Atherosclerotic cardiovascular disease is a major world-wide cause of morbidity and premature death. Despite this, the pathogenesis of atherosclerosis remains incompletely understood, although changes in arterial stiffness and disruption of normal vascular endothelial function are thought to play an important part (1,2). Platelets also play a prominent role in atherogenesis, adhering to areas of endothelial damage, aggregating, and releasing mitogenic and procoagulation factors (2). Platelet activation is also a key event initiating the formation of coronary thrombus, which frequently occurs at sites of atherosclerotic plaque rupture as a prelude to vessel occlusion and myocardial infarction (2).
The endothelium releases nitric oxide (NO), which is a potent vasodilator and has important antiatherogenic actions including inhibition of smooth-muscle cell proliferation, monocyte adhesion, and platelet adhesion and aggregation (3). The effects of endothelium-derived NO on platelets are supplemented by NO synthesised directly in platelets (4). Recent evidence also suggests that NO is important in regulating arterial stiffness (5,6). Oxygen free radicals, such as the superoxide anion, have a number of important proatherogenic actions including inactivation of NO (7) and oxidation of low-density lipoproteins (LDLs) (8). Oxidized LDL damages the vascular endothelium and promotes foam cell formation (2). Oxidant stress is increased in conditions such as hypercholesterolaemia and diabetes mellitus, which are major cardiovascular risk factors (9,10). Vitamins C and E scavenge oxygen free radicals and are likely to have complex antiatherogenic properties, including the protection of NO against oxidant inactivation (11). Indeed, vitamin C inhibits LDL oxidation (12), stimulates NO synthesis (13), increases NO bioavailability (14), and reduces peripheral blood pressure (15,16), although its effects on other aspects of vascular function have been less fully investigated. The Cambridge Heart Antioxidant Study (CHAOS) study demonstrated that supplementation with vitamin E, in patients with coronary artery disease, reduced the frequency of cardiovascular events, although overall mortality was unaltered (17).
This study was designed to test the hypothesis that short-term oral administration of vitamin C can reduce arterial stiffness and inhibit platelet aggregation.
PATIENTS AND METHODS
Eight healthy male volunteers, mean age 29 years (range, 20-42 years), were recruited from a community database held at the Western General Hospital, Edinburgh. The local research Ethics Committee had previously approved the study, and written informed consent was obtained from all participants. Subjects with cardiovascular risk factors, including diabetes mellitus, hypercholesterolaemia (total cholesterol >6.5 mM), hypertension (blood pressure, >160/95 mm Hg), or clinical evidence of cardiovascular disease, were excluded. All subjects were nonsmokers and not receiving medication. After an overnight fast, studies were carried out in a quiet, temperature-controlled room (22 ± 2°C). Alcohol and caffeine were excluded for the 24 h preceding each visit. The study was conducted in a double-blind, randomized, placebo-controlled manner, and two visits were made 1 week apart.
After 45-min supine rest, duplicate measurements were made of blood pressure in the dominant arm (HEM-705CP; Omron Corporation, Tokyo, Japan) and cardiac index (CI) by using transthoracic electrical bioimpedence (NCCOM3-R7; BioMed, Irvine, CA, U.S.A.). Total peripheral vascular resistance index (TPVR) was calculated as mean arterial pressure (MAP) divided by CI, and was expressed in arbitrary units (AU). Ascending aortic pressure and augmentation index (AIx) were determined by using pulse-wave analysis (PWA) (18). Venous blood was then taken through a cannula inserted in the antecubital fossa of the dominant arm for platelet aggregation in response to 1-8 μM adenosine diphosphate (ADP; 30 ml in trisodium citrate tubes), determination of plasma vitamin C (5 ml in lithium-heparin tubes), and total serum cholesterol (5 ml in EDTA tubes). Subjects then received either 2 g of vitamin C orally (dispersible tablets) or matching placebo, and all measurements were repeated 6 h later.
Plasma vitamin C measurements were made in plasma from venous blood samples taken at t = 0 h and t = 6 h by using a published method (19) (Cobas Bio centrifugal analyzer equipped with fluorescence attachment).
The noninvasive technique of PWA relies on the principle of applanation tonometry to record pressure waveforms from a peripheral artery. These are then subject to a generalized transfer factor to derive the corresponding central arterial waveforms (18,20). From the latter, central pressure and AIx-a measure of systemic arterial stiffness-can be calculated (5,18). Arterial pressure waveforms were recorded from the nondominant radial artery by using a high-fidelity micromanometer (SPC-301; Millar Instruments, Houston, TX, U.S.A.) with an experienced operator. Data were collected directly into a portable microcomputer equipped with SphygmoCor software (SCOR; PWV Medical, Sydney, Australia). After 20 sequential waveforms had been acquired, the integral software was used to generate an averaged peripheral and corresponding central waveform, which were then subject to further analysis-determination of AIx and ascending aortic pressure. All measurements were made in duplicate.
Venous blood (30 ml) was collected into evacuated tubes containing trisodium citrate (0.8 ml/10 ml) and centrifuged (120 g; 10 min) to obtain platelet-rich plasma (PRP), which was aspirated and prewarmed to 37°C before aggregation studies by using a standard optical technique (Chronolog Ca560 aggregometer; Labmedics, Stockport, England). The maximal aggregation attained within 7 min of ADP treatment was recorded and expressed as a percentage of that seen in response to the maximal concentration of ADP (8 μM) at t = 0 h for each subject. Platelet counts were made in whole blood samples and PRP at t = 0 h and t = 6 h in both placebo and vitamin C phases of the studies (Beckman-Coulter Act.8 Coulter Counter, Beckman-Coulter, High Wycombe, England).
Data are presented as mean ± SEM, unless otherwise stated, and were analysed as change from baseline by using Student's paired t tests. Repeated-measures analysis of variance (ANOVA) was used to identify differences in response between vitamin C and placebo (SPSS for Power Macintosh). Results were considered significant at p < 0.05.
Plasma vitamin C
Baseline plasma vitamin C concentrations (42 ± 8 μM) were all above the accepted level for deficiency (17 μM) (21). The concentration increased after oral administration of vitamin C to 104 ± 8 μM (p < 0.001), but not after placebo (52 ± 12 μM, t = 0 h; 55 ± 14 μM, t = 6 h; p = 0.63).
Baseline variables did not differ between the two visits except for AIx, which was significantly lower at the vitamin C visit (p = 0.04). There was no change in any measured parameter after administration of placebo (Table 1). After vitamin C there was a reduction in AIx of 9.6 ± 3.0% (−1.3 ± 4.5% vs. −10.9 ± 3.4% at 6 h; p = 0.016), peripheral MAP (88 ± 2 vs. 84 ± 3 mm Hg; p = 0.041), and central MAP (84 ± 2 vs. 79 ± 3 mm Hg; p = 0.019). There was no change in either CI or heart rate, although TPVR did decrease from 27.2 ± 2.1 to 25.7 ± 1.9 AU (p = 0.025). When the responses to vitamin C were compared directly with the response to placebo, the only significant change was a reduction in AIx (9.6 ± 3.0% after vitamin C, and 0.0 ± 3.3% after placebo; p = 0.001). The haemodynamic changes are summarized in Table 1 and Fig. 1a.
Platelet aggregation in response to ADP (2-8 μM) was significantly reduced in PRP derived from subjects 6 h after vitamin C administration (Fig. 1b; p = 0.046, area under the curve), but not 6 h after placebo (p = 0.66). Platelet counts were not significantly altered in samples taken at t = 0 h and t = 6 h in either placebo (442 ± 27 and 415 ± 17 × 109 platelets/L, respectively; p = 0.09) or vitamin C (441 ± 20 and 413 ± 19 × 109 platelets/L, respectively; p = 0.23) phases of the study.
The main findings of this study were that a single oral dose of 2 g of vitamin C reduces AIx and platelet aggregation in healthy subjects compared with placebo. AIx provides a useful noninvasive index of systemic arterial stiffness, as it is integrally dependent on three main factors: pulse-wave velocity, the site of wave reflection, and the amplitude of the reflected pressure wave (22). Stiffening of the smaller arteries increases the amplitude of the reflected wave and shifts the site of wave reflection proximally. As the large arteries stiffen, pulse-wave velocity increases. Together these effects result in a larger reflected wave reaching the ascending aorta earlier, augmenting central systolic pressure (22).
Changes in blood pressure and heart rate influence AIx, confounding the effect of stiffness on AIx. There was no significant change in heart rate in either phase of the study, and although MAP decreased significantly during the vitamin C phase, this was not significant when compared with the placebo phase, in which there was also a nonsignificant decrease in MAP. Previous studies with vitamin C alone, and in combination with other antioxidants, including vitamin E, have demonstrated a blood pressure-reducing effect (15,23). However, the extent of the reduction was relatively small (5-7 mm Hg) and may not have been reproduced in our study because of our small sample size. Interestingly, neither study reported a significant change in diastolic pressure, but only a reduction in systolic pressure (15,23). Pulse pressure is a surrogate marker of arterial stiffness, and the reduction in pulse pressure in these studies suggests that arterial stiffness was reduced. However, to our knowledge, this study is the first to demonstrate that vitamin C reduces an index of systemic arterial stiffness. The magnitude of the reduction in AIx produced by vitamin C is comparable to that produced by glyceryl trinitrate (24). As shown in Fig. 1a, there is no trend for the magnitude of the effect to depend on baseline AIx.
ADP-induced platelet aggregation was also significantly reduced after oral vitamin C administration, with maximal aggregation in response to 8 μM ADP reduced by 25%. This is a similar reduction to that seen after 3 days of therapy with the glycoprotein IIb/IIIa antagonist, ticlopidine (250 mg/day), and marginally less than that seen with the ADP-receptor antagonist, clopidogrel (50-100 mg/day) (25). Aggregation was not reduced after placebo, and platelet counts were not significantly different in PRP from blood taken at t = 0 h and t = 6 h. Our results show for the first time that short-term oral administration of vitamin C inhibits platelet aggregation and support a previous study showing that platelet aggregation is inhibited by addition of high concentrations of vitamin C to the sample in vitro, immediately before aggregation (26). Calzada et al. (27) did not demonstrate any effect of vitamin C, given for 8 weeks, on platelet aggregation. However, they used a lower dose of vitamin C than in our study (250 mg daily), and plasma vitamin C was not directly measured. It is unclear, therefore, whether supplementation significantly increased plasma levels. Our observed effects of vitamin C on platelet function are surprising in view of the fact that this was an ex vivo study, and the platelets were no longer under the influence of the endothelium. Antioxidant effects of vitamin C in the in vitro environment are likely to be exerted within the platelets themselves, acting to protect platelet-derived NO. However, it is also possible that, like other antioxidants (28), vitamin C might alter eicosanoid production.
In conclusion, oral vitamin C reduces arterial stiffness and platelet aggregation in healthy subjects. One potential explanation for these effects is increased bioavailability of NO through scavenging of oxygen free radicals, although further studies are necessary to confirm the mechanism(s) involved. If similar beneficial effects are seen in patients with atherosclerosis or cardiovascular risk factors, then vitamin C may be a useful therapeutic agent to reduce left ventricular load and mass, atheroma formation, and ultimately mortality (5).
Acknowledgment: The Sir Stanley & Lady Davidson Fund, the Urquhart Trust, and the High Blood Pressure Foundation jointly supported this study. Professor Webb is currently the recipient of a Research Leave Fellowship from the Wellcome Trust (WT 0526330). We also thank Mr. N. Johnston, Ms. S. McCall, and Ms. M. Millar, for technical assistance, and Prof. R. Riemersma for advice.
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