We recruited 28 participants for the study (18 females). Of these, 17 were white (11 female), 7 were black (3 females), 3 were Asian (all female), and 1 described her race as other. All participants were 21–37 years of age. We excluded one participant who had a baseline (preexposure before first site visit) salivary cotinine concentration of 5.25 ng/mL—a concentration characteristic of very high SHS exposure or occasional smoking. This individual lived with a smoker. Otherwise, the range of preexposure salivary cotinine concentrations in samples provided before the first study visit confirmed that prestudy SHS exposures were low (0.011–0.480 ng/mL).
Biomarker levels according to sample and site. One participant did not provide next-day saliva and urine samples. Also, 11 of the 242 samples collected did not have enough saliva for cotinine analysis (n = 1 preexposure, n = 7 postexposure, and n = 3 next-day) and 1 sample was below the LOD (0.015 ng/mL). Geometric means of preexposure, postexposure, and next-day salivary cotinine levels are presented in ; quartiles and 95th percentiles of salivary cotinine for these sampling time points are presented in . Although salivary cotinine levels remained flat after participants visited the control site, salivary cotinine levels increased, as expected, after restaurant and bar visits, with greater increases after bar visits. Salivary cotinine concentrations in postexposure and next-day samples were similar. One participant’s postexposure – preexposure change in salivary cotinine reached 0.4 ng/mL after the bar visit.
Salivary cotinine and urinary NNAL in nonsmokers after visits to outdoor locations.
Figure 1 Salivary cotinine (ng/mL) for control, restaurant, and bar (A, B, and C, respectively), urinary uncorrected NNAL (pg/mL) for control, restaurant, and bar (D–F), and creatinine-corrected NNAL (pg/mg creatinine) for control, restaurant, and bar (more ...)
For the 27 participants at baseline, NNAL was measured above the LOD in 9 (33.3%), below the LOD in 10 (37.0%), and not detected in 8 (29.6%). Overall, of 242 urine samples collected during the study period from 27 participants (81 preexposure, 81 postexposure, and 80 next-day samples, 1 did not provide a next-day urine sample), urinary NNAL was measured above the LOD in 56.2% (n = 136), below the LOD in 14.9% (n = 36), and not detected in 28.9% (n = 70). Geometric means for uncorrected and creatinine-corrected preexposure, postexposure, and next-day urinary NNAL are presented in , and quartiles and 95th percentiles are illustrated in . Urinary NNAL concentrations remained flat after visits to the control site. There was also a lack of change between previsit and postvisit restaurant and bar samples, respectively, in contrast to changes noted for next-day restaurant and bar samples. As expected, larger changes in urinary NNAL levels were observed in next-day samples after visits to the bar site.
Differences in biomarker responses between sites. Regression models to test differences in biomarkers after exposure between sites excluded two participants whose samples were not collected according to their preassigned sequence because of personal scheduling conflicts, and one participant who did not complete all three visits. As a result, we had eight complete Latin squares with three participants per square (24 participants total).
Mean differences in postexposure versus preexposure salivary cotinine concentrations were significantly greater after visits to the bar location [0.115 ng/mL (95% CI: 0.105, 0.126)] and restaurant location [0.030 ng/mL (95% CI: 0.028, 0.031)] relative to changes observed after visiting the control site [–0.004 ng/mL (95% CI –0.005, –0.003)] (), with p < 0.001 for both comparisons based on regression models (). We obtained similar results when we looked at next-day versus preexposure concentrations (, ).
Figure 2 Saliva-cotinine and urinary-NNAL changes after a 3-hr visit to outdoor seating areas of a restaurant and a bar where outdoor smoking was allowed and to an open-air control location with no smokers. (A) Salivary cotinine postexposure minus preexposure (more ...)
Test of effect of exposure location and pair-wise comparisons between location types.
As noted above, there were no significant differences in urinary (uncorrected and creatinine-corrected) NNAL concentrations for samples collected immediately after site visits compared with previsit samples, regardless of site (). Consistent with expectations, there also were no significant differences in changes observed between sites (). However, contrasts between next-day and previsit uncorrected and creatinine-corrected urinary NNAL concentrations were significantly greater after visits to the bar location (p < 0.001) and restaurant location (p = 0.006) compared with the control site (, ). Further, when we analyzed preexposure, postexposure, and next-day NNAL as repeated measures (Friedman's nonparametric chi-square test), location type had a significant effect on the distribution of urinary NNAL (uncorrected: χ2 = 7.16, p = 0.028; creatinine corrected: χ2 = 13.9, p = 0.001). These results are consistent with those presented above.
Differences in weekly preexposure biomarkers (crossover effect). Compared with baseline average salivary cotinine levels [0.038 ng/mL (95% CI: 0.029, 0.049)], week-3 preexposure salivary cotinine was significantly higher [0.055 ng/mL (95% CI: 0.043, 0.071); p = 0.006]. Average week-2 preexposure salivary cotinine levels [0.045 ng/mL (95% CI: 0.035, 0.058)] were not significantly different from baseline (p = 0.416). Preexposure urinary NNAL did not differ significantly during the 3 weeks of the study (uncorrected, p = 0.778; creatinine corrected, p = 0.169). Weeks 1, 2, and 3 geometric means and 95% CI for preexposure creatinine-corrected NNAL were as follows: 0.5 (95% CI: 0.3, 0.7), 0.8 (95% CI: 0.5, 1.2), and 0.7 (95% CI: 0.5, 1.1) pg/mg creatinine, respectively.
Biomarkers versus cigarette counts. On average, we counted a higher number of lit cigarettes over the 3-hr sampling period outside the bar (mean ± SD, 144.5 ± 39.9) than outside the restaurant (mean ± SD, 33.5 ± 28.0). No lit cigarettes were observed at the control site. The effect of cigarette count on postexposure minus preexposure changes in salivary cotinine [0.0032 (0.0003), presented as effect estimate and SE] and on next-day minus preexposure changes in salivary cotinine [0.0032 (0.0004)] were significant (both p <0.001). Just as with location type, cigarette count was not associated with postexposure minus preexposure changes in urinary uncorrected and creatinine-corrected NNAL but was significantly associated with next-day minus preexposure changes in uncorrected NNAL [0.0003 (0.0007)] and creatinine-corrected NNAL [0.0032 (0.0008)] (both p = 0.018).
Salivary cotinine versus urinary NNAL correlations and ratios.
Changes in salivary cotinine were generally moderately correlated to changes in urinary NNAL (uncorrected and creatinine corrected); higher correlations were observed between changes in postexposure – preexposure and next-day – preexposure salivary cotinine and in next-day – preexposure changes in NNAL (). Although the ratios of urinary NNAL to salivary cotinine did not differ significantly across location type, ratios were lower in postexposure samples compared to preexposure and next-day samples [see Supplemental Material, Table 2
Spearman rank correlation coefficients between changes measured in salivary cotinine and urinary NNAL.