Nicotine
Inhalation of drugs is a very effective means of delivery because inhaled drugs avoid first-pass metabolism by the liver, which rapidly metabolizes nicotine through the enzyme cytochrome P450 (CYP2A6). The main metabolite of nicotine is cotinine, and cotinine concentrations in blood, urine, or saliva are often used as biomarkers to evaluate tobacco use and exposure to environmental tobacco smoke [
4‐
6]. Cotinine has a longer half-life (average 18-20 hours) than does nicotine (2-3 hours) [
5], making it a more feasible marker of tobacco use than nicotine. Cotinine is further metabolized to trans-3’-hydroxycotinine (3 HC). Although glucuronidation (a process by which glucuronic acid is conjugated with a substrate) is usually a minor metabolic pathway for nicotine and cotinine, it can be a major determinant of nicotine clearance in people who have low CYP2A6 activity. That is, in people who metabolize nicotine more slowly through the CYP2A6 pathway, the glucuronidation pathway may metabolize a larger share of nicotine. Thus, glucuronidation may become a more significant factor in overall nicotine metabolism. Slower metabolism of nicotine means that levels of nicotine in the body remain elevated for a longer period of time, allowing a longer timeframe for nicotine to interact with nicotinic receptors all over the body.
The authors of a small cross-over study of seven Black/African American and seven White smokers found that menthol cigarette smoking resulted in slower nicotine metabolism and slower total nicotine clearance. Overall, there were no significant racial/ethnic differences in the disposition kinetics of nicotine [
7]. Menthol cigarette smoking was associated with reduced nicotine metabolism through a decrease in CYP2A6 enzyme activity and a substantial reduction in glucuronidation. These findings were supported in an in vitro study using human microsomes that reported that menthol inhibits the CYP2A6 enzyme, resulting in inhibition of nicotine metabolism [
8].
Using data from a study of 755 Black/African American smokers who smoked fewer than 10 cigarettes a day (so-called “light” smokers), Ho and colleagues [
9] found that smokers who smoke menthol cigarettes had slower metabolism of cotinine as compared to non-menthol smokers.
Clark et al [
10] studied the effects of menthol cigarettes on biochemical markers of smoke exposure among 161 adult Black/African American and White smokers in a cross-sectional study. There were also racial/ethnic differences, with African American smokers having significantly higher cotinine per cigarette ratios, but it is unknown if this is due to differences in metabolism, smoking behavior, or other reasons. After adjusting for race, cigarettes per day and average amount of each cigarette smoked, serum cotinine levels were significantly higher among menthol cigarette smokers than among smokers of non-menthol cigarettes, suggesting greater exposure to nicotine. [
10] Numerous other reports [
4,
10‐
15] have found that Black/African American smokers are more likely than White smokers to smoke fewer cigarettes per day (CPD) yet have substantially higher cotinine levels. Because Black/African American individuals are considerably more likely to smoke menthol cigarettes, menthol may have been a mitigating factor; however, the potential impact of menthol was not evaluated separately in these studies.
In a between-subjects study of 95 female adult smokers stratified by race/ethnicity and menthol/non-menthol cigarette preference, Ahijevych and Parsley [
11] reported that smokers of menthol cigarettes had higher cotinine levels. This finding would be expected, given menthol’s inhibition of CYP2A6 metabolism and glucuronidation [
8,
16,
17]. As was found with Clark et al [
10], Black/African American subjects had higher cotinine per cigarette levels, suggesting greater exposure to nicotine.
Data from the National Health and Nutrition Examination Survey (NHANES) were used to compare the serum cotinine levels of more than 1,500 smokers. The data from this large, nationally representative sample compared menthol and non-menthol smokers. Menthol smokers were found to have significantly higher serum cotinine levels (1333.8 ± 40.1 nmol/L) as compared to non-menthol smokers (1230.3 ± 24.5 nmol/L) [
6].
In an inpatient research study by Ahijevych and colleagues [
4], plasma samples were taken while the subjects were smoking as desired, followed by several days of smoking abstinence. Cotinine half-life and did not significantly differ between menthol (23.1 ± 7.9 hours) and non-menthol smokers (18.1 ± 8.1 hours) [
4]. However, as has been discussed previously [
10,
11], there was a main effect for race, with Black/African American smokers having greater cotinine levels as compared to white smokers. Again, this suggests that these people had greater exposure to nicotine on a per cigarette basis.
A study by Patterson et al (2003) of 190 treatment-seeking smokers (29 menthol smokers, 161 non-menthol smokers) failed to find significant differences in nicotine boost (an individualized measure of how much nicotine has been extracted from smoking a cigarette) produced following the smoking a preferred-brand cigarette (p < 0.10). However, the authors note that “the absence of racial differences in boost and a lack of association with cigarette characteristics (e.g., menthol) may be attributable to the relatively small number of African Americans in the sample” [
18]. Consistent with other studies [
4,
10,
11], being Black/African American was significantly associated with greater levels of blood nicotine following a single cigarette. [
18]
Mustonen et al [
19] investigated possible associations between cotinine/CPD ratios in subgroups varying by gender and race/ethnicity in a randomized clinical trial. Consistent with other studies [
4,
10,
11,
18], Black/African American smokers smoked fewer CPD, but did not have significantly lower cotinine levels, suggesting a higher cotinine per cigarette ratio. Although there was a pattern toward higher cotinine levels in smokers of menthol cigarettes, it was not statistically significant. There was, however, a significant gender by race by menthol interaction on salivary cotinine level as well as cotinine/CPD ratio. Black/African American menthol cigarette smokers and Black/African American non-menthol cigarette smoking women had higher cotinine/CPD ratios than did White smokers. These findings suggest that the relationship between number of cigarettes consumed and salivary cotinine is complex. This study was limited in that puffing rate, depth of inhalation and length of cigarette smoked could not be controlled for in the study sample. The authors concluded that type of cigarette, race/ethnicity, and gender need to be evaluated concurrently [
19].
Wang et al [
20] investigated the effects of menthol cigarettes on adult smokers’ exposure to nicotine in a large, cross-sectional study. The menthol cigarette smokers were more likely to be Black/African American and more likely to be female, which is consistent with the demographics in other studies (for review, see Ahijevych and Garrett 2004 [
21]). There were no significant differences in nicotine equivalents (nicotine and five major nicotine metabolites) per cigarette, leading the researchers to conclude that smoking menthol cigarettes does not increase daily exposure to smoke constituents. No significant differences were found in serum cotinine levels between menthol and non-menthol cigarette smokers, suggesting that menthol had no effect on the metabolism of nicotine in the study. Consistent with other studies [
4,
10,
11,
18,
19], although the Black/African American smokers smoked fewer cigarettes per day, there was no different in serum cotinine levels, suggesting a higher nicotine/cigarette ratio. One limitation of the study was that only a small proportion of the Black/African American smokers smoked non-menthol cigarettes.
In a tobacco industry-associated parallel arm study designed to investigate whether moderately heavy (≥ 15 CPD) smokers of menthol cigarettes had different biomarker levels than non-menthol cigarette smokers, the researchers failed to find significant differences in urine levels of nicotine or glucuronidated nicotine metabolites. Unlike other studies [
4,
10,
11,
18,
19], they found no significant racial/ethnic differences in metabolism [
22]. A limitation of the study was the small number of Black/African American non-menthol cigarette smokers compared with the number of White non-menthol cigarette smokers.
Using a stored sample from 255 current smokers from the Southern Community Cohort Study (65 Black/African American men, 65 Black/African American women, 63 White men, 62 White women), comparisons of serum cotinine levels of menthol and non-menthol smokers were made. There were significant interactions between gender and race/ethnicity, but no significant differences were found between menthol and non-menthol groups [
23]. Consistent with previous studies [
4,
10,
11,
18,
19], after adjustment for CPD differences, Black/African American smokers had higher cotinine levels as compared to white smokers.
In a study of more than 700 Black/African American light smokers (≤ 5 CPD), menthol smokers did not differ in plasma cotinine levels when compared to their non-menthol smoking counterparts. This may be due to a wide range between the minimum and maximum plasma cotinine levels across smokers in the study: 5.0 ng/mL versus 937.8 ng/mL [
9].
A study by Williams and colleagues [
24] compared cotinine levels of menthol- and non-menthol-smoking patients with and without schizophrenia. The laboratory study of 142 people assessed blood cotinine levels during a typical smoking day, two minutes after smoking a usual-brand cigarette. There were no significant differences when comparing the schizophrenic smokers with non-schizophrenic smokers. However, menthol smokers had significantly higher serum cotinine levels as compared to non-menthol smokers (294.3 ng/ml and 238.8 mg/ml, respectively; p = .041). Menthol smokers also had significantly higher serum nicotine levels (27.2 mg/ml and 22.4 mg/ml, respectively; p = 0.01) an effect that appears to be driven by schizophrenic smokers having significantly higher serum nicotine levels as compared to non-schizophrenic smokers (p < 0.05). The authors suggest that the elevated levels observed in menthol smokers may be due in part to increased intake of smoke or menthol-mediated inhibition of nicotine metabolism [
24].
Carbon monoxide
Like the nicotine/cotinine data, the data from studies measuring expired air carbon monoxide (CO) and/or levels of blood carboxyhemoglobin (a measure of CO exposure), which are often used as biomarkers to indicate level of exposure to tobacco smoke, are not consistent. Some investigators have found that menthol cigarette smoking increased CO (as measured by expired CO, CO boost, or blood carboxyhemoglobin) as compared to non-menthol cigarettes smoke [
10,
24‐
26], whereas other studies, including one associated with the tobacco industry, have found either no difference in CO exposure [
22,
26‐
29], or a lower level among menthol smokers [
30,
31]. Ahijevych et al (1996) that found that menthol smokers had lower CO measurements as compared to non-menthol smokers, reporting statistically significant differences for the Black/African American menthol participants of this women-only study [
30]. Possible reasons for the inconsistency of the findings include the possibility that physiologic variables, such as mucous layers in mucosal cold nerve endings or differences in how the cigarette burns (menthol pyrolysis) may affect CO levels [
30,
32]. Also, menthol’s effects on biomarkers such as blood carboxyhemoglobin and cotinine are not linear and, as has been noted by publicly available internal tobacco industry documents, are affected by other chemicals in the smoke [
33].
Tobacco-specific nitrosamines
Toxins present in tobacco, such as tobacco-specific nitrosamines (TSNAs), which are known carcinogens, are also used as biomarkers of tobacco smoke exposure. Menthol may alter glucuronidation metabolism of some TSNAs, such as the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; (NNAL), which goes through a glucuronic metabolic pathway to form NNAL-Glucuronide (NNAL-Gluc) [
16]. Thus, inhibition of the glucuronidation process may result in adverse effects, such as an accumulation of NNAL.
As a parallel to the findings by MacDougall et al [
8] in an experimental
in vitro study, Muscat et al [
16] found that menthol inhibited glucuronidation of NNAL in human microsomes; however, in an
in vivo study of rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, or NNK (a TSNA that is metabolized into the TSNA NNAL), those that received oral menthol showed increased levels of NNAL metabolites. This suggests enhanced metabolism. Orally administered menthol delivered in the absence of the other constituents of tobacco smoke, as well as generalizing from rats to humans, makes generalizations regarding the possible effects of menthol tobacco smoke difficult.
In an
in vivo component of the Muscat et al [
17] study mentioned above, urinary ratios of NNAL/NNAL-Gluc in adult smokers were measured. Smokers of menthol cigarettes had lower urinary ratios of NNAL/NNAL-Gluc than smokers of non-menthol cigarettes, suggesting that menthol inhibited NNAL glucuronidation [
17]. Although these findings provide additional support that menthol generally inhibits glururonidation [
7,
8] a tobacco industry associated study failed to find any inhibition of glucuronidation of NNAL [
22]. This parallel-arm study, which measured levels of total NNAL and NNAL-gluc in the urine of moderately heavy (≥ 15 CPD) smokers of “light” cigarettes (7–15 mg Federal Trade Commission [FTC] “tar”), failed to find significant differences when comparing levels in menthol versus non-menthol smokers [
21]. Differences in urinary metabolites of NNAL between Black/African American and White smokers have been found in other studies, but menthol was not specifically investigated as the cause of these differences [
34].