Prolonged, physiologically relevant nicotine concentrations in the airways of smokers

Abstract

Nicotine from cigarette smoke is a biologically active molecule that has pleiotropic effects in the airway, which could play a role in smoking-induced lung disease. However, whether nicotine and its metabolites reach sustained, physiologically relevant concentrations on airway surfaces of smokers is not well defined. To address these issues, concentrations of nicotine, cotinine, and hydroxycotinine were measured by mass spectrometry (MS) in supernatants of induced sputum obtained from participants in the subpopulations and intermediate outcome measures in COPD study (SPIROMICS), an ongoing observational study that included never smokers, former smokers, and current smokers with and without chronic obstructive pulmonary disease (COPD). A total of 980 sputum supernatants were analyzed from 77 healthy never smokers, 494 former smokers (233 with COPD), and 396 active smokers (151 with COPD). Sputum nicotine, cotinine, and hydroxycotinine concentrations corresponded to self-reported smoking status and were strongly correlated to urine measures. A cutoff of ∼8-10 ng/mL of sputum cotinine distinguished never smokers from active smokers. Accounting for sample dilution during processing, active smokers had airway nicotine concentrations in the 70-850 ng/mL (∼0.5-5 µM) range, and concentrations remained elevated even in current smokers who had not smoked within 24 h. This study demonstrates that airway nicotine and its metabolites are readily measured in sputum supernatants and can serve as biological markers of smoke exposure. In current smokers, nicotine is present at physiologically relevant concentrations for prolonged periods, supporting a contribution to cigarette-induced airway disease.

Keywords: COPD; cotinine; sputum.

Graphical abstract

Figure 1.

Nicotine metabolites in sputum and urine. A: sputum nicotine was elevated in active smokers relative to never or former smokers. B: sputum cotinine was elevated in active smokers relative to never or former smokers. C: sputum hydroxycotinine was elevated in active smokers relative to never or former smokers. D: urine cotinine as measured through the SPIROMICS core laboratory for comparison. Data are depicted in violin plots as nanograms per milliliter, with lines indicating median and IQR. Note scale differences in A. **P < 0.01 by ANOVA with Tukey’s posttest analysis. IQR, interquartile range.

Figure 2.

Sputum vs. urine cotinine. A: strong correlation was observed between sputum and urine cotinine values (r = 0.95, P < 0.0001). B: frequency analysis suggested two populations of sputum cotinine concentrations with a cut-off at 0.8 log units (6 ng/mL). C: frequency analysis of urine cotinine suggested a cut-off between populations of 2.0 log units (100 ng/mL).

Figure 3.

Nicotine metabolites and recent smoking. A: sputum nicotine was increased in active smokers who reported smoking >10 cigarettes within 24 h before the study visit (AS >10, n = 227) vs. active smokers who reported no smoking within 24 h before the study visit (AS 0, n = 20). Sputum nicotine was increased in both groups relative to never smokers (NSs). B: similar pattern for sputum cotinine, with increases in both the AS 0 and AS >10 groups relative to NS, with the AS >10 group higher than the AS 0. C: the pattern for urine cotinine was similar to that for sputum cotinine. D: subjects in the AS 0 group reported fewer average cigarettes smoked per day than those in the AS >10 group. ***P < 0.001 by ANOVA with Tukey’s posttest (A–C) or Student’s t test (D). Cig, cigarettes.

Figure 4.

Relationships of sputum nicotine to other measures in active smokers with preserved spirometry. A: a modest but statistically significant correlation was observed between sputum nicotine and reported cigarettes smoked in the 24 h before the study visit (Spearman r = 0.28, P < 0.0001). B–D: weak but statistically significant correlations were noted between sputum nicotine and sputum adenosine (r = 0.18, P < 0.02; B); hypoxanthine (r = 0.26, P < 0.001; C), and xanthine (r = 0.32, P < 0.001; D). Cig, cigarettes.