Salivary cotinine concentrations in daily smokers in Barcelona, Spain: a cross-sectional study

Nicotine, the main alkaloid of tobacco, is responsible for its addictive effect. It is readily absorbed from tobacco smoke, and its concentration rises over 6-8 hours during the day in regular smokers [1]. About 70 to 80% of nicotine is metabolized to cotinine [2]. As the primary metabolite of nicotine, cotinine has been widely used as a specific biomarker of tobacco exposure because its half-life in the body (12-20 hours) is longer than that of nicotine (3-4 hours) [1, 2]. Cotinine in biological materials is suitable for assessment of doses over short periods of time (from 1 to 10 days, in urine, plasma, or saliva) or longer periods (weeks or months, in hair or nails). Consequently cotinine concentration is feasibly used as a biomarker in epidemiological studies [3–5].

Cotinine measurements have been used to describe and compare patterns of tobacco consumption in smokers in different countries to establish if addiction and smoking patterns vary across populations [6–8]. These studies show that the number of cigarettes smoked is the main determinant of salivary cotinine concentrations [9]. These studies have been conducted in selected samples from a variety of countries at different stages of the tobacco epidemic, such as China, Mexico, Brazil, and Poland [9, 10]. However, there is scant information about the relation between cotinine measurements and smoking patterns in samples from the general population.

Spain is currently in an advanced stage of the tobacco epidemic [10, 11]. Data from the 2006 Spanish National Health Interview Survey show prevalence rates of daily smokers of 31.6% and 21.5% in adult men and women, respectively [12]. In men, a steady increase in smoking occurred during the first half of the twentieth century, reaching a peak prevalence rate of 59.1% in the nineteen-seventies. This peak was followed by a decade of stabilization and a continued decrease of smoking until the present. Uptake of smoking in women was delayed, with a prevalence rate of 5% through the nineteen-seventies. This was followed by a substantial increase throughout the next two decades (22.5% by 1995), which only recently stopped [11, 13, 14].

An understanding of cotinine concentration and smoking patterns at the population level is potentially useful to design suitable strategies for cessation. The aim of this study is to describe and characterize the distribution of salivary cotinine concentration in a representative sample of adult (>16 years old) daily smokers in Barcelona, Spain.

As shown in Table 2, we found no statistically significant differences in the salivary cotinine concentration by type of cigarettes, use of filter, frequency, or depth of inhalation. Median cotinine concentration was higher among smokers of black tobacco (180.2 ng/ml) than among smokers of blond tobacco (137.0 ng/ml; p = 0.043). This association was confounded by the higher median consumption by smokers of black tobacco (Table 1), and by the predominance of men (70%) among users of black tobacco. There was no association between the type of tobacco smoked and cotinine concentration within strata of number of cigarettes smoked (1-9, 10-19, and = 20 cigarettes in the last 24 hours) or of sex (data not shown).

The distribution of salivary cotinine concentration in relation to the number of cigarettes smoked during the 24 hours prior to saliva collection is shown in Fig 1. In the simple unadjusted linear model the number of cigarettes smoked in the last 24 hours was a predictor of cotinine concentrations (R ² = 0.339; solid line). A significant improvement of the fit was obtained with a quadratic model, in which the number of cigarettes smoked accounted for almost 39% (adjusted R ²) of the variance, and the exposure-response relationship leveled-off near 20 cigarettes (Fig. 1; dashed line). [see Additional file 1]

Due to differences found in cotinine concentrations between men and women, we analyzed these groups separately (Table 4). We observed an increment of 15.7 and 11.2 ng/ml in cotinine concentration per one cigarette smoked at a consumption of 1 cigarette per day in the quadratic model for men and for women, respectively. Further adjustment for age, educational level, and BMI in the quadratic model showed the best fit of the data with a similar increase in the cotinine concentration by cigarettes smoked (Table 4). Adjustment for other potential confounders identified in the bivariate analysis did not improve the model fit and hence these variables were not included in the final models (data not shown). Fig 2 shows the regression lines from linear and quadratic models for men and women. We verified the final models for error specification, normality, homoscedasticity, multicollinearity, outliers and self-correlation, and all diagnostics showed that the chosen models fulfilled the assumption [see Additional file 1]

This is the first study on smoking behavior in a Spanish adult population using both a questionnaire and a biomarker of tobacco exposure. Salivary cotinine concentration was associated with the number of cigarettes smoked in the last 24 hours. This relation was better explained with a quadratic function and in separate strata for men and women. The greater the number of cigarettes smoked, the greater the cotinine concentration in a linear scale up to 20 cigarettes per day, after which the association plateaus. A similar relation between salivary [6–9, 20] or serum [21] cotinine concentrations and number of cigarettes smoked has been observed in other studies. Abrams et al. dichotomized tobacco consumption, and found that for smokers of less than 25 cigarettes per day, salivary cotinine concentration was highly correlated with tobacco consumption, while among heavier smokers the relation was not evident [22].

Other studies have found that cotinine concentration plateaus at different numbers of cigarettes: 25 cigarettes per day [23], 5 cigarettes per day [24], and 4 cigarettes per day in adolescent smokers [25]. The evidence suggests that cotinine concentration rises in a non-linear fashion with increasing number of cigarettes smoked, but the point where concentrations level off may vary across different populations. This finding suggests a difference in how people regulate their intake of nicotine to reach the desired dose [2], even for adolescents, who may be more susceptible to nicotine than adults and require only 4-5 cigarettes per day to satisfy their nicotine cravings [25].

We observed that cotinine concentrations differed by sex, regardless of the number of cigarettes smoked. Some studies reported similar findings of higher cotinine concentrations in men than in women [7, 23, 26, 27], but other studies did not find differences by sex [6, 28]. Association between urinary cotinine and cigarettes smoked according to sex was found in a study in the USA: urinary cotinine concentrations in men increased up to 34 cigarettes per day and then declined, while no flattening was observed in women [29]. The differences we observed by sex could reflect not only differences in tobacco consumption by sex, i.e., men usually smoke more cigarettes than women, but also a difference in the metabolism of nicotine between men and women [30, 31].

Our data showed a higher cotinine concentration in smokers of black tobacco that did not persist with control for the number of cigarettes smoked. Whereas uptake of carcinogens is higher among black tobacco smokers [32–34], differences in nicotine uptake by type of tobacco smoked have not been reported [35]. In our study, use of filter, frequency, and depth of inhalation were not related to cotinine concentrations. An explanation of these results could be that smokers tend to maintain the same intake level of nicotine by drawing in more smoke per cigarette when they try to smoke fewer cigarettes. Benowitz et al. reported that among people who reduced from 37 to 5 cigarettes per day on average, the intake of tobacco toxins per cigarette increased roughly threefold [36]. This could also explain how cotinine concentrations level-off in smokers of more than 20 cigarettes per day, when a certain intake of nicotine is achieved [8, 9].

The role of age in cotinine concentrations is still not clear, since our results, as well as previous studies [7, 37], indicated no association between cotinine concentrations and age, while others have found a significant association [6, 18, 29, 38]. Some studies have modeled the relation between cotinine concentrations and cigarette consumption by taking into account several of these variables. The fit of the multivariate model improved once age, BMI, educational level, and a quadratic term for cotinine were included.

Cotinine concentration differed by sex and increased up to consumption of 20 cigarettes per day and then flattened at higher levels of smoking. Further investigation may help to better understand the relationship between number of cigarettes smoked, age, sex, weight, subjects' levels nicotine or cotinine concentrations, and the degree of nicotine dependence that may have implications in smoking cessation.