study (Table 1). The ABPM values did not significantly change during the study, and central BP levels measured by PWA likewise remained unchanged. After antioxidant supplementation, FMD was significantly (P < .001) improved as compared to placebo, whereas response to GTN was not modified (Fig. 2). After antioxidant supplementation, the outgoing pressure wave (P1) increased, but not significantly (from 33.5 to 34.3 mm Hg; P = not significant [NS]), time to first systolic peak (T1) was not modified, whereas time to the second systolic peak (T2) increased significantly (from 234 to 241 msec; P < .01). The AIx was reduced, although not significantly (P = .09), whereas CPWV was significantly (P < .01) lowered after antioxidant supplementation (Fig. 3).

Table 1

Clinical characteristics of study population

 Baseline Vitamins C and E Placebo 
Body mass index (kg/m227.2 ± 2.2 27.1 ± 2.3 27.3 ± 2.3 
24-h systolic BP (mm Hg) 135 ± 10 134 ± 10 134 ± 10 
24-h diastolic BP (mm Hg) 87 ± 7 86 ± 7 87 ± 6 
Central systolic BP (mm Hg) 139 ± 13 139 ± 15 138 ± 13 
Central diastolic BP (mm Hg) 90 ± 5 89 ± 7 89 ± 7 
Total cholesterol (mmol/L) 5.38 ± 0.96 5.47 ± 0.93 5.34 ± 0.98 
HDL-cholesterol (mmol/L) 1.27 ± 0.37 1.31 ± 0.31 1.26 ± 0.37 
LDL-cholesterol (mmol/L) 3.38 ± 0.96 3.48 ± 0.96 3.42 ± 0.95 
Triglycerides (mmol/L) 2.95 ± 2.0 3.03 ± 1.9 2.95 ± 2.0 
Plasma glucose (mmol/L) 2.50 ± 0.26 2.53 ± 0.27 2.47 ± 0.25 
Hematocrit (%) 42.3 ± 4.8 43.6 ± 3.5 44.6 ± 3.5 
Hemoglobin (g/L) 15.9 ± 1.2 15.7 ± 1.0 15.6 ± 1.3 
 Baseline Vitamins C and E Placebo 
Body mass index (kg/m227.2 ± 2.2 27.1 ± 2.3 27.3 ± 2.3 
24-h systolic BP (mm Hg) 135 ± 10 134 ± 10 134 ± 10 
24-h diastolic BP (mm Hg) 87 ± 7 86 ± 7 87 ± 6 
Central systolic BP (mm Hg) 139 ± 13 139 ± 15 138 ± 13 
Central diastolic BP (mm Hg) 90 ± 5 89 ± 7 89 ± 7 
Total cholesterol (mmol/L) 5.38 ± 0.96 5.47 ± 0.93 5.34 ± 0.98 
HDL-cholesterol (mmol/L) 1.27 ± 0.37 1.31 ± 0.31 1.26 ± 0.37 
LDL-cholesterol (mmol/L) 3.38 ± 0.96 3.48 ± 0.96 3.42 ± 0.95 
Triglycerides (mmol/L) 2.95 ± 2.0 3.03 ± 1.9 2.95 ± 2.0 
Plasma glucose (mmol/L) 2.50 ± 0.26 2.53 ± 0.27 2.47 ± 0.25 
Hematocrit (%) 42.3 ± 4.8 43.6 ± 3.5 44.6 ± 3.5 
Hemoglobin (g/L) 15.9 ± 1.2 15.7 ± 1.0 15.6 ± 1.3 

24-h = 24-h ambulatory; BP = blood pressure.

Graphs show differences in augmentation index (AIx) and central pulse wave velocity (CPVW) at baseline (white bars), after 8 weeks of vitamin antioxidant supplementation (black bars) as compared to placebo (gray bars). *Denotes a significant difference (P < .01).

Graphs show differences in flow-mediated dilation (FMD) and response to sublingual glyceryl trinitrate (GTN) at baseline (white bars), after 8 weeks of vitamin antioxidant supplementation (black bars) as compared to placebo (gray bars). *Denotes a significant difference (P < .001).

Changes in markers of oxidative stress and antioxidant status are shown in Table 2. Compared to the placebo group, plasma of vitamins C and E, and FRAP levels increased significantly. After antioxidant supplementation, plasma levels of MDA decreased, although not significantly, whereas levels of LOOH were significantly reduced as compared to placebo. The increase in vitamin C levels was significantly related to the changes in FRAP and LOOH (R = 0.37; P < .05, R = −0.40, P < .05, respectively). Changes in FRAP were related to changes in MDA (R = −0.37, P < .05).

Table 2

Markers of oxidative stress and antioxidant status

 Baseline Vitamins C and E Placebo 
MDA (μmol/L) 2.33 ± 0.84 2.03 ± 0.97 2.35 ± 0.92 
LOOH (μmol/L) 3.12 ± 2.6 2.06 ± 1.7* 3.10 ± 2.3 
FRAP (μmol/L) 789 ± 141 831 ± 139 783 ± 137 
Plasma vitamin C (μmol/L) 39.12 ± 22.43 52.35 ± 32.65* 33.95 ± 22.83 
Plasma vitamin E (μmol/L) 20.41 ± 8.68 25.50 ± 9.36 19.81 ± 7.55 
 Baseline Vitamins C and E Placebo 
MDA (μmol/L) 2.33 ± 0.84 2.03 ± 0.97 2.35 ± 0.92 
LOOH (μmol/L) 3.12 ± 2.6 2.06 ± 1.7* 3.10 ± 2.3 
FRAP (μmol/L) 789 ± 141 831 ± 139 783 ± 137 
Plasma vitamin C (μmol/L) 39.12 ± 22.43 52.35 ± 32.65* 33.95 ± 22.83 
Plasma vitamin E (μmol/L) 20.41 ± 8.68 25.50 ± 9.36 19.81 ± 7.55 

FRAP = Ferric reducing ability of plasma; LOOH = lipoperoxides; MDA = malondialdehyde.

*

P < .05 v baseline

P < .05 v placebo.

At baseline, FMD was related to BAD (R = −0.47; P < .01), levels of vitamin C (R = 0.40; P < .05), and FRAP (R = 0.36; P < .05). The AIx correlated significantly with age (R = 0.50; P < .01) and inversely with FRAP (R = −0.42; P < .05). The CPWV was related to systolic BP (R = 0.40; P = .03), age (R = 0.38; P < .05), LOOH (R = 0.38; P < .05), and levels of MDA (R = 0.39; P < .05). Multivariate analysis including other possible confounders (age, BP values, LDL- and HDL-cholesterol) showed that for FMD only BAD (P = .02) remained a significant variable (R2 = 0.44). For AIx, age (P < .01) was found to be a significant predictor (R2 = 0.32) and systolic BP (P = .03) for CPWV (R2 = 0.40).

Changes in FMD after antioxidant supplementation were not related to changes in CPWV (r = 0.14; P = NS). Changes in FMD showed a negative, but nonsignificant correlation with changes in MDA (R = −0.31, P = .07). Changes in CPWV after antioxidant supplementation were related to changes in MDA levels (R = 0.43; P < .05) and exhibited a borderline relation to changes in levels of FRAP (R = −0.30, P = .06). Changes in AIx were significantly related to the observed differences in levels of MDA (R = 0.41; P < .05). When multivariate analysis was performed, taking into consideration possible confounding variables including age, BP values, LDL- and HDL-cholesterol, changes in MDA remained significantly related to changes in CPWV (R2 = 0.31) and AIx (R2 = 0.29).

Discussion

The present study demonstrates that oral supplementation with vitamins C and E for 8 weeks improved arterial stiffness and endothelial function in untreated essential hypertensive patients. Supplementation with vitamins increased total plasma antioxidant capacity, vitamin C and vitamin E levels, and reduced the plasma oxidative markers LOOH and MDA, indicating a beneficial modulation of systemic markers of oxidative stress.

To our knowledge no previous study has investigated the effect of antioxidant vitamins on both arterial stiffness and endothelial function in essential hypertensive patients. Data concerning arterial stiffness available so far are not consistent, showing either a beneficial8,23 or no effect24 of different kinds of antioxidant supplementation on parameters of arterial stiffness in apparently healthy subjects. However, an improvement in central aortic AIx was found after 500 mg/d oral vitamin C supplementation for 1 month in a population with type 2 diabetes.25

In the present study, oral supplementation with combined vitamins significantly improved CPWV. Importantly, the improvement in CPWV showed a relation to a decreased level of oxidative stress markers. Furthermore, change in MDA was the only significant predictor of changes in CPWV in multivariate analysis. The AIx, an integrated index of arterial stiffness and arterial wave reflection, proved to be lower after antioxidant supplementation, but not significantly. Because central BP levels remained unchanged, the change in AIx can be most likely explained by the observed increase in the outgoing pressure wave (P1). A significant increase in T2 was also observed. Thus, supplementation with antioxidants seems to have a beneficial effect on central large artery function, as demonstrated by the results obtained with CPWV. These findings suggest that oxidative stress contributes to inducing arterial stiffness in untreated hypertensive patients. Because an 8-week treatment period is not sufficient to achieve any significant change in vascular structure, it is conceivable that functional mechanisms account for the improvement in arterial stiffness, such as an improvement in endothelial function26 and a possible beneficial effect of antioxidants on protein cross-linking in conduit arteries, including the aorta.27

The FMD improved after antioxidant supplementation, whereas no significant changes in the response to GTN were observed. This suggests that treatment with combined vitamins selectively improves endothelium-dependent vasodilation of the brachial artery. The improvement in FMD was not significantly related to changes in antioxidant status or oxidative stress after vitamin supplementation. However, it has already been pointed out that evaluation of plasma markers of oxidative stress may not accurately reflect oxidative status at the level of endothelial cells.18 Previous studies have shown that the beneficial effect of ascorbic acid administration on endothelial function in hypertensive patients was only achieved at supra-physiologic doses by intra-arterial administration,6,9 whereas prolonged oral administration (500 mg/d for 1 month) was not effective.10 In contrast, a beneficial effect of a similar treatment with ascorbic acid has been described in patients with congestive artery disease (CAD).28 Finally, in apparently healthy older subjects, 10 weeks of daily supplementation with vitamin E (1000 IU/d) failed to show a beneficial effect on endothelial function.11 It is conceivable that the beneficial effect of chronic oral antioxidant treatment on endothelial function, at least in patients with relatively low cardiovascular risk such as our population of untreated hypertensive patients, requires the administration of combined vitamin C and vitamin E at appropriate dosages. Because vitamin C can reverse the pro-oxidant status of vitamin E in turning α-tocopheroxyl back into α-tocopherol,14 combined treatment can more effectively counteract the NO scavenging action of oxidative stress, but can also increase NO production per se.29 Thus, ascorbic acid can improve tetrahydrobiopterin availability in the vasculature and α-tocopherol exerts a direct stimulatory effect on e-NOS activation, an effect that is amplified by ascorbic acid. This suggests that the two compounds may act synergistically in optimizing endothelial NO synthesis.29

Our results may have clinical implications as both endothelial dysfunction and arterial stiffness are early mechanisms of vascular alterations and independent predictors of cardiovascular diseases.4,5 The majority of controlled studies evaluating the effect of vitamin supplementation on vascular lesions and cardiovascular events show inconclusive results.30–33 One of the possible explanations could reside in the clinical conditions of the study populations in which the presence of advanced vascular alterations or associated clinical conditions might negatively influence the beneficial effect of antioxidant vitamins. An alternative explanation could lie in the long-term use of vitamin E (α-tocopherol) as the sole antioxidant supplement in some of the studies.11,31,32,34 Thus, our results may indicate that early combined treatment with vitamins C and E in uncomplicated essential hypertensive patients can have beneficial effects in the prevention of vascular alterations and cardiovascular events.

In our study clinic, ambulatory and central BP levels were not changed by antioxidant vitamin supplementation. Although one study suggested that treatment with oral vitamin C (500 mg/d, 30 days) was associated with a reduction in BP values,35 a recent review revealed that no real evidence exists for an effect of antioxidant supplementation in preventing or treating high BP.36

One possible limitation of the present study is the fact that no measurements of intravascular oxidative stress were performed. Therefore, our conclusions on the associations between differences in oxidative stress and vascular parameters could be overestimated or underestimated. Furthermore, the results of the present study do not allow conclusions to be drawn with regard to an in vivo interaction of the combined supplementation, as the effects of vitamin C or E alone were not evaluated.

In conclusion, this randomized study demonstrates that compared to placebo, combined oral antioxidant treatment with vitamins C and E for 8 weeks has beneficial effects on arterial stiffness and endothelial function in untreated essential hypertensive patients. The improvements in arterial stiffness and possibly in endothelial function seem to be due to an effective increase in antioxidant defenses, leading to a subsequent decrease in oxidative stress. Further research is still needed to study the potential long-term benefit of vitamin antioxidant supplementation on prevention of early vascular alterations.

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