Quantitative analysis of menthol in human urine using solid phase microextraction and stable isotope dilution gas chromatography-mass spectrometry.
ABSTRACT: To accurately measure menthol levels in human urine, we developed a method using gas chromatography/electron ionization mass spectrometry with menthol-d4 stable isotope internal standardization. We used solid phase microextraction (SPME) headspace sampling for collection, preconcentration and automation. Conjugated forms of menthol were released using β-glucuronidase/sulfatase to allow for measuring total menthol. Additionally, we processed the specimens without using β-glucuronidase/sulfatase to quantify the levels of unconjugated (free) menthol in urine. This method was developed to verify mentholated cigarette smoking status to study the influence of menthol on smoking behaviour and exposure. This objective was accomplished with this method, which has no carryover or memory from the SPME fiber assembly, a method detection limit of 0.0017μg/mL, a broad linear range of 0.002-0.5μg/mL for free menthol and 0.01-10μg/mL for total menthol, a 7.6% precision and 88.5% accuracy, and an analysis runtime of 17min. We applied this method in analysis of urine specimens collected from cigarette smokers who smoke either mentholated or non-mentholated cigarettes. Among these smokers, the average total urinary menthol levels was three-fold higher (p<0.001) among mentholated cigarette smokers compared with non-mentholated cigarette smokers.
Project description:In the U.S., more than 80% of African-American smokers use mentholated cigarettes, compared to less than 30% of Caucasian smokers. The reasons for these differences are not well understood. To determine if genetic variation contributes to mentholated cigarette smoking, we performed an exome-wide association analysis in a multiethnic population-based sample from Dallas, TX (N = 561). Findings were replicated in an independent cohort of African Americans from Washington, DC (N = 741). We identified a haplotype of MRGPRX4 (composed of rs7102322[G], encoding N245S, and rs61733596[G], T43T), that was associated with a 5-to-8 fold increase in the odds of menthol cigarette smoking. The variants are present solely in persons of African ancestry. Functional studies indicated that the variant G protein-coupled receptor encoded by MRGPRX4 displays reduced agonism in both arrestin-based and G protein-based assays, and alteration of agonism by menthol. These data indicate that genetic variation in MRGPRX4 contributes to inter-individual and inter-ethnic differences in the preference for mentholated cigarettes, and that the existence of genetic factors predisposing vulnerable populations to mentholated cigarette smoking can inform tobacco control and public health policies.
Project description:The purpose of this study was to estimate black/white differences in cotinine levels for current smokers of both sexes, and to explore the potential contribution of mentholated cigarettes to these differences. Sera from 255 current smokers sampled from Southern Community Cohort Study participants (65 black men, 65 black women, 63 white men, 62 white women) were analyzed for cotinine, and linear regression was used to model the effect of race on cotinine level, adjusting for the number of cigarettes smoked within the last 24 hours, use of menthol vs. non-menthol cigarettes, exposure to environmental tobacco smoke, and age. Black smokers smoked fewer cigarettes than white smokers, yet had crude mean cotinine levels nearly as high or higher than white smokers. After multivariate adjustment, cotinine levels were an average of 50 ng/ml higher among black than white women (p=0.008) and non-significantly 12 ng/ml higher among black than white men (p=0.52). We observed no increase in cotinine levels associated with menthol cigarette use. We conclude that differences in cotinine levels among smokers suggest racial variation in exposure to and/or metabolism of tobacco smoke constituents, but our findings do not support a role for menthol preference in this disparity.
Project description:In the United States, cigarette flavorings are banned, with the exception of menthol. The cooling effects of menthol could facilitate the absorption of tobacco toxicants. We examined levels of biomarkers of tobacco exposure among U.S. smokers of menthol and nonmenthol cigarettes.We studied 4,603 White, African-American, and Mexican-American current smokers 20 years of age or older who participated in the National Health and Nutrition Examination Survey (NHANES) from 1999 through 2010 and had data on cigarette type and serum cotinine, blood cadmium, and blood lead concentrations. Urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) (NNAL) was studied in 1,607 participants with available measures.A total of 3,210 (74.3%) participants smoked nonmenthol cigarettes compared with 1,393 (25.7%) participants who smoked menthol cigarettes. The geometric mean concentrations comparing smokers of nonmenthol with menthol cigarettes were 163.1 versus 175.9 ng/mL for serum cotinine; 0.95 versus 1.02 ?g/L for blood cadmium; 1.87 versus 1.75 ?g/dL for blood lead; and 0.27 versus 0.23 ng/mL for urine NNAL. After multivariable adjustment, the ratios [95% confidence interval (CI)] comparing smokers of menthol with nonmenthol cigarettes were 1.03 (0.95-1.11) for cotinine, 1.10 (1.04-1.16) for cadmium, 0.95 (0.90-1.01) for lead, and 0.81 (0.65-1.01) for NNAL.In a representative sample of U.S. adult smokers, current menthol cigarette use was associated with increased concentration of blood cadmium, an established carcinogen and highly toxic metal, but not with other biomarkers.These findings provide information regarding possible differences in exposure to toxic constituents among menthol cigarette smokers compared with nonmenthol cigarette smokers.
Project description:<h4>Background</h4>US mentholated cigarette sales have increased considerably over 50 years. Preference for mentholated cigarettes is markedly higher in Black people. While menthol itself is not genotoxic or carcinogenic, its acute respiratory effects might affect inhalation of cigarette smoke. This possibility seems consistent with the higher lung cancer risk in Black men, despite Black people smoking less and starting smoking later than White people. Despite experimental data suggesting similar carcinogenicity of mentholated and non-mentholated cigarettes, the lack of convincing evidence that mentholation increases puffing, inhalation or smoke uptake, and the similarity of lung cancer rates in Black and White females, a review of cigarette mentholation and lung cancer is timely given current regulatory interest in the topic.<h4>Methods</h4>Epidemiological studies comparing lung cancer risk in mentholated and non-mentholated cigarette smokers were identified from MedLine and other sources. Study details were extracted and strengths and weaknesses assessed. Relative risk estimates were extracted, or derived, for ever mentholated use and for long-term use, overall and by gender, race, and current/ever smoking, and meta-analyses conducted.<h4>Results</h4>Eight generally good quality studies were identified, with valid cases and controls, and appropriate adjustment for age, gender, race and smoking. The studies afforded good power to detect possible effects. However, only one study presented results by histological type, none adjusted for occupation or diet, and some provided no results by length of mentholated cigarette use.The data do not suggest any effect of mentholation on lung cancer risk. Adjusted relative risk estimates for ever use vary from 0.81 to 1.12, giving a combined estimate of 0.93 (95% confidence interval 0.84-1.02, n = 8), with no increase in males (1.01, 0.84-1.22, n = 5), females (0.80, 0.67-0.95, n = 5), White people (0.87, 0.75-1.03, n = 4) or Black people (0.90, 0.73-1.10, n = 4). Estimates for current and ever smokers are similar. The combined estimate for long-term use (0.95, 0.80-1.13, n = 4) again suggests no effect of mentholation.<h4>Conclusion</h4>Higher lung cancer rates in Black males cannot be due to their greater preference for mentholated cigarettes. While some study weaknesses exist, the epidemiological evidence is consistent with mentholation having no effect on the lung carcinogenicity of cigarettes.
Project description:In the U.S. menthol remains the sole permitted characterizing cigarette flavor additive in part because efforts to link menthol cigarette use to increased tobacco-related disease risk have been inconclusive. To perform definitive studies, cigarettes that differ only in menthol content are required, yet these are not commercially available. We prepared research cigarettes differing only in menthol content by deposition of L-menthol vapor directly onto commercial nonmenthol cigarettes, and developed a method to measure a cigarette's menthol and nicotine content. With our custom-mentholation technique we achieved the desired moderately high menthol content (as compared to commercial brands) of 6.7 ± 1.0 mg/g (n = 25) without perturbing the cigarettes' nicotine content (17.7 ± 0.7 mg/g [n = 25]). We also characterized other pertinent attributes of our custom-mentholated cigarettes, including percent transmission of menthol and nicotine to mainstream smoke and the rate of loss of menthol over time during storage at room temperature. We are currently using this simple mentholation technique to investigate the differences in human exposure to selected chemicals in cigarette smoke due only to the presence of the added menthol. Our cigarettes will also aid in the elucidation of the effects of menthol on the toxicity of tobacco smoke.
Project description:One-third of smokers primarily use menthol cigarettes and usage of these cigarettes leads to elevated serum nicotine levels and more difficulty quitting in standard treatment programmes. Previous brain imaging studies demonstrate that smoking (without regard to cigarette type) leads to up-regulation of ?(2)*-containing nicotinic acetylcholine receptors (nAChRs). We sought to determine if menthol cigarette usage results in greater nAChR up-regulation than non-menthol cigarette usage. Altogether, 114 participants (22 menthol cigarette smokers, 41 non-menthol cigarette smokers and 51 non-smokers) underwent positron emission tomography scanning using the ?(4)?(2)* nAChR radioligand 2-[(18)F]fluoro-A-85380 (2-FA). In comparing menthol to non-menthol cigarette smokers, an overall test of 2-FA total volume of distribution values revealed a significant between-group difference, resulting from menthol smokers having 9-28% higher ?(4)?(2)* nAChR densities than non-menthol smokers across regions. In comparing the entire group of smokers to non-smokers, an overall test revealed a significant between-group difference, resulting from smokers having higher ?(4)?(2)* nAChR levels in all regions studied (36-42%) other than thalamus (3%). Study results demonstrate that menthol smokers have greater up-regulation of nAChRs than non-menthol smokers. This difference is presumably related to higher nicotine exposure in menthol smokers, although other mechanisms for menthol influencing receptor density are possible. These results provide additional information about the severity of menthol cigarette use and may help explain why these smokers have more trouble quitting in standard treatment programmes.
Project description:Nicotine is a major oral irritant in smokeless tobacco products and has an aversive taste. Mentholated smokeless tobacco products are highly popular, suggesting that menthol increases their palatability and may facilitate initiation of product use. While menthol is known to reduce respiratory irritation by tobacco smoke irritants, it is not known whether this activity extends to oral nicotine and its aversive effects.The two-bottle choice drinking assay was used to characterise aversion and preference in C57BL/6 mice to a range of menthol concentrations (10-200?µg/mL). Then, effects of menthol on oral nicotine aversion were determined. Responses were compared with those in mice deficient in the cold/menthol receptor, TRPM8, expressed in trigeminal sensory neurons innervating the oral cavity.Mice showed aversion to menthol concentrations of 100?µg/mL and above. When presented with a highly aversive concentration of nicotine (200?µg/mL), mice preferred solutions with 50 or 100?µg/mL menthol added over nicotine alone. In contrast to wild-type mice, Trpm8-/- showed a strong aversion to mentholated (100?µg/mL) nicotine (200?µg/mL) and preferred nicotine alone. Trpm8-/- mice show aversion to lower concentrations of menthol than wild-type mice.Oral menthol can reduce the aversive effects of oral nicotine and, at higher concentrations, acts as an irritant by itself. Menthol's effects in relation to nicotine require TRPM8, the cool temperature sensing ion channel that activates analgesic and counterirritant mechanisms. These mechanisms may underlie preference for menthol-containing smokeless tobacco products and may facilitate initiation of product use.
Project description:<h4>Introduction</h4>Menthol cigarette use remains a serious public health problem, prompting the consideration of tobacco regulatory efforts to ban menthol cigarettes. The current study uses a novel empirical design to model the potential effects of a ban of menthol cigarettes on smoking behaviour among current menthol smokers.<h4>Methods</h4>29 non-treatment-seeking adults who smoked menthol cigarettes were recruited in Connecticut in 2017-2018 (n=15 female; n=17 Black, n=10 White, n=5 Hispanic). Repeated-measures analyses examined within-person changes in smoking behaviour when participants were switched from smoking their usual brand menthol cigarettes to a matched-brand non-menthol cigarette for 2 weeks to model a potential ban of menthol cigarettes.<h4>Results</h4>Participants smoked significantly fewer non-menthol (vs menthol) cigarettes per day (mean decrease=2.2 cigarettes, SD=3.2, p<0.001), confirmed by significant reductions in urine cotinine levels (p=0.013). After switching to non-menthol cigarettes, participants had significantly lower nicotine dependence scores (reduced by >18%, p<0.001) and greater increases in quitting motivation and confidence (rated 1-10) (motivation: mean increase=2.1, SD=2.8, p<0.001; confidence: mean increase=1.3, SD=3.3, p=0.04). Exploratory analyses indicated significant interactions by race (p=0.004); Black smokers had greater reductions in cigarettes per day (mean decrease=3.5 cigarettes, SD=2.8) versus non-Black smokers (mean decrease=0.2, SD=2.6).<h4>Conclusions</h4>Banning menthol as a characterising flavour in cigarettes may decrease smoking and reduce the addictive potential of cigarettes among current smokers. Results provide additional support for tobacco regulatory policies banning menthol flavour in an effort to improve public health.<h4>Trial registration</h4>NCT03075839.
Project description:INTRODUCTION:Characterizing flavors are widely available in e-cigarettes and motivate initiation and continued use. Flavors may enhance appeal and facilitate development of addiction to tobacco products through modulation of tobacco products' reinforcing or aversive actions. Palatable flavors (eg, fruit) may increase appeal through primary reinforcing properties. Menthol's cooling and anesthetic effects may increase appeal by counteracting nicotine's aversive effects. Genetics provide a method for modeling individual differences in sensitivity to nicotine's effects. A common polymorphism, rs16969968, encoded in the ?5 nicotinic acetylcholine receptor subunit gene (CHRNA5), is a well-recognized marker for smoking risk and reduces sensitivity to nicotine aversiveness. METHODS:This pilot study tested how flavors impacted e-cigarette appeal and self-administration. In a single testing day, cigarette smokers (N = 32; 94% menthol-smokers) self-administered e-cigarettes containing e-liquids differing in nicotine level (0 mg/mL, 24 mg/mL) and flavor (unflavored, menthol, fruit-flavored) within directed and ad libitum e-cigarette paradigms. Subjective drug effects, number of puffs, rs16969968 genotype, plasma nicotine, and menthol glucuronide levels were collected. RESULTS:Menthol partially ameliorated nicotine aversiveness; fruit did not. In nicotine's absence, fruit flavor increased self-reported preference and ad libitum use relative to menthol-containing or unflavored e-liquids. Individuals with high-smoking-risk rs16969968 genotype (N = 7) reported greater craving alleviation following directed administration of nicotine-containing e-liquids, showed a trend rating nicotine-containing e-liquids as less harsh, and self-administered more nicotine during ad libitum compared to individuals with low-smoking-risk genotype (N = 23). CONCLUSIONS:While menthol countered aversiveness of nicotine-containing e-liquids, fruit flavor increased appeal of nicotine-free e-liquids. These preliminary findings suggest menthol and fruit flavor increase e-cigarettes' appeal through distinct mechanisms. IMPLICATIONS:This study provides a detailed characterization of the effects of flavors (unflavored, menthol, fruit), nicotine (0 mg/mL, 24 mg/mL) and their interactions on the subjective drug effects and ad libitum self-administration of e-cigarettes. Genetics were used to assess these effects in higher-smoking-risk (diminished sensitivity to nicotine aversiveness) and lower-risk groups. Findings could inform impact of regulation of flavors or nicotine in e-cigarettes, and their impacts on vulnerable sub-populations.
Project description:<h4>Introduction</h4>Some, but not all, studies suggest that menthol cigarette smokers have more difficulty quitting than non-menthol cigarette smokers. Inconsistent findings may be a result of differences in smoker characteristics (eg, daily vs. non-daily smokers) across studies. This study examines the relationship between menthol cigarette use, cessation, and relapse in a longitudinal, nationally representative study of tobacco use in the United States.<h4>Aims and methods</h4>Data come from four waves of the Population Assessment of Tobacco and Health Study. Waves 1-4 were conducted approximately annually from September 2013 to January 2018. Generalized estimating equation models were used to prospectively examine the relationship between menthol cigarette use, cessation, and relapse in non-daily and daily adult (18+) smokers. Cessation was defined as smokers who had not used cigarettes within the past 30 days at their subsequent assessment. Relapse was defined as cessation followed by past 30-day smoking in the next assessment.<h4>Results</h4>Among daily smokers (n = 13 710), 4.0% and 5.3% of menthol and non-menthol smokers quit after 1 year, respectively. In an adjusted model, menthol smokers were less likely to quit compared with non-menthol smokers (odds ratio [OR] = 0.76 [0.63, 0.91]). When the sample was stratified by race/ethnicity, African American (OR = 0.47 [0.24, 0.91]) and White (OR = 0.78 [0.63, 0.97]) daily menthol users were less likely to have quit. Among non-daily smokers (n = 3608), there were no significant differences in quit rates. Among daily and non-daily former smokers, there were also no differences in relapse rates between menthol and non-menthol smokers.<h4>Conclusions</h4>Menthol cigarette use is associated with lower odds of cessation.<h4>Implications</h4>Findings from this study suggest that menthol cigarette use is associated with lower odds of cessation, but not relapse. Removing menthol cigarettes from the market may improve cessation rates.