Relationship Between Nicotine Dependence Scores and Nicotine, Cotinine, 3′-Hydroxycotinine and Nicotine Metabolite Ratio in Chinese Male Smokers

Nicotine dependence (ND) is an extremely complex phenomenon with nicotine intake, genetic variability, personality traits, social pressure and environmental determinants (11, 12, 14, 25). The complex mechanisms of ND make comprehensive and accurate assessment of smokers’ dependence challenging. From different perspectives, multiple approaches have been used to measure ND (11), such as the smokers’ self-administered questionnaire method (standardized questionnaires), the objective method (tests the smokers’ actual exposure to nicotine directly). The best-known measures are those developed by Fagerström (7). The Fagerström Test for Nicotine Dependence (FTND), which derives from the Fagerström Tolerance Questionnaire (FTQ), is a six-item self-reporting questionnaire reflecting a continuum of dependence. It is widely used to assess ND (21, 16, 17, 20). Derived from this questionnaire, the ‘Heaviness of Smoking Index’ (HSI) is a shorter validated version (7). Measurement of tobacco exposure biomarkers in plasma, urine and saliva is an objective approach by which ND can be assessed (14, 32, 19). Nicotine is mostly metabolised to cotinine and about 15% cotinine is excreted from the body in the urine (6). Nicotine is metabolised to cotinine by the major nicotine-metabolising CYP2A6 enzyme, and 3′-hydroxycotinine (3HC) is the main metabolite of cotinine, which accounts for about 50% of cotinine excretion (6, 18). NMR is a biomarker of CYP2A6 activity (31). As a genetically influenced biomarker of nicotine clearance, lower NMR appears to increase likelihood of successful cessation (22). Previous studies already found that NMR is related to cigarette consumption (5), quitting the smoking habit (18), and smoking behaviours (34). There is no generally accepted relationship between NMR and ND (29, 28, 32, 34). However, ND may depend on the ethnicity of various smoking populations (1). The value of NMR may be different in African, Northern European, and East Asian populations (18, 36). Some studies examined ND among Chinese smokers by use of only a self-reporting questionnaire (8, 11, 26, 35, 37, 38). To our knowledge, there are limited data available regarding NMR determined in urine and its relation to ND in Chinese male smokers. The aims of the current study were (a) to investigate the relationship between ND as measured by the FTND and biomarkers of tobacco exposure of daily smokers and (b) to determine whether the NMR was associated with ND in Chinese smokers.

Table 1 presents the general characteristics of participants and biomarkers of exposure levels. Of the total participants (N = 289), the mean score of the FTND, nicotine, cotinine, 3HC concentrations and NMR were 3.2, 1282.7 ng/mg creatinine, 1549.4 ng/mg creatinine, 3859.1 ng/mg creatinine, and 2.8, respectively. In terms of three age groups, 86 participants were younger than 35 (29.8%), 106 participants were aged from 35 years to 50 years (36.7%), and 97 participants were older than 50 years (33.6%). Each age group accounted for approximately one third of the participants. Among the three age groups, participants had a mean FTND score of 2.4, 3.3 and 3.6, respectively. Nicotine concentrations were 913.9, 1374.0 and 1510.1 ng/mg creatinine, respectively. FTND scores and nicotine concentrations were substantially different among the three age groups. However, none of the cotinine, 3HC and NMR showed statistical significance among the three age groups. We observed significant differences in nicotine and cotinine concentrations connected to different BMIs, with the lowest value in the obese group. No statistically significant differences in FTND scores, 3HC or NMR were found between the different BMI groups. In terms of groups by number of smoking years, both the FTND scores and the nicotine concentration levels showed significant differences, with the highest FTND scores and nicotine concentration levels in the group of smoking years ≥ 31. No statistically significant differences in cotinine and 3HC concentration by smoking years were found. FTND scores and nicotine, cotinine and 3HC levels showed statistical significance among different CPD groups (P < 0.05). Participants who smoked more cigarettes per day had higher FTND scores and higher nicotine, cotinine and 3HC levels. No statistical significance in the NMRs was obtained among the four groups.

Correlation coefficients between measured biomarkers and variables obtained from questionnaires are shown in Table 2. There were significant correlations between FTND scores and nicotine, cotinine and 3HC concentrations. Each of the three biomarkers was considerably correlated with CPD, age and smoking years. Significant correlations were also observed between NMR and nicotine, but no correlations between NMR and other variables were found.

The geometric means (GMs) of nicotine, cotinine and 3HC concentrations, according to the single FTND items, are shown in Table 3. The lowest concentrations were found in the zero-score groups of the six items. The lowest concentrations were observed in smokers whose time to their first cigarette of the day was longer than 60 min after rising, who had no difficulty refraining from smoking, who least hated to give up the first one in the morning, who smoked fewer than 10 cigarettes per day, who smoked less in the first hours of the day and who had no difficulty refraining from smoking when ill or forbidden, although some of these differences were not statistically significant. Except for item 2 (difficulty refraining), which showed no differences in nicotine, cotinine or 3HC concentrations, most of the FTND items showed higher concentrations at higher single scores.

We sought to compare the relationship between the FTND scores and cotinine, nicotine and 3HC levels in human urine. The FTND scores were categorised as low (score: 0–4) or high (score: 5 – 10) based on previously published studies (10). Of the 289 subjects, 197 (68.2%) were classified in the low-nicotine dependence group, and 101 (34.9%) in the high-nicotine dependence groups. Cotinine, nicotine and 3HC concentrations in the urine of participants with high FTND scores were significantly higher than in those with low FTND scores (P < 0.05) (Figure 1 and Table 4).

Figure 1

We found that the FTND scores differed significantly among different age, CPD and smoking-years groups. The higher the age/CPD/smoking years, the higher the FTND scores in this study population of daily smokers. We also found significant correlations between FTND scores and urinary biomarker concentrations (nicotine, cotinine, 3HC). The concentrations of nicotine, cotinine and 3HC in the urine of smokers with high FTND scores were higher than those with low FTND scores. All items except item 2 (difficulty refraining) showed differences in nicotine, cotinine and 3HC concentrations, with higher concentrations at higher single scores. And there was no association between the NMR and either the FTND or general characteristics such as basic information of participants, e.g., age, BMI, smoking years, number of cigarettes smoked per day, etc. These findings indicated that there were relationships between ND scores and nicotine, cotinine and 3HC. NMR possibly has no impact on ND in Chinese male smokers.

The current study shows differences in FTND scores based on age and years of smoking. This study suggests that those who started smoking at a younger age and had smoked for more years were more dependent on nicotine. One study in China demonstrated that FTND scores largely increased with age among daily smokers and also earlier smoking commencement was associated with higher ND (38). We also found that FTND scores showed statistical significance among different CPD groups. This conclusion is consistent with the results of Becoņa et al. (2) who reported that Fagerström scores increased significantly with a rise in age and cigarette consumption in a Spanish population. The result of the FTND scores of smokers across different BMI groups showed that there was no statistically significant difference, which agrees with the conclusions of other studies (33). The mean FTND score in our study – 3.2 – is similar to that observed in other studies (32, 39). We also found that the NMR was not related to age, BMI, years of smoking and CPD, same with other studies (29, 34). Among the three age groups, nicotine concentrations were substantially different, whereas cotinine and 3HC showed no statistical significance. Previous studies (13, 30) found that the role of age in cotinine concentrations was still not clear, indicating no association between cotinine concentrations and age, whereas others found cotinine levels increased with increasing age (9, 24).

In the correlation analysis, correlation coefficients between the FTND scores and nicotine, cotinine and 3HC concentrations showed positive relationships; the higher the FTND scores, the higher the nicotine/cotinine/3HC concentrations. The correlation coefficients between the FTND scores and nicotine, cotinine and 3HC were 0.301, 0.371, 0.365, respectively, suggesting that FTND scores displayed a positive association with the levels of nicotine/cotinine/3HC. These results were consistent with previous studies (27, 32). NMR, as a reliable measure of CYP2A6 activity, has a role in ND (5, 18, 29). However, in our study, we did not find a significant correlation between FTND scores and NMRs, just in accordance with results reported in a previous study (34).

The data shows that the time between rising and smoking the first cigarette of the day, hating most to give up smoking, number of cigarettes smoked per day, smoking more in the first hours of the day and smoking when ill are associated with nicotine, cotinine and 3HC concentrations. The shorter the time between waking up and smoking the first cigarette of the day, the higher the concentrations of nicotine, cotinine and 3HC in the urine. These results agree with the findings described by Fu et al. (15).

After adjustment for the number of cigarettes smoked in the previous 24 h, the time to the first cigarette of the day is associated with cotinine concentration (15). The higher the number of cigarettes smoked, the higher the cotinine concentration. One likely reason is that smokers regulate their smoking behaviour to compensate for variations of nicotine levels in the body (3). Thus, smokers in the highly dependent group may smoke more cigarettes per day and during the first minutes of the day, may hate most to give up the first cigarette in the morning and smoke even when ill, resulting in higher nicotine, cotinine and 3HC concentrations compared with low-dependent smokers. The concentrations of nicotine, cotinine and 3HC in the urine of smokers of high FTND scores were higher than in those with low FTND scores.

In conclusion, our data confirm the association between FTND scores and levels of nicotine, cotinine and 3HC in Chinese male smokers. Smokers’ ND may not be affected by the NMR. FTND item 2 (difficulty refraining) was not found to be related to ND in Chinese smokers. Further studies are necessary to investigate the potential role of NMR and FTND item 2 (difficulty refraining) in large numbers of Chinese smokers.