In this population of African-American light smokers, where approximately one-third consume ≤5 CPD, two commonly used biomarkers of cigarette smoke exposure, expired CO and plasma COT, were significantly correlated with self-reported CPD. However, the strength of the correlations were relatively weak (r ~ 0.31 – 0.37), and in a multiple regression model, only ~17% of the variance in CPD was explained by plasma COT and expired CO. This is in contrast to heavy smoking Caucasian populations, where correlation coefficients from 0.3 – 0.8 have been reported (4
). Self-reported number of cigarettes smoked per day is a limited indicator of exposure as there is a nonlinear relationship between biomarkers and CPD, with a plateau observed at higher levels of consumption (>20 – 25 CPD) (28
). Heavy smokers reporting consumption at these levels appear to smoke each cigarette with less intensity (25
). Thus, self-reported measures of CPD may not be representative of exposure particularly at extremely high or low levels of smoking.
It would have been ideal to compare our findings with a matched group of Caucasian light smokers from a clinical trial (e.g. treatment seekers) to determine whether the weaker correlations between the biomarkers and self-reported cigarette consumption in this study were reflective of variables that were specific to African-Americans, or resulted from the narrow range in cigarettes consumed. However, established light smoking patterns among adults is less common among Caucasians, and the clinical trial from which participants in the current study was drawn is the only published one to date to have recruited specifically light smokers (≤10 CPD) (29
). To partially address this issue, we analyzed a subset of Caucasian smokers that reported ≤ 10 CPD in our previously published biomarkers paper (25
). Despite the considerably smaller numbers in this subset analyses (N = 40 vs. 152) the correlation coefficients appeared higher in the Caucasian light smokers. Specifically, the Pearson’s correlation coefficient between CO with CPD (r = 0.37, p = 0.02) was similar, while the correlations between COT with CPD (r = 0.51, p < 0.001), and CO with COT (r = 0.77, p < 0.001) were stronger than in African American light smokers (r = 0.32, 0.39 and 0.60 respectively, ). The correlations between CO and CPD were improved when the total sample of Caucasians in that study (n = 152, mean CPD = 19.4) (25
) was examined (r = 0.60, p < 0.001), although the relationships between COT and CPD (r = 0.53, p < 0.001) and CO with COT (r = 0.74, p < 0.001) remained similar. Thus, it appears that COT may be a poorer biomarker of cigarette consumption in African-American light smokers compared to Caucasians, and as expected CO appears poorly correlated with CPD in all light smokers. Furthermore, in a subset of heavy-smoking, treatment-seeking African-Americans, in which these variables were available, recruited for a clinical trial testing the efficacy of bupropion (n = 93) (30
), both CO and COT were poorly correlated with self-reported cigarette consumption (r = 0.20, p = 0.05 for CO with CPD, and r = 0.05, p = 0.62 for COT with CPD). Together this suggests that CO is a poor correlate of cigarette consumption in light smokers in general, while COT is a poor correlate of cigarette consumption among African-American smokers.
Traditional cutoff levels of expired CO and plasma COT for differentiating between smokers and nonsmokers have previously been determined primarily in heavy smoking Caucasian populations (2
). Our results suggest that using expired CO ≤ 10 ppm to verify smoking status in light smokers may result in misclassification of smokers as nonsmokers, as ~40% of our treatment-seeking sample of smokers had expired CO levels below this limit. In contrast, very few individuals (3.1%) had plasma COT levels below the traditional cutoff of 14 ng/ml. This cutoff value was determined more than 20 years ago when there were high levels of secondhand smoke (2
). Recently, it was suggested that the plasma COT cutoff should be further reduced to 3 ng/ml, with optimal cutoff revised to 6 ng/ml for African-Americans (32
). This revised cutoff of 6 ng/ml would misclassify only 2.5% of smokers as nonsmokers in this sample. While further studies will be needed to precisely determine the optimal cutoff points for expired CO and plasma COT among African-American light smokers, our study suggests plasma COT may be a better indicator of smoking status than expired CO.
The second objective of this study was to determine whether other variables (i.e. CYP2A6 activity, gender, age, BMI, smoking mentholated cigarettes) influence biomarkers levels (expired CO, plasma COT) or their relationships to self-reported CPD. Individuals with slow CYP2A6 activity, as indicated by genotype and 3HC/COT, had significantly higher plasma COT levels despite similar intake as represented by self-reported CPD and expired CO values. COT clearance rates were previously found to be reduced by ~35% in individuals with CYP2A6
genetic variants (33
), and in this study, COT levels are ~20 – 30% higher in individuals with slow CYP2A6 activity. Despite its substantial effect on COT levels however, CYP2A6 activity did not greatly alter the correlations between NIC or its metabolites with self-reported CPD or expired CO levels, similar to what was observed in our previous study of Caucasian heavy smokers (25
Individuals with higher BMI were found to have significantly lower plasma COT levels (r = −0.19), as well as lower NIC (r = −0.16) and 3HC (r = −0.26) in this study. A negative correlation between BMI and COT levels has been previously reported (r = −0.16 to −0.36) (5
). It is possible that differences in BMI may result in altered volumes of distribution for NIC and its metabolites, thus resulting in altered plasma levels. The volume of distribution for NIC and COT have been correlated with total body weight and lean body mass (r = 0.23 – 0.67), although no significant correlation was found between the volume of distribution with adipose mass (14
). BMI has also been negatively associated with the 3HC/COT ratio (15
), suggesting obesity and rate of CYP2A6 activity may be related, although this has not been examined explicitly. While the lower plasma COT levels in individuals with higher BMI may also be interpreted as lower exposure to cigarette smoke, this is unlikely as BMI was not significantly associated with expired CO or CPD in multiple regression analyses. As such, it is yet unclear whether the relationship between BMI and COT represents altered rates of COT metabolism (altered CYP2A6 activity), or volume of distribution, or a combination of both. Interestingly, expired CO appeared to be a better measure of exposure in obese individuals (BMI ≥30.0) as the correlation to CPD appeared stronger in these individuals.
It has been proposed that the cooling sensation associated with smoking mentholated cigarettes allows for increased smoke intake. Thus, this may contribute to the higher COT levels and disproportionately higher incidences of tobacco-related illnesses among African-Americans, who because of the influence of marketing campaigns in the 1960s, predominantly smoke mentholated cigarettes (36
). However, while some cross-sectional and experimental studies have found higher CO and COT levels among mentholated cigarette smokers, this has not been consistently replicated (16
). Studies finding an effect were generally of small sample size, and included both heavy-smoking Caucasians and African-Americans. In this current study of African-American light smokers, mentholated cigarette users did not have significantly higher expired CO or plasma COT levels despite our large sample, with 131 non-menthol smokers examined. Thus, cigarette mentholation did not appear to contribute to increased intensity of cigarette smoking or increased absorption of nicotine in our sample of African-American light smokers.
No differences in CPD, expired CO or plasma COT were found by age, in contrast to previous findings where older individuals had higher COT levels (20
). In general, drug metabolism is thought to decrease by age (37
), and while NIC clearance rates are reduced in the elderly (age >65), this has been attributed to age-related changes in hepatic blood flow as no differences in CYP2A6 protein by age have been observed (38
). The renal clearance of COT is also reduced in the elderly, although pharmacokinetic parameters such as area-under-the-curve and elimination half-lives are not altered (39
). Accordingly, the relationships between NIC or its metabolites with CPD or expired CO did not greatly differ among mentholated cigarettes users or by age.
We did not find any gender differences in CPD, expired CO, or plasma COT. However, in the present study the proportion of variance in CPD explained by expired CO was more than tripled in females compared to males (14.4% vs. 4.4%, respectively). This was unlikely due to differences in the type of cigarettes smoked, as there was no difference in prevalence of mentholated cigarettes use by gender. A number of studies have reported gender differences in smoking topography, with males taking larger and longer puffs compared to females (40
). However, we did not observe any differences in CO/cigarette or COT/cigarette by gender in this population (p > 0.10). Among African-American light smoking males, there may be more variability in the manner in which cigarettes are smoked, resulting in weaker relationships between self-reported CPD and expired CO.
One limitation of our study is that this was secondary analyses performed on participants that were originally recruited for a clinical trial on smoking cessation, and may not be representative of African-American light smokers in the general population. Thus, biochemical measures were collected randomly from ad libitum
smoking and the time of the last cigarette may have been a significant source of variation, particularly for expired CO where the half-life is short. It is possible that this treatment-seeking sample may have attempted to stop smoking prior to the start of the trial; however, we excluded from these analyses any participants that reported smoking zero cigarettes within the past seven days prior to the collection of biochemical data. That said, we cannot exclude the possibility that there may have been a selection bias as participants were light smokers who were highly motivated to quit smoking and had difficulty quitting in the past. Thus, they may be more dependent or smoke cigarettes differently from other non-treatment seeking light smokers. In addition, participants may have underreported their cigarette consumption to meet the inclusion criteria for the clinical trial. The average plasma COT levels derived from ad libitum
smoking (244 ng/ml) is similar to those found previously in a heavier smoking population of African-Americans (292 ng/ml) recruited for a clinical trial of smoking cessation at the same community health centre as the current study, with an inclusion criteria of smoking at least 10 CPD (mean = 17 CPD) (30
). It is also possible that self-reported CPD is a poor measure of average cigarette consumption among individuals at this low level of smoking, which may in part explain their weak correlations to the biomarkers. In a previous study of an African-Canadian light smoking population (median of 8 CPD), we found some discrepancies of cigarette consumption when it was reported as cigarettes per day, versus cigarettes per week (CPW), versus cigarettes per month (CPM)(Mwenifumbo JC, Tyndale RF, personal communications). For example, one individual reported consuming 6 CPD, but 18 CPW and 60 CPM. Thus, in light smokers where daily smoking is variable and smoking may occur at irregular intervals, CPD may be a poor indicator of consumption and alternative measures of self-report, such as timeline follow-back procedures (43
), needs to be tested. It is also notable that a large portion of the participants (45%) reported puffing or inhaling as far as the throat only. While self-reported measures of depth of inhalation may not be representative of actual smoking behaviors (44
), this may be another source of variability in cigarette exposure among these light smokers.
In summary, the results from this study suggest that the commonly used biomarkers of cigarette smoke exposure, expired CO and plasma COT, are significantly but weakly correlated with self-reported CPD. Furthermore, these relationships are not greatly altered by variables that were previously reported to have an influence on these parameters, such as CYP2A6 activity, smoking mentholated cigarettes or age, although the relationships may differ slightly by gender and BMI. The proportion of variance in CPD explained by expired CO and plasma COT was generally lower than that observed in heavy Caucasians smokers even after accounting for these variables, suggesting these biomarkers are limited as indicators of cigarette smoke exposure among African-American light smokers.
Our study suggests that expired CO may be a poor indicator of smoking status as many smokers had expired CO levels below the traditionally defined cutoff level. While plasma COT may be useful in ascertaining smoking status in this population, the level is highly influenced by the rate of CYP2A6 activity, and it is also a poor indicator of the levels of smoke exposure. This suggests the rate of CYP2A6 activity needs to be considered when COT is used as a biomarker of intake in African-American populations, where there are higher proportions of individuals with reduced rates of CYP2A6 activity compared to Caucasians. A number of other biomarkers such as thiocyanate or the tobacco alkaloids anabasine and anatabine have also been proposed (1
); however, these also have their own set of limitations in terms of specificity, sensitivity and cost for detection. Validated biomarkers are important for ascertaining smoking status before recruitment into research studies or clinical trials for smoking cessation, or for verifying abstinence among light smokers. In addition, validated biomarkers of cigarette smoke exposure are also necessary for the proper assessment of the dose-related risk of smoking and health outcomes in epidemiological studies of African-Americans, a population that have been reported to have a disproportionately elevated risk of developing tobacco-related illnesses despite lower levels of self-reported cigarette consumption (45