Blood Pressure Circadian Rhythm and Endothelial Function in Heavy Smokers: Acute Effects of Transdermal Nicotine

Abstract

Nicotine replacement therapy appears to be safe when used by healthy patients to aid in smoking cessation; however, the immediate acute effects of nicotine replacement therapy on the circadian rhythm of blood pressure (BP) and endothelial function in heavy smokers are not well understood. Twenty‐six heavy smokers were requested to stop smoking for 48 hours. BP and heart rate were recorded over 48 hours by ambulatory BP monitoring, with beat‐to‐beat changes being monitored for the first 10 hours by a noninvasive finger device. The reactivity of the brachial artery was evaluated using flow‐mediated dilation immediately after smoking cessation, before the application of a 21‐mg nicotine patch or placebo patch, and 24 hours after patch placement. Transdermal nicotine caused a mild but significant elevation in BP in the early morning in 21 of 26 volunteers. The decrease in nocturnal BP was attenuated in patients with the nicotine patch compared with the placebo patch; this was associated with impaired endothelium‐dependent vasodilation.

Cigarette smoking, the most preventable risk factor for cardiovascular disease, causes atherosclerosis and premature cardiovascular death associated with peripheral arterial disease, coronary heart disease, aortic aneurysms, and stroke. ¹ This risk factor, when associated with hypertension, increases the incidence of coronary heart disease by approximately four‐fold. ² , ³ , ⁴

Despite epidemiologic evidence demonstrating a strong link between smoking and cardiovascular disease, the mechanisms by which cigarette smoking promotes atherosclerotic disease are not fully understood. Direct evidence that cigarette smoking can cause endothelial injury was reported by Pittilo and colleagues ⁵ in the 1980s. Recent reports have also demonstrated that smoking is associated with damage to the blood vessel wall and endothelial injury. ⁶ , ⁷ , ⁸ Cigarette smoke is a complex mixture of chemical substances that includes carbon monoxide, free radicals, polycyclic aromatic hydrocarbons, and nicotine; however, it is still unclear whether nicotine has a direct effect on atherosclerosis. The acute cardiovascular effects of smoking are related to the hemodynamic actions of nicotine. IV infusions, nasal sprays, and chewing gum that contain nicotine cause acute increases in heart rate (HR) and blood pressure (BP). ⁶ More recent studies have shown that cigarette smoke can morphologically and functionally alter the vascular endothelium. ⁵ Smoking cessation, however, may be followed by fast recovery of the accompanying endothelial dysfunction. ⁹

Nicotine is the main compound responsible for addiction to tobacco. ¹⁰ Nicotine replacement therapy (NRT) using chewing gum, nasal sprays, buccal inhalers, lozenges, or transdermal patches is effective when used as part of a strategy for smoking cessation. ¹¹ , ¹² Although other medications and a combination of two or more methods of providing nicotine have been proposed, ¹³ nicotine by itself has been the mainstay of pharmacotherapy for tobacco addiction. ¹¹ Whereas NRT is safe in normotensive ¹² and mildly hypertensive ¹⁴ smokers, some studies have shown that nicotine contributes to endothelial dysfunction in veins ¹⁵ and arteries. ¹⁶ Since vascular reactivity is impaired in hypertensive individuals ¹⁶ , ¹⁷ , ¹⁸ and smokers, ¹⁹ , ²⁰ , ²¹ it is possible that NRT may alter the normal circadian pattern of arterial BP.

In this study, we investigated whether the acute administration of nicotine patches (NPs) caused alterations in the circadian rhythm of BP and vascular responsiveness in heavy smokers 24 hours after smoking cessation. We also measured plasma nitrite/nitrate levels as an indicator for nitric oxide (NO) formation and thromboxane B₂ (TXB₂), a product of arachidonic acid metabolism, as a biochemical marker for increased thromboxane biosynthesis by activated platelets. Both of these mediators are associated with endothelial dysfunction in smokers.

The hypothesis tested in this work is that endothelial dysfunction exists in heavy ex‐smokers who use NPs to stop smoking.

METHODS

Subjects

The subjects were 26 healthy men aged 35–52 years (mean age, 43.9 years) who smoked more than 30 cigarettes per day (average, 36; range, 30‐44) and had smoked for more than 15 years (range, 8–22 years). Written informed consent was obtained from each subject, and the study was approved by the institutional Ethics Committee of the Faculty of Medical Sciences at the State University of Campinas (UNICAMP). The volunteers provided a complete medical history and underwent a physical examination, an electrocardiogram (ECG), and laboratory analysis to exclude individuals with dyslipidemia, diabetes mellitus, and evidence of hepatic, renal, or hematologic dysfunction.

Experimental Protocol

The study had a placebo‐controlled, parallel, single‐blind design. The subjects were asked to stop smoking for 24 hours on two separate days. On these occasions, the volunteers were randomized to treatment with either 21‐mg transdermal nicotine (Nicoderm, Alza, Palo Alto, CA) or placebo patches (Pps) after they had remained supine for at least 30 minutes. The patches were placed at 8:00 a.m. An indwelling venous catheter was inserted for blood sample collection, and the subjects remained supine thereafter. The first 10 hours of the study was done in the hospital, after which the volunteers went back to their daily routine and returned to the hospital the following morning (Figure 1).

Figure 1.

Experimental protocol. ABPS=ambulatory blood pressure system; TXB₂=thromboxane B₂; FMD=flow‐mediated dilation

Noninvasive BP Monitoring

The beat‐to‐beat mean BP (MBP) and HR were monitored with a noninvasive finger Finapres 2300 device (Ohmeda, Englewood, CO) during the first 10 hours of each 24‐hour study. Briefly, this device, which is based on the arterial volume clamp method, measures BP through a small finger cuff wrapped around the middle finger of the hand. ²² The MBP and HR values were recorded every 10 seconds throughout the study period. The average values recorded for 5 minutes around predetermined time points were used to compare the MBP and HR results. Simultaneously, an ambulatory BP system (Spacelabs, Issaquah, WA) was used to check the Finapres measurements every 15 minutes. After the 10th hour, the BP was monitored only by the ambulatory BP system (Figure 1).

Measurement of Plasma Nitrite/Nitrate and TXB₂

Venous blood samples (10‐15 mL) were collected in tubes containing ethylenediaminetetraacetic acid at the time of smoking cessation, immediately before placement of the NPs or Pps (patch type was determined randomly), and 24 hours later (end of protocol) (Figure 1). The plasma was separated by centrifugation and stored at −20°C until assayed. Nitrite and nitrate levels were used as markers for the activity of NO synthase and NO biosynthesis. The assay was based on the determination of nitrite using the Griess reaction. Nitrate was measured as nitrite after enzymatic conversion by nitrate reductase as described by Green et al. ²³ Plasma samples were extracted using C₁₈ reverse‐phase cartridges (Waters Co., Milford, MA) and the TXB₂ levels were determined using a commercial enzyme immunoassay (Cayman Chemical Co., Ann Arbor, MI). ²⁴

Flow‐Mediated Dilation Technique

Arterial endothelial and vascular smooth muscle function was assessed by examining the brachial artery responses to endothelium‐dependent (flow‐mediated dilation [FMD]) and flow‐independent (nitroglycerin [NTG]‐mediated) stimuli, using a modification of the technique described by Celermajer et al. ²⁵ and according to the guidelines report of the International Brachial Artery Reactivity Task Force published in 2002 by Corretti et al. ²⁶ This method presents a negligible intraobserver and interobserver bias (0.05% FMD) and, compared with manual measurements, the difference is 1.05% of the FMD. ²⁷ The subjects rested quietly for 15 minutes before the first scan and remained in the supine position throughout the study. The brachial artery was scanned longitudinally 5–10 cm above the elbow and, when a satisfactory transducer position had been found, a special probe holder designed specifically for the study was fixed around the arm to secure the ultrasound transducer, which was then held in the same position throughout the study.

Arterial diameter measurements were made at end‐diastole (R‐wave peak of the ECG) using ultrasound equipment and a high‐resolution linear vascular transducer 7–12 MHz ATL HDI system (Advanced Technology Laboratories, Seattle, WA), coupled with computer‐assisted analysis software (MEDWARE, Brasilia, Brazil) utilizing a computerized edge detection system for multiple measurements of the brachial artery diameter.

The FMD responses were used as a measure of endothelium‐dependent vasodilation. A specific polyurethane cuff (Hokanson, Inc., Bellevue, WA) placed above the transducer was inflated around the arm to a pressure of 250 mm Hg for 5 minutes and then rapidly deflated. The resulting FMD in the arm and the increased brachial artery diameter was recorded from 15 to 90 seconds after cuff deflation. Changes in the brachial artery diameter in response to endothelium‐dependent NO‐mediated vasodilation were expressed as the percentage change relative to the vessel diameter immediately before cuff inflation. After allowing 10‐15 minutes for the brachial artery to recover, another baseline scan was taken. Subsequently, 0.4 mg of NTG (Nitrostat, Parke‐Davis, Morris Plains, NJ) was given sublingually and 5 minutes later the brachial artery was imaged. The response to NTG was used as a measure of endothelium‐independent vasodilation and expressed as the percentage change relative to the vessel diameter immediately before drug administration.

These data were obtained no more than 15 minutes before or after patch placement or removal, respectively. Evaluation of the computer images were made by independent observers blinded to the assignment. ²⁷

Statistical Analyses

The results were expressed as the mean ± SD. The sample size for shear stress‐mediated vasodilation was calculated to achieve a power of 0.80. Baseline MBP, HR, and nitrite/nitrate and TXB₂levels were compared using one‐way analysis of variance (ANOVA) followed by the Student‐Newman‐Keuls post hoc test. The changes in MBP, HR, and nitrite/nitrate and TXB₂ levels from baseline values within each group were analyzed using one‐way analysis of variance (ANOVA) for repeat measurements followed by Dunnett's test. A probability value of p<0.05 indicated significance.

RESULTS

Characteristics of the Study Group

Table I summarizes the basic characteristics of the study subjects. Some of these results were derived from a questionnaire used to assess addiction to smoking.

Table I.

Clinical Characteristics of the Study Participants

Parameter	Value
Age (yr)	43.9±8.1
Weight (kg)	75.5±14.4
Height (cm)	163.5±11.5
Body mass index (kg/m2)	29.0±2.7
Systolic blood pressure (mm Hg)	127±15
Diastolic blood pressure (mm Hg)	81±12
Heart rate (bpm)	70±10
Smoking (cigarettes/d)	36.3±6.2
Glycemia (mg/dL)	91.6±16.3
Glycosylated hemoglobin (%)	5.5±0.3
Total cholesterol (mg/dL)	189.1±34.0
Low‐density lipoprotein cholesterol (mg/dL)	93.5±26.4
Triglycerides (mg/dL)	160.0±70.4
Number of subjects	26
All values are expressed as mean ± SD.

Arterial BP Recordings

Data from the Finapres system showed similar changes when compared with those from the ambulatory BP system and were used to analyze the MBP and HR responses during the first 10 hours (Figure 2A and 2B). The 24‐hour use of a PP did not change the MBP and HR in heavy smokers compared with the same parameters described previously for nonsmokers. ¹²

Figure 2.

A) Mean blood pressure (MBP) (left) and heart rate (right) responses during the first 10 hours after smoking cessation (N=26). The points are the mean±SD. B) MBP (left) and heart rate (right) responses during the first 10 hours after randomization to the placebo (n=13) or nicotine (n=13) patches. *p<0.05

Transdermal nicotine (21 mg) caused a mild but significant (p<0.05) elevation in systolic BP in the morning (6–7 a.m.) in 11 of 13 volunteers (132±5 mm Hg vs. 112±4 mm Hg at 5 a.m.) when compared with the increase seen with the PP. Using the ambulatory BP method, the drop in nocturnal BP was attenuated in the NP group compared with the PP group (12.4% vs. 33.1%; p<0.01) (Figure 3A and 3B). Neither of the BP monitoring systems revealed any significant changes in the HR in any phase of the protocols.

Figure 3.

The 24‐hour blood pressure (BP) recordings after placement of the placebo (A) and nicotine (B) patches. The points are the mean±SD. The drop in nocturnal BP was attenuated in the nicotine (B) compared with the placebo (A) patch group. 12.4% vs. 33.1%. SBP=systolic BP; DBP=diastobc BP; *p<0.01

Vascular Responsiveness

The results for the endothelium‐dependent and NTG‐induced vasodilation are summarized in Table II. Twenty‐four hours after the interruption of smoking, the vascular responsiveness was normal in heavy smokers, but NPs impaired the FMD (endothelium‐dependent) vasodilation 24 hours after patch application. In contrast to the endothelium‐dependent response, endothelium‐independent vasodilation was normal in the NP group when compared with the PP group.

Table II.

Flow‐Mediated (Endothelium‐Dependent) and Glyceryl Trinitrate‐Induced Vasodilation of the Brachial Artery in Heavy Smokers (N=26), Before and After Smoking Cessation, and Placebo (n=13) or Nicotine Patch Randomization (n=13)

Brachial Artery Dilation	Before Smoking Cessation		After Smoking Cessation		After Patch Randomization
	Placebo	Nicotine	Placebo	Nicotine	Placebo	Nicotine
Flow‐mediated	8.0±1.2	7.6±1.1	14.1±1.3*	15.6±1.2*	13.4±1.0	9.1±0.8**,†
Glyceryl trinitrateinduced	22.3±2.0	23.9±2.5	24.6±3.0	22.9±2.5	25.9±2.7	24.8±3.1
All values are expressed as mean ± SD. *p<0.05 vs. before smoking cessation; **p<0.05 vs. after smoking cessation; † p0.01 vs. after patch randomization

Plasma Nitrite/Nitrate and TXB₂ Levels

The interruption of smoking was followed by an increase in the plasma nitrite/nitrate levels and a decrease in TXB₂ Treatment with the Pps and NPs reduced the plasma nitrite/nitrate levels. After placement of the NPs, the TXB₂ concentrations returned to the levels seen on the first day after smoking cessation (Figure 4).

Figure 4.

Plasma nitrite (NO₂)/nitrate (NO₃) and thromboxane B₂ (TXB₂) levels before and after smoking cessation (24 hours) and after randomization of the placebo (n=13) and nicotine (n=13) patches. The columns are the mean±SD. *p<0.01 vs. before smoking cessation; **p<0.05 vs. placebo patch

DISCUSSION

In this study, the use of 21‐mg NPs altered the BP circadian rhythm in apparently healthy heavy smokers and was associated with impaired endotheliumdependent vasodilation. In addition, transdermal nicotine had no effect on nitrite and nitrate levels and restored the plasma TXB₂ concentrations to the level seen on the first day after smoking cessation.

Some studies have shown that a damaged vascular endothelium and enhanced platelet aggregation play a role in the development of premature atherosclerosis caused by smoking, although it is uncertain whether nicotine is responsible for platelet activation in cigarette smokers. ²⁸ , ²⁹ In addition to oxidant gases from cigarette smoke, ³⁰ nicotine may have an important role in the cardiovascular effects of smoking because it interacts with the endothelium ⁸ , ³¹ and decreases endothelium‐dependent vasodilation in arteries ¹⁶ , ¹⁷ and veins, ³² thereby potentially contributing to the endothelial dysfunction associated with smoking. ³³ On the other hand, cessation of smoking and the acute interruption of cigarette consumption reverses smoking‐induced endothelial dysfunction, ⁹ and this could partly explain the rapid reduction in the risk of cardiovascular mortality after quitting smoking.

The pharmacologic effects of nicotine on the cardiovascular system are well known and include direct and indirect increases in the BP and HR due to sympathomimetic effects on the heart and because it stimulates catecholamine release. ³⁴ The mechanisms involved in acute cardiac events in smokers include hypercoagulability, prothrombotic state, and hemodynamic alterations caused by nicotine. ³⁵ The latter changes include an increase in HR and BP, which, in turn, increase myocardial work and oxygen demand. Sympathetic neural activation is also associated with coronary vasoconstriction and a decrease in blood flow and oxygen supply to the heart in individuals with coronary artery disease. ⁶

The safety of high‐dose transdermal nicotine for smoking cessation has been evaluated in healthy men, with an assessment of dose‐related effects. No significant changes in the HR or BP were observed with doses of nicotine up to 63 mg/d or with combined transdermal nicotine and smoking compared with nicotine alone, which suggested that the treatment of heavy smokers with highdose transdermal nicotine is safe. ³⁶

The present study revealed two important effects of nicotine on the circadian rhythm of BP, namely, a slight increase in BP in the early morning and a well‐defined attenuation of the normal decrease in arterial pressure at night, both of which were possibly related to nicotine‐induced endothelial dysfunction. These findings are of clinical relevance because they suggest that healthy heavy smokers who interrupt tobacco consumption and simultaneously start NRT may have a higher risk of developing cardiac events. ³⁷ However, the risk associated with NRT is considerably lower than that associated with the continuation of smoking. ¹² The endothelium‐dependent dysfunction induced by the withdrawal of smoke‐derived nicotine may contribute to vascular hyperreactivity and is probably sustained by high levels of nicotine seen in smokers. ³⁶ , ³⁷ , ³⁸ , ³⁹ , ⁴⁰

Although the sympathomimetic actions of nicotine and its complex cardiovascular effects are believed to involve the central nervous system and the stimulation of autonomic ganglia, including the release of noradrenaline from sympathetic nerve endings and of adrenaline from the adrenal medulla, ⁴¹ the absence of an elevation in HR argues against this possibility. Nicotine receptor down‐regulation could explain alterations in HR and BP in heavy smokers. ⁴² , ⁴³ , ⁴⁴ Although NPs produce only minor disturbances of autonomic regulation, ⁴⁵ , ⁴⁶ and are therefore considered to be clinically safe, these minor effects should not be considered irrelevant. ⁴⁷

The stable plasma TXB₂ and nitrite/nitrate levels seen in heavy smokers after the use of transdermal nicotine suggest that NRT does not increase their risk of cardiovascular events any further. ⁴⁸ , ⁴⁹ Although heavy smokers have high plasma TXB₂ concentrations compared with nonsmokers, the administration of 21 mg of transdermal nicotine does not alter the urinary excretion of TXB₂. ⁵⁰ In our study, heavy smokers who received NPs had similar basal TXB₂levels at the start of smoking interruption and at the end of the 24‐hour patch placement periods, in agreement with a previous report. ¹⁴ This finding probably reflected the relatively long half‐life of nicotine and could explain why plasma TXB₂ levels of heavy smokers were similar before and after the NP. Increasing nicotine plasma levels could be associated with endothelial and platelet dysfunctions, both reflected by a TXB₂ increase.

The finding that plasma nitrite/nitrate levels were similar in heavy smokers during the periods with the Pps and NPs probably reflected the controversial effects of nicotine on endothelial cells and expression of endothelium‐dependent relaxation and constricting factors. The low plasma levels of NO reported in long‐term habitual smokers is probably related to the rapid scavenging of NO by reactive oxygen species present in cigarette smoke. ³³ , ³⁴ Our finding was consistent with other reports that have investigated NO levels during 24 hours of smoking interruption and related this to the short plasma half‐life of reactive oxygen species.

CONCLUSION

Since disturbances in the BP circadian rhythm are clearly associated with cardiac events, the main finding of this study was the close relationship between the attenuated nocturnal decrease in BP and the impaired endothelial function in heavy smokers who were about to initiate NRT. Clinically, physicians and other professionals need to provide adequate instruction in the use of NRT, especially with regard to the adequate monitoring of the interval between the interruption of smoking and the placement of NPs. It is important to advise heavy smokers who suffer from coronary artery disease or hypertension and who are intending to cease smoking with the help of NPs of the possible dangers and to monitor these individuals closely.

Acknowledgments and disclosure: The authors thank Joaquim Francisco do Prado and David A. Hewitt for their technical assistance. This study (98/05298–6) and SEM (01/09584–8) were supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, SP, Brazil). H. Moreno Jr, M.C. Irigoyen, and E.A. Nogueira were supported by research fellowships from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil).