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Department of Epidemiology and Public Health (E.B., M.K.), and International Institute for Society and Health (M.K.), University College London, London WC1E 6BT, United Kingdom; and Department of Biological Psychology (C.K.), Technical University of Dresden, D-01062 Dresden, Germany
Address all correspondence and requests for reprints to: Ellena Badrick, B.Sc., M.Sc., Department of Epidemiology and Public Health, University College London, London WC1E 6BT, United Kingdom. E-mail: e.badrick{at}ucl.ac.uk.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Objective: The objective of the study was to assess the relationship between smoking status and salivary cortisol.
Design: This was a cross-sectional study of smoking status and cortisol secretion from phase 7 (2002–2004) of the Whitehall II study.
Setting: An occupational cohort was originally recruited in 1985–1987.
Participants: The study population consisted of 3103 men (1514 never-smokers, 1278 ex-smokers, and 311 smokers) and 1128 women (674 never-smokers, 347 ex-smokers, and 107 smokers). Information was collected on smoking status, average number of cigarettes smoked, and additional covariates.
Outcome Measures: Saliva samples were taken on waking; waking + 0.5, 2.5, 8, and 12 h; and bedtime for the assessment of cortisol.
Results: Smoking status was significantly associated with increased salivary cortisol release throughout the day (P < 0.001) adjusted for covariates; this was apparent for the cortisol awakening response (P < 0.001) when examined separately. Compared with never-smokers, smokers had higher release of total cortisol (P = 0.002), whereas no difference was observed between never-smokers and ex-smokers (P = 0.594): mean release per hour (nanomoles per liter), never-smokers, 4.13 [confidence interval (CI) 4.02–4.24]; ex-smokers, 4.21 (CI 4.08–4.35); smokers, 4.63 (CI 4.35–4.93). There was no significant relationship between number of cigarettes smoked and total cortisol release. However, a difference was observed for the cortisol awakening response: mean release by tertiles of cigarettes smoked (nanomoles per liter): high, 13.49 (CI 10.74–16.23); medium, 9.58 (CI 7.40–11.76); low, 8.49 (CI 5.99–10.99), P = 0.029.
Conclusion: Salivary cortisol is increased in current smokers, compared with nonsmokers; no differences were observed between ex-smokers and never-smokers, suggesting that smoking has a short-term effect on the neuroendocrine system.
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Introduction |
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Studies have also shown a direct acute affect of smoking on cortisol levels (7), effects that have been attributed to activation of central nicotinic receptors. Activation of the HPA axis is reported to require acute nicotine exposure (consumption of two high-strength cigarettes in quick succession), and some studies have shown a dose response action (8, 9, 10). It is believed the HPA axis is altered in smokers (11); however, the reported effects of habitual smoking on cortisol levels in everyday life are mixed with some studies indicating no relationship (12, 13, 14), whereas others report that smoking is associated with increased cortisol secretion (15, 16, 17, 18, 19). The differences in the findings of these studies may be attributed to small study sample (19), differences in sample design (measurement of cortisol in urine or plasma), and differences in timing of the sample collection (15).
Cortisol has a diurnal profile, which is characterized by a substantial increase in cortisol secretion after awakening, peaking at about 30 min, and a subsequent decline over the remainder of the day. It is hypothesized that the cortisol awakening response (CAR) and release across the day are under different control mechanisms. There is evidence that the CAR is greater in smokers, compared with nonsmokers (19), and smokers have greater cortisol production in assessment of samples over the rest of the day (15).
Cortisol can be measured in saliva, urine, or plasma. The use of saliva sampling allows reliable unobtrusive measurement in a naturalistic setting, and can be designed to capture the diurnal profile of cortisol (20, 21). The collection of salivary cortisol is not awkward for participants or subject to white coat syndrome, and a person can participate in their normal daily activities.
We examined the association between smoking status and salivary free cortisol secretion in a large cohort of middle-aged men and women. We hypothesize that cortisol released throughout the day will be raised in smokers, compared with never smokers and ex-smokers. We investigated a possible dose-response action by assessing cortisol release of smokers by number of cigarettes smoked. We examined differences in the CAR and cortisol release throughout the day. Our study had the advantage of being large enough to take into account correlates of cortisol secretion in the analysis and allowed us to assess differences among never-, ex-, and current smokers.
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Subjects and Methods |
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Data reported here are from phase 7 (2002–2004) of the Whitehall II study. The Whitehall II cohort was initially recruited between 1985 to 1988 (phase 1) from 20 London-based civil service departments; 10,308 people participated. Details of the clinical assessment and cohort profile have been reported elsewhere (22). The number participating at phase 7 was 6941; of these, 93.4% had a clinical assessment; of those eligible for cortisol assessment, 90.1% (n = 4609) returned samples. In analyses reported here, fewer participants were in the lowest grades, compared with phase 1 of the study; however, this difference was small. Ethical approval for the Whitehall II study was obtained from the University College London Medical School committee on the ethics of human research. Informed consent for involvement in the study was gained from every participant.
Cortisol collection and analysis
To collect a saliva sample, participants used a device called a salivette (Sarstedt, Leicester, UK). Participants were instructed to provide six samples over the course of a normal weekday at waking, waking +30 min, waking +2.5 h, waking +8 h, waking +12 h, and bedtime. Participants were instructed to record the time of sample collection, take samples as soon as they woke, avoid caffeine and acidic drinks in the first 30 min, and not brush teeth or eat or drink anything for 15 min before a sample collection. An instruction booklet was given that acted as a logbook to record information on wake time, mood at time of sampling, smoking, alcohol consumption, exercise, and stressful events on the day of sampling. The salivettes and logbook were returned via post. Salivette devices were centrifuged at 3000 rpm for 5 min, resulting in a clear supernatant of low viscosity. Salivary cortisol levels were measured using a commercial immunoassay with chemiluminescence detection (CLIA; IBL-Hamburg, Hamburg, Germany). The lower concentration limit of this assay was 0.44 nmol/liter; intra- and interassay coefficients of variance were less than 8%. Any sample over 50 µg was repeated.
Smoking status was assessed by the following questions: 1) Do you smoke cigarettes now (i.e. not cigars/pipe), with responses yes, no, or social/occasional smoker; 2) if not a current cigarette smoker, did you smoke in the past; and 3) do you currently smoke cigars or a pipe, with yes or no response options. We used the logbook question, did you use any tobacco today? (e.g. cigarettes, cigars, pipes, chewing tobacco), yes or no. From these questions a person was assigned to be a current, ex-, or never-smoker; if any nicotine replacement therapy was used, they were assigned a smoker (n = 4). For ex-smokers the response to the question, how old were you when you stopped smoking? with their age at the screening was used to calculate the length of time since quitting. Participants were asked to record how many cigarettes they smoked on the day of sampling; this was categorized into tertiles of low, medium, and high numbers of cigarettes smoked. This was calculated separately for men and women, resulting in cut points of 6 and 15 cigarettes for men and 8 and 15 cigarettes for women.
Covariates
Social position was determined by job grade if still in the civil service or on last grade if they had left the service. For our analysis, civil service grades were collapsed into three categories. Low income was defined as earning less than £15,000 a year. Wealth was assessed by the question, if you sold all the assets your household owns for example (list of assets) and cashed in savings and investments and paid off all your debts (including your mortgage), how much money do you think you would have? (low wealth defined as between £0 and £99,000). More details on the definitions of income and wealth can be found in the report by Martikainen et al. (23).
Financial insecurity was assessed using the question, thinking of the next 10 yr, how financially secure do you feel? (with secure, fairly secure, fairly insecure, and insecure the possible answers). Participants responding feeling insecure or fairly insecure were defined as feeling financial insecurity, as in the report by Ferrie et al. (24).
Depression was measured using the General Health Questionnaire (GHQ) depression subscale using four items; respondents scoring 0–2 were considered noncases and those scoring 3 or more, GHQ depression cases. This is not a clinical definition but one validated to be used for the 30-item GHQ scale (25).
Diabetes was assessed if participants stated they were diabetic when asked or had a postload glucose 11.1 mmol/liter or greater (if missing a fasting glucose 7.0 mmol/liter or greater).
Poor health was assessed by the question, in general, would you say your health is (with five response items) excellent, very good, good, fair, or poor? Fair or poor was categorized as having poor health.
Alcohol consumption was calculated using the units consumed in a week; high alcohol intake was assigned if greater than 21 U for men and 14 for women. The logbook provided alcohol consumed on the day of sample collection: did you drink alcoholic beverages today? Number of alcoholic drinks was recorded; high daily alcohol consumption was defined as more than four drinks a day for men and more than three drinks a day for women.
Body mass index (BMI) was assessed by measurement of height and weight in the standard way at the clinical assessment. BMI was calculated as weight/height squared.
Stress on the day of sample collection was assessed with the question, we would like to know if this was a typical day for you, compared with your usual workdays (or weekends), in terms of how busy, pressured, or stressed you felt. Response options were: today was typical/greater/lower in terms of my workload or stress level. Participants were asked about the most stressful event and categories were: not at all stressed, somewhat stressed, moderately stressed, very stressed, or the most stressed I have ever felt. Participants were classified as having a stressful experience if they responded that they were very stressed or the most stressed I have ever felt.
Statistical techniques
Gender has been shown to have an effect on smoking status (26) and cortisol levels. However, there was no interaction between smoking status and gender after adjustment for age and last known employment grade; analyses were therefore combined. Participant characteristics by smoking status were examined by 
2 analysis for categorical variables and linear regression for continuous variables. Due to nonnormal distribution, cortisol values were log transformed for analysis; normal data are presented in the figures. For each of the cortisol samples, outliers were removed (sample 1, n = 4; sample 2, n = 5; sample 3, n = 5; sample 4, n = 4; sample 5, n = 4; sample 6, n = 3). For missed samples, imputations were performed for samples taken at time 3 (n = 16), 4 (n = 37), and 5 (n = 36). No statistical differences were observed; therefore, imputed results are presented. The cortisol release over the course of the day was analyzed using a general linear model for repeated measures. The CAR was calculated as the ratio of cortisol in sample 1 to cortisol in sample 2.
Analyses examining the association between smoking status and cortisol secretion were first run with adjustment for age, gender, and employment grade as a measure of social position. Effect sizes were calculated to determine the size of the association. Models were then run with additional adjustments for other potential confounding or mediating factors. These analyses included participants with complete data on all variables included in the models. The association with number of cigarettes smoked and cortisol secretion was also assessed by regression. All data were analyzed using SPSS (version 13.0; SPSS Inc., Chicago, IL). Bonferroni correction was used for multivariate analysis.
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Results |
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The participant characteristics by smoking status are shown in Table 1
. Smokers were younger (ANOVA, P < 0.001), more likely to be in the lower employment grades (P < 0.001), lowest income (P = 0.008), and lowest wealth (P 
0.001) groups. Smokers had a higher weekly alcohol consumption (P < 0.001) and higher consumption of alcohol on the day of sampling (P < 0.001) and were more likely to have a BMI greater than 30 (P = 0.02). Smokers reported poorer self-rated health (P < 0.001) and more depressive symptoms (P = 0.02) and felt more financially insecure (P < 0.001).
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; smokers had a larger production of cortisol over the course of the day in repeated-measures analysis (P < 0.001). This is confirmed when total cortisol production over the day was examined (overall AVOVA, P = 0.008; Fig. 2). Cortisol secretion was higher in smokers compared with never-smokers (P = 0.002), and no significant difference was observed between never- and ex-smokers (P = 0.594).
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illustrates that the increased release of cortisol in smokers, compared with never-smokers, which is robust to adjustment; thus, cortisol levels were raised in smokers [mean release per hour in nanomoles per liter per hour: 4.64 (CI 4.36–4.95), P = 0.005] for between-subjects effects in a repeated-measures ANOVA. No difference was observed between never-smokers [4.14 (CI 4.03–4.26)] and ex-smokers [4.20 (CI 4.07–4.34), P = 0.594] after adjustment for age, gender, employment grade, BMI, high alcohol consumption, poor health, depression, and financial insecurity. The effect size between smokers and never-smokers was 0.28. The CAR was larger in smokers (P < 0.001, adjusted for age, gender, grade, and time of waking); CAR in smokers was 9.73 (CI 8.42–11.04) nmol/liter, compared with never-smokers, 7.52 (CI 6.96–8.09) nmol/liter. This effect size was 0.15. In assessment of the CAR, time of waking (P < 0.001) and gender (P = 0.012) had independent relationships with the size of CAR.
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. There was no relationship between tertiles of number of cigarettes smoked and release of cortisol in the course of the day [repeated measures assessment of cortisol (P = 0.646) or mean cortisol produced over the day (P = 0.095)]. The CAR was assessed by increasing tertile of cigarettes smoked [low, 8.49 (CI 5.99–10.99); medium, 9.58 (CI 7.40–11.76); and high, 13.49 (CI 10.74–16.23)] and shows a positive trend (P = 0.029). Linear regression was performed for number of cigarettes smoked on the day including age, gender, and last known grade as covariates, with mean cortisol per hour of the day as the dependent variable. There was no significant relationship with number of cigarettes smoked (P = 0.231, ß = 1.078, n = 270) and mean cortisol.
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), difference was significant only for high tertile consumption group (low, P = 0.416; medium, P = 0.417; high, P = 0.001). Linear regression analysis suggested that there was a linear association between tertiles of cigarettes smoked and total cortisol secretion (ß = 1.07, P = 0.003) after adjustment for age, gender, and last known grade.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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These results support some (15, 27) but not all (13, 28) previous reports of an association between cigarette smoking and increased cortisol secretion. These findings may reflect methodological differences in study design or differences in sample size. The size of the study allowed comparisons to be made with ex-smokers in relation to never- and current smokers. Two lines of evidence suggest that the effect of smoking on the HPA axis is not due to the chronic effect of smoking on health: first, once smoking has ceased, the diurnal pattern of cortisol release returns to that of a never-smoker, and second, adjustment for health measures failed to alter our results. Investigation of the dose-response association with numbers of cigarettes indicated that the differences are driven largely by the heavy smoking group, supporting evidence that smoking has a short-term effect on the HPA axis.
The effect size of smoking on cortisol secretion is small to moderate (0.28), but this consistent effect every day over many years may potentially have large consequences on downstream endocrine function. For example, evidence suggests that the moderately raised cortisol levels observed in smokers would potentially have large impacts on glucose and insulin metabolism (28, 29), and small increases in cortisol are associated with reduced bone mineral density (30); this is a potential link between smoking and development of osteoporosis.
The potential pathways by which smoking affects the HPA axis are numerous, and the literature is unclear about the mechanisms that could be mediating the effect. Activation of central nicotinic receptors to raise systemic cortisol levels has been posited. However, this effect is not observed in users of nicotine replacement; cortisol is usually decreased in people attempting to quit (31). There is evidence of desensitization to the effects of nicotine in animal models (32); however, it is unclear whether this applies for humans especially if they have been smoking for more than 40 yr. The mode of ingestion of nicotine could be relevant in activation of receptors or the influence of other chemicals and compounds in cigarette smoke that activate the HPA axis. If this were the case, it is unclear why we observed differences in the CAR rather than release throughout the day. Evidence indicates that nicotine diminishes in the body within a few hours but other metabolites of smoking are present for longer periods (days) (33), and these may mediate the changes in the CAR. Alternately, an immune action induced by inflammation of the lung tissue due to smoke inhalation or more generalized effect mediated by low systemic inflammation could explain the increase cortisol for smokers observed for the CAR and measures over the rest of the day. The final possibility is there is no direct affect of smoking on the HPA axis; it is confounding by other social factors or underlying illness not accounted for in these analyses.
The positive and negative aspects of our study need to be considered. The percentage of smokers in this study is smaller than that of other studies (15); however, the overall size of the study is greater, had an excellent response rate, and has allowed assessment of the HPA axis in ex-smokers. Indicators suggest that the participants understood the instructions and took samples in the correct manner, confirming the reliability of the data set. A large number of possible confounders are measured and adjusted for; the results are still significant. However, these are cross-sectional analyses that make it difficult to assess the direction of causation. Thus, it is possible that a stressful environment raises cortisol levels and encourages people to smoke or continue to smoke. This latter theory is not well supported by the literature. The Whitehall II study is an occupational cohort of U.K. civil servants and therefore may not be representative of the population, which may reduce the generalizability of the results. The rate of smoking in this cohort is 9.2%; in the U.K. population, it is 25% in this age group (34). The effect smoking has on cortisol levels means the smaller number of smokers in this population does not affect the ability to detect HPA axis changes in response to smoking. We were unable to determine the length of time between consumption of a cigarette and when a saliva sample was taken. We believe that knowledge of this information would not change our overall conclusion that there are no long-term effects of smoking on cortisol secretion. Smokers are known to have poorer oral health (35), and leakage of plasma cortisol into the saliva could artificially raise salivary cortisol levels in smokers. Evidence suggests blood leakage into the saliva can have a slight effect on cortisol levels (Granger, D., personal communication); however, oral microinjury has been shown not to significantly alter salivary cortisol levels (36), and studies showing increased plasma cortisol in smokers would dispute this theory (15). There were too few participants taking nicotine replacements to assess the role of nicotinic activation, compared with other chemicals in cigarette smoke and inflammatory activation due to smoke inhalation.
In conclusion, these results indicate that activation of the HPA axis occurs in smokers and affects diurnal rhythm of cortisol as illustrated by differences observed for the CAR and whole-day assessment. The mechanisms by which these associations occur remain to be determined but appear not to involve confounding by social position or mediated by psychosocial stress pathways. The large group of ex-smokers available enabled comparison of this group, and no differences were observed with never-smokers, indicating no long-term effect of smoking on the HPA axis.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online December 19, 2006
Abbreviations: BMI, Body mass index; CAR, cortisol awakening response; CI, confidence interval; GHQ, General Health Questionnaire; HPA, hypothalamic-pituitary-adrenal.
Received October 2, 2006.
Accepted December 12, 2006.
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