Smoking is a major risk factor for coronary and peripheral vascular disease (1-3). Although the precise mechanism by which smoking accelerates atherosclerosis is not known, long-term cigarette smoking has been shown to be associated with an impaired endothelium-dependent vasodilation (EDV) in the coronary circulation (4) and the brachial artery (5) by some, but not all groups (6,7). Short-term cigarette smoking has recently been found to impair flow-mediated vasodilation in the brachial artery in healthy subjects (8,9). Because endothelial dysfunction is an early event in the development of atherosclerosis (3), these data suggest that the endothelium may be a target for the deleterious effects of smoking.
Although the components of cigarette smoke responsible for these effects are not known, some clinical and experimental observations have suggested a potential role of the generation of oxygen free radicals by smoke (9-12). Cigarette smoke also is known to contain, among other things, carbon monoxide and nicotine, which may also, directly or indirectly, affect endothelial function. Nicotine chewing gum is commonly used during the cessation period from smoking. The chewing gum releases nicotine to raise the circulating levels of this drug to the levels seen shortly after smoking a cigarette. Although the hemodynamic effects of nicotine chewing gum have been explored (13), the effects on EDV have not been studied.
This study was designed to investigate the short-term effects of smoking and nicotine chewing gum, to evaluate the role of nicotine alone on endothelium-dependent vasodilation in the forearm of young, healthy habitual smokers using forearm plethysmography. The hypothesis evaluated was that an increase in circulating nicotine levels could impair EDV.
MATERIALS AND METHODS
The study sample consisted of 16 young healthy students (seven men and nine women) who ranged in age from 20 to 25 years and were regular smokers. None of the subjects was taking regular medication or had a history of any disease known to affect the cardiovascular system. No subjects with a history of any metabolic or other serious diseases were included in the study. Four of the subjects smoked more than 10 cigarettes a day, six fewer than five cigarettes a day, and six between five and 10 cigarettes a day. The mean duration of smoking was 5 ± 3 (SD) years.
The studies were performed in the morning after an overnight fast. All subjects abstained from smoking for ≥8 h before the study. The subjects were supine in an air-conditioned room at a constant temperature (20°C). An arterial catheter was inserted into the brachial artery for regional infusion of metacholine (MCh; evaluating endothelium-dependent vasodilation, EDV) or sodium nitroprusside (SNP; evaluating endothelium-independent vasodilation, EIDV). The infused dosages were 4 μg/min for MCh and 10 μg/min for SNP. Our previous studies have shown that these infusion rates do not induce any alterations in systemic hemodynamics or in forearm blood flow (FBF) in the contralateral arm, and induce similar increments in FBF in the infused arm (14).
Thirty minutes after the insertion of the catheter, resting FBF was measured. Thereafter, one of the vasodilatory drugs (MCh or SNP) was given locally in the forearm for 5 min. During min 4, FBF was recorded. This procedure was repeated with the other vasodilator after 15 min of washout. The order of the two vasodilators was randomized.
Protocol 1: acute and semiacute effects of smoking
To examine the effects of cigarette smoking on EDV and EIDV, 10 subjects smoked one cigarette (0.9 mg nicotine) over 4-5 min after the evaluation of the baseline recordings, standing in an open window close to the examination bed. Five minutes after the completion of smoking, MCh or SNP was infused for 5 min. During the fourth minute, FBF was recorded (early phase, evaluating EDV or EIDV at peak concentration of nicotine). Pilot studies without smoking, but using a similar procedure, showed that central hemodynamics and FBF had returned to baseline levels at this time. This procedure was repeated for the same vasodilatory drug 20 min later (plateau phase, evaluating EDV or EIDV after nicotine levels had reached a plateau). After 15 min of washout, the other vasodilatory drug was infused, and FBF was determined. After another 15 min of washout, to evaluate the effect of peak concentrations of nicotine on the second vasodilatory drug, the subjects were asked to smoke half a cigarette during 4-5 min, increasing the nicotine levels to a comparable peak as seen after the first cigarette. After 5 min, infusion of the second drug was started, and FBF was recorded during the 4th minute of infusion of the second drug. The order of the two vasodilatory drugs was randomized.
Protocol 2: acute effects of nicotine gum
To examine the effects of nicotine on EDV and EIDV, six subjects were instructed to chew a 4-mg nicotine gum (Nicorette; Pharmacia & Upjohn, Uppsala, Sweden) for 30 min after similar baseline recordings were performed as described for the smoking protocol. After 30 min, when the plasma nicotine levels were supposed to reach maximum, EDV and EIDV were determined in a random order with a washout phase of 15 min between the drugs while the subjects continued chewing the gum.
Hemodynamic variables were monitored, and blood samples for the assay of nicotine levels were gathered at the time of measurements of EDV and EIDV in the two protocols.
FBF was measured by venous occlusion plethysmography. A mercury in Silastic strain gauge was placed at the upper third of the forearm, which rested comfortably slightly above the level of the heart. The strain gauge was coupled to a calibrated plethysmograph. Venous occlusion was achieved by a blood pressure cuff applied proximal to the elbow and inflated to 40 mm Hg by a rapid cuff inflator. Determinations of FBF were made by the mean of five consecutive recordings.
We have previously shown the short-term (2 h) and long-term (3 weeks) variability of FBF during vasodilation with MCh and SNP with this method to be <7% (14). In this study, a control protocol was performed in four young healthy subjects who underwent FBF and other hemodynamic measurements according to a time table and manner similar to that in the two other groups, with the exception that they did not smoke.
Blood pressure was monitored with an automatic device (OMRON HEM 705C, Tokyo, Japan). Mean arterial pressure (MAP) was calculated as pulse pressure divided by 3, added to the diastolic blood pressure.
Cardiac output and heart rate were measured with a thoracic bioimpedance-cardiograph (BioMed NCCOM 3 cardiodynamic monitor; Irvine, CA, U.S.A.). Cardiac output was divided by body surface area to obtain cardiac index (CI). Total peripheral resistance index (TPRI) was obtained by the formula: 80 × (MAP - 3)/CI. In this equation, the central venous pressure was regarded to be 3 mm Hg in all subjects.
As a change in blood pressure was expected during the investigation, forearm vascular resistance (FVR) was calculated as MAP divided by FBF, and the FVR values obtained during infusions of MCh and SNP were regarded as our main measurements of EDV and EIDV, respectively. To obtain the relative contribution of the endothelium to the vasodilation, the ratio between FVR during SNP and during MCh infusion, the index of endothelial function (14) was calculated.
For the assay of nicotine, 5 ml venous blood was collected in a venoject VT-100H and centrifuged at 1,400 g for 10 min. The plasma was then separated, transferred to a cryogenic vial, and frozen within 4 h. Samples were analyzed with a gas-liquid chromatography method (22) at Pharmacia & Upjohn, Helsingborg, Sweden.
The study was approved by the Ethics Committee at Uppsala University, and each participant gave informed consent.
Differences within each protocol were evaluated by means of analysis of variance (ANOVA) for repeated measurements with Dunnett's post hoc test. Relations between the continuous variables were investigated using univariate and multiple regression analysis. A value of p < 0.05 was regarded as significant.
Mean values for heart rate, CI, MAP, TPRI, and resting FBF at baseline, early, and plateau phases of cigarette smoking are presented in Table 1. In Table 2 the corresponding effects of chewing the nicotine gum are given. Serum nicotine levels are presented in Fig. 1. The serum levels of nicotine were similarly elevated 10 min after the first cigarette and 10 min after the later smoked half of a cigarette.
MAP, CI, and resting FBF changed significantly during smoking. An increase was seen during the early phase of smoking, but not the plateau phase (p < 0.01 for all variables compared with baseline). Heart rate was increased, compared with baseline, at both the early and the plateau phases of smoking (p < 0.01 and p < 0.05, respectively). Resting FVR and TPRI remained unchanged during both the early and the plateau phases (for details, see Table 1).
Nicotine gum increased MAP, heart rate, and CI significantly (p < 0.05 for all), but not resting FBF, resting FVR, or TPRI (for details, see Table 2). FVR during infusion with MCh changed significantly during both smoking and chewing gum, increasing from 4.6 ± 1.4 to 5.9 ± 2.1 mm Hg/ml/min/100 ml tissue at the early and to 5.0 ± 1.6 mm Hg/ml/min/100 ml tissue at the plateau phase of smoking (p < 0.01 for both vs. baseline, and p < 0.05 between early and late effect) and from 4.5 ± 1.6 to 5.2 ± 1.6 mm Hg/ml/min/100 ml tissue after chewing the nicotine gum (p < 0.01; Fig. 2). The impairments in EDV seen after cigarette smoking and chewing gum were highly reproducible and were seen in all of the subjects investigated. The order of infusions of MCh and SNP did not influence the results. As baseline EDV and EIDV did not differ significantly between the two intervention groups, only the baseline values for the smoking group are given in the figures. No significant changes in FVR during infusion with SNP were seen after smoking or after chewing the nicotine gum (Fig. 2).
Consequently, the index of endothelial function, here defined as the ratio between FVR during infusion with SNP and metacholine, changed significantly during smoking, as well as after chewing the nicotine gum (p < 0.05 for the plateau and p < 0.01 for the early phase of smoking and nicotine gum vs. baseline and p < 0.05 between early and plateau phases of smoking). These changes in the endothelial function index were seen in all investigated subjects.
Neither MAP, heart rate, CI, and TPRI, nor resting FBF or FVR during MCh or SNP infusions changed significantly during the control protocol (see Table 3). When all data were analyzed together (baseline, early phase, plateau phase, and chewing gum), there were significant relations between plasma nicotine levels and mean arterial pressure (r = 0.60, p < 0.001) or heart rate (r = 0.71, p < 0.001). Furthermore, plasma nicotine levels, MAP, and heart rate were all univariate predictors of the index of endothelial function (r = −0.57, p < 0.001, r = −0.59, p < 0.001, and r = −0.39, p < 0.01, respectively, see Fig. 3). These relations were significant also when the baseline values were excluded. In multiple regression analysis, only MAP and plasma nicotine levels remained as significant predictors of the endothelial function index.
This study showed that both smoking and nicotine chewing gum impaired EDV ≤30 min after exposure in young habitual smokers. Furthermore, the degree of impairment in endothelial function achieved by smoking and chewing gum was independently related to both the circulating levels of nicotine and the blood pressure reaction.
Several investigators have studied the differences in EDV between smokers and nonsmokers in a cross-sectional way. Using this approach, an impaired, an unchanged, or even an enhanced EDV has been reported in long-term smokers (4-7,16). However, the two studies published so far evaluating the short-term effects of smoking, using the ultrasound technique to evaluate flow-mediated vasodilation, both found a deleterious effect of smoking shortly after smoking a cigarette (8,9).
Our investigation extends these observations on short-term smoking with measurements of EDV and EIDV performed both at 10 min after the start of the smoking and also after 30-50 min by the use of local infusion of metacholine and SNP in the forearm. It was then found that although EDV was mainly attenuated shortly after smoking, the smoking-induced impairment in EDV was still present 30-50 min after smoking a cigarette. Thus the deleterious effect of smoking on EDV is not only a short transient effect seen when the nicotine levels are at maximal levels.
The mechanisms whereby smoking impairs EDV are not known. It has repeatedly been shown that smoking acutely increases blood pressure, heart rate, and cardiac output by an increased catecholamine release, predominantly epinephrine (17). Furthermore, cigarette smoke contains a considerable amount of carbon monoxide and oxygen free radicals (18). It is evident that at least free radical generation is involved in the pathophysiologic events induced by smoking, as the levels of the antioxidant vitamin C were decreased and the levels of a marker for lipid peroxidation (TBAR) were increased in smokers.
Furthermore, addition of vitamin C improved flow-mediated vasodilation and decreased TBAR in the brachial artery in smokers (9).
In our study the deleterious effects of smoking were independently related to both the achieved circulating levels of nicotine and the increase in blood pressure. Although the mechanism by which nicotine affects the endothelium is not known, changes in structural and mitotic activity of aortic endothelial cells (19) and an increased aortic endothelial cell death (20) have been shown in nicotine-treated animals, indicating a direct effect of nicotine on endothelial cells.
Although it has been shown that essential hypertension is associated with an impaired EDV (21), it is not known if the attenuated EDV found in these patients is due to the high blood pressure or if an impaired EDV is involved in the development of hypertension. However, in a recent study, we were able to show that a short-term elevation of blood pressure in otherwise normotensive subjects rapidly (within minutes) impaired EDV (22). Thus the short-term impairment in EDV induced by smoking might be mediated by the acute increase in blood pressure seen while smoking. On the other hand, as long-term smoking generally is not associated with an elevated blood pressure the long-term effects of smoking on EDV might be mediated by other factors.
Chewing a nicotine gum is often used to avoid nicotine abstinence during the period of withdrawal from smoking. The time to reach the maximal nicotine levels by gum is slower than that during cigarette smoking, and therefore EDV and EIDV were assessed after 30 min. Chewing the nicotine gum resulted in an increase in both nicotine levels and blood pressure with values between the early and late response of smoking. The effect on EDV was between the early and late response of smoking, suggesting a dose-response relation between the exposure to nicotine and the impairment in EDV. This is further supported by the regression analysis showing a close relation between endothelial function and nicotine levels, in which the subjects exposed to chewing gum fitted the regression line, as well as those smoking a cigarette.
In this study, blood pressure was changed by the intervention, and therefore FVR rather than FBF was used for the calculation of EDV and EIDV. We also calculated the endothelial function index, an index independent of changes in blood pressure, and similar to EIDV, it was only marginally changed by the intervention. Both of these approaches to evaluate endothelium-dependent vasodilation yielded similar results.
During the experiments, baseline FBF increased. This might theoretically alter the vasodilatory responses to MCh and SNP as it might alter the local pharmacokinetics of the vasodilators. However, we have previously found that this is not the case if baseline FBF is not grossly changed. In a previous study, baseline FBF was not related to EDV or EIDV (14), and in other yet unpublished studies, interventions that changed baseline FBF to a similar extent as in our study did not influence the magnitude of EDV and EIDV. In our study, this is exemplified by the fact that EIDV was not altered, although baseline FBF was increased by smoking.
A limitation of this study is that it was performed with a small number of subjects. Although the findings were highly reproducible and all participants responded in the same way, it cannot be concluded that every habitual smoker would respond in the same way. The design of the investigation was an open study with a separate time-control group. The ideal schedule would have been to reinvestigate the same subjects with placebo smoking or chewing a placebo gum. It should also be emphasized that these results were found in habitual smokers and that the results cannot be extrapolated to nonregular smokers or to passive smoking.
In conclusion, cigarette smoking induced a dose-dependent attenuation in EDV, reaching a maximum shortly after initiation of smoking and persisting up to 30-50 min. Chewing nicotine gum also induced a similar impairment in EDV. This unwarranted effect of both smoking and the chewing gum was independently related to both the achieved level of nicotine and the increase in blood pressure.
Acknowledgment: This study was supported by a grant from Pharmacia & Upjohn. We also thank Anna Lindberg at Pharmacia & Upjohn for skillful analysis of the nicotine samples.
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