Background Chronic smoking is associated with endothelial dysfunction, an early stage of atherosclerosis. It has been suggested that endothelial dysfunction may be a consequence of enhanced degradation of nitric oxide secondary to formation of oxygen-derived free radicals. To test this hypothesis, we investigated the effects of the antioxidant vitamin C on endothelium-dependent responses in chronic smokers.
Methods and Results Forearm blood flow responses to the endothelium-dependent vasodilator acetylcholine (7.5, 15, 30, and 60 μg/min) and the endothelium-independent vasodilator sodium nitroprusside (1, 3, and 10 μg/min) were measured by venous occlusion plethysmography in 10 control subjects and 10 chronic smokers. Drugs were infused into the brachial artery, and forearm blood flow was measured for each drug before and during concomitant intra-arterial infusion of the antioxidant vitamin C (18 mg/min). In control subjects, vitamin C had no effect on forearm blood flow in response to acetylcholine and sodium nitroprusside. In contrast, in chronic smokers the attenuated forearm blood flow responses to acetylcholine were markedly improved by concomitant administration of vitamin C, whereas the vasodilator responses to sodium nitroprusside were not affected.
Conclusions The present studies demonstrate that the antioxidant vitamin C markedly improves endothelium-dependent responses in chronic smokers. This observation supports the concept that endothelial dysfunction in chronic smokers is at least in part mediated by enhanced formation of oxygen-derived free radicals.
Chronic cigarette smoking represents a major risk factor for the development of atherosclerosis associated with coronary and peripheral vascular disease.1 Cigarette smoke is known to contain a large number of oxidants, and it has been hypothesized that many of the adverse effects of smoking may result from oxidative damage to critical biological substances.2 Previous reports have demonstrated abnormal endothelial function in chronic smokers.3 4 Endothelial dysfunction, in turn, has been proposed to play a pathogenetic role in the initiation of vascular disease.5 Although smoking-induced endothelial dysfunction is very likely multifactorial, more recent clinical and experimental observations strongly point to a potential role of oxygen-derived free radicals in mediating this phenomenon.6 7
Vitamin C is a strong reducing agent known to act as an antioxidant in vitro and in vivo.8 Vitamin C very effectively protects lipids in human plasma against peroxidative damage by scavenging oxygen-derived free radicals.9 In addition, epidemiological studies have shown that smokers have a significantly lower plasma level of vitamin C compared with nonsmokers.10
Therefore, the present study was designed to investigate whether the antioxidant vitamin C can improve endothelium-dependent vasodilation in chronic smokers.
Ten control subjects (mean age, 51±3 years) and 10 chronic smokers (mean age, 55±3 years) were studied (Table⇓). Chronic smokers were included if they had a history of 20 or more pack-years (1 pack-year defined as smoking 20 cigarettes per day for 1 year or the equivalent). No participant had a history of hypercholesterolemia, arterial hypertension, diabetes mellitus, or any other systemic disease predisposing them to endothelial dysfunction. Further exclusion criteria were current use of antioxidants or vasoactive medications. All participants gave written informed consent, and the study protocol was approved by the local Ethics Committee of the University of Freiburg.
All studies were performed in a temperature-controlled room in the postabsorptive state. Under local anesthesia and sterile conditions, a 20-gauge polyethylene catheter was inserted into the brachial artery of the nondominant arm for infusion of drugs. Forearm blood flow was measured by venous occlusion plethysmography as recently described.3 Systolic, diastolic, and mean arterial pressures and heart rate were determined at the contralateral arm with a Dinamap (845 oscillometric) blood pressure recorder. Forearm vascular resistance was calculated as the ratio of mean blood pressure to forearm blood flow and expressed as units reflecting mm Hg·mL−1·min−1·100 mL tissue−1.
Baseline measurements were obtained during intra-arterial infusion of 0.9% saline at a rate of 1.66 mL/min. To assess endothelium-dependent vasodilation, acetylcholine chloride was administered at increasing concentrations (7.5, 15, 30, and 60 μg/min). Sodium nitroprusside was infused as an endothelium-independent vasodilator (1, 3, and 10 μg/min). Each concentration of acetylcholine and sodium nitroprusside was infused for a 5-minute period. Forearm blood flow was measured during the last 2 minutes of the infusion period.
After another rest period of at least 40 minutes, vitamin C (sodium ascorbate, Merck) was administered intra-arterially to test the effects of vitamin C on forearm blood flow response to acetylcholine and sodium nitroprusside. Concentration and infusion rate of vitamin C (total dose about 1 g) was chosen to reach local forearm concentrations, which have been demonstrated to inhibit superoxide anion-mediated lipid peroxidation.11 Moreover, a similar concentration of vitamin C has been reported recently to improve endothelial function in patients with diabetes mellitus.12
We also measured plasma cholinesterase activity in both groups by using a cholinesterase activity assay (kinetic color test, Merck) to address a potentially confounding influence of cholinesterase to our results (normal range, 2300 to 7200 U/L).
All values are reported as mean±SEM. Group comparisons with respect to baseline characteristics were performed by unpaired t test. To test for differences in the overall dose-response relationship in response to acetylcholine and sodium nitroprusside with and without vitamin C, a two-way ANOVA for repeated measures was applied. Statistical significance was accepted at a level of α=5%.
The clinical characteristics of the study groups are provided in the Table⇑. All subjects included in this study were men. There was no difference in terms of age, blood pressure, lipid profile, forearm length, forearm volume, and cholinesterase activity. Chronic smokers had a history of 39±7 pack-years. The basal forearm blood flow and forearm vascular resistance were similar in both groups. In a separate group (n=4 smokers), the effect of vitamin C on cholinesterase activity was measured. Cholinesterase activity before and after administration of vitamin C (total dose of 1 g) was virtually identical (5054±193 versus 5037±199 U/L).
Effects of Intra-arterial Acetylcholine and Sodium Nitroprusside on Forearm Blood Flow in Control Subjects and Chronic Smokers
Increasing concentrations of acetylcholine dose-dependently increased forearm blood flow in control subjects from 3.2±0.2 to maximally 19.9±0.5 mL·min−1·100 mL−1 and decreased forearm vascular resistance from 29.7±2.4 to maximally 4.6±0.1 mL·min−1·100 mL−1. The vasodilator responses to acetylcholine were significantly (P<.05) attenuated in chronic smokers compared with nonsmoking control subjects (increase maximally from 3.0±0.28 to 12.2±1.1 mL·min−1·100 mL−1). In contrast, the increases in forearm blood flow in response to the endothelium-independent nitrovasodilator sodium nitroprusside were comparable in both groups (Figs 1⇓ and 2⇓).
Effects of Vitamin C on Forearm Blood Flow Responses to Acetylcholine and Nitroprusside
In control subjects, concomitant infusion of vitamin C did not alter responses to either acetylcholine or nitroprusside. In contrast, in chronic smokers vitamin C markedly improved endothelium-dependent vasodilation in response to acetylcholine (P<.05) while having no effect on vasodilation induced by the endothelium-independent vasodilator sodium nitroprusside (Figs 1⇑ and 2⇑). Since we did not observe significant changes in mean arterial pressure, changes in forearm vascular resistance mirrored changes in forearm blood flow.
The present studies demonstrate that impaired endothelium-dependent vasodilation in chronic smokers can be markedly improved by acute concomitant administration of the antioxidant vitamin C. This observation strongly supports the concept that increased production and/or activity of oxygen-derived free radicals contribute to endothelial dysfunction in chronic smokers.
With these studies we found a marked attenuation of endothelium-dependent vasodilation in forearm resistance vessels in chronic smokers compared with healthy control subjects. This observation is in agreement with previous observations showing in chronic smokers blunted endothelium-dependent vasodilation of large and small arteries in response to pharmacological (such as acetylcholine3 ) and/or mechanical stimuli (such as shear stress4 ). The precise mechanism of smoking-related endothelial dysfunction is not well understood but is probably multifactorial. Blunted responses to endothelium-dependent vasodilators may be explained in general by a nonspecific attenuation of the vasodilator capacity of the vessel at the vascular smooth muscle level, by diminished nitric oxide formation within endothelial cells, or may be secondary to enhanced degradation of nitric oxide as the result of endothelial and/or smooth muscle production of oxygen-derived free radicals.
A nonspecific attenuation of the vasodilator capacity of forearm resistance arteries is not very likely, since the vasodilator responses to the endothelium-independent nitrovasodilator nitroprusside were preserved. We also can exclude a confounding influence of the cholinesterase activity on our results, since the cholinesterase activity did not differ between smokers and nonsmokers and vitamin C per se had no stimulatory effect on the cholinesterase activity (see “Results”).
More recent experimental and clinical data strongly demonstrate evidence for increased oxidative stress being responsible for endothelial dysfunction in chronic smokers.6 7 Cigarette smoke (especially the gas phase) contains large amounts of free radicals and prooxidants such as NO, NO2, peroxinitrite, transition metals, phenols, epoxides, and nitrosamines.2 The particulate phase contains high concentrations of lipophilic quinones, which in biological systems undergo redox cycling to produce oxidant species such as O2−· and H2O2.2 These two compounds can form the highly reactive OH· radical at site-specific locations where catalytic iron is available.2 Furthermore, O2−· from cigarette smoke may reach the vascular endothelium and can then react with NO·,13 reducing the vasoactive levels of NO· and diminishing the response to endothelium-dependent vasodilators via formation of peroxynitrite anion (ONOO−), a highly reactive intermediate with strong cytotoxic potency.13 14 Thus, the damaging free radicals in cigarette smoke may cause either direct arterial wall injury or more may initiate and/or accelerate secondary processes including depletion of antioxidants (such as vitamin C or vitamin E9 ), protein peroxidation,15 and activation of phagocyte-platelet-endothelial cell interactions.16 17 Oxygen-derived free radicals within the vasculature may initiate a vicious cycle by oxidizing lipids or lipoprotein(a), which in turn may inhibit endothelium-dependent vasodilation directly18 or indirectly by the formation of superoxide anions.19 Recently, we demonstrated a close inverse relationship between antibody titers against oxidized LDL and acetylcholine-induced changes in forearm blood flow in chronic smokers, suggesting that oxidized LDL may be involved in endothelial dysfunction in chronic smokers.3 Further imbalance between prooxidants and antioxidants within the vasculature also may be operative in smokers, since plasma levels of vitamin C are in general lower compared with healthy control subjects,10 a phenomenon that appears to reflect increased metabolism as the result of oxidant load rather than decreased food intake.20
In the present study, the acute administration of the antioxidant vitamin C markedly improved endothelium-mediated vasodilatation in chronic smokers while having no significant effects on endothelium-independent vasodilation induced by sodium nitroprusside. This finding strongly suggests that the antioxidant vitamin C decreases oxidative stress within the vasculature of chronic smokers by directly scavenging oxygen-derived free radicals11 such as superoxide anions or hydroxyl radicals. Further antioxidant mechanisms of vitamin C may include an inhibition of smoke-induced leukocyte adhesion and aggregation to endothelial cells,21 protection from oxidized LDL-induced leukocyte adhesion to vascular endothelium, and protection of LDL against atherogenic modification9 22 (see Fig 3A⇓). The chemical reaction between superoxide and vitamin C is a protonation of superoxide leading to the perhydroxyl radical (HO2· [Reference 23]) or eventually to H2O2 (Reference 24) (see Fig 3B⇓).
The present study demonstrates that acute administration of vitamin C almost completely reverses endothelial dysfunction in chronic smokers. Whether or not daily oral intake of vitamin C may limit the cardiovascular consequences of chronic smoking requires further clinical investigation.
- Received April 15, 1996.
- Accepted May 7, 1996.
- Copyright © 1996 by American Heart Association
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