Journal of Exposure Analysis and Environmental Epidemiology (2004) 14, S65S70 r 2004 Nature Publishing Group All rights

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Relationship between environmental tobacco smoke and urinary cotinine levels in passive smokers at their residence
HYOJIN KIM,a YOUNGWOOK LIM,b SEOKJU LEE,a SOUNGEUN PARK,c CHANGSOO KIM,d CHEINSOO HONG,e AND DONGCHUN SHINd
a

The Graduate School of Health Science and Management, Yonsei University, Seoul, Korea Department of Environmental Health, Seonam University, Korea c Envioneer Co., Ltd, Seoul, Korea d Department of Preventive Medicine, College of Medicine, Yonsei University, Seoul, Korea e Department of Internal Medicine, Institute of Allergy, College of Medicine, Yonsei University, Seoul, Korea
b

Studies of the health effects of environmental tobacco smoke (ETS) using measured air concentrations are subject to bias. Cotinine, a nicotine metabolite detected in urine, has been recommended as a quantitative measure of nicotine intake and thus as a marker for ETS exposure in humans. The aim of this study was to correlate home indoor ETS levels with passive smokers urinary cotinine levels. The urinary cotinine concentrations of 57 non-smoking women who spend 419 h a day at home and the nicotine levels in their living room air were measured over a period of 24 h. Nicotine and urinary cotinine levels were analyzed using GC/MS and HPLC/UV, respectively. In addition, information was collected regarding the smoking habits of the subjects families. A signicant correlation was found between the nicotine levels in indoor air and the urinary cotinine to creatinine ratio of the passive smokers. The smoking habits of the subjects family members were also correlated to the urinary cotinine levels of the passive smokers. Journal of Exposure Analysis and Environmental Epidemiology (2004) 14, S65S70. doi:10.1038/sj.jea.7500360

Keywords: biomarker, cotinine, ETS, indoor air, passive smoker.

Introduction
Human health effects caused by direct and passive smoking are a potential public health problem. Environmental tobacco smoke (ETS) contains many toxic substances not otherwise present in indoor air (nicotine, acrolein, formaldehyde, polycyclic aromatic hydrocarbons (PAHs), and dimethylnitrosamine), as well as carcinogens, cocarcinogens, ciliotoxins, irritants, etc. (John, 1991; US Department of Health, Education, and Welfare, 1997). Accordingly, ETS is likely harmful to the health of passive smokers. In 1997, Brownson reported that passive smoking increases the risk of pulmonary cancer (Brownson et al., 1997), and increased incidences of chronic coughing, chronic wheezing, and respiratory infections are also observed in children exposed to parental smoking (Zmirou et al., 1990). Nicotine, carboxyhemoglobin (Sillett et al., 1978), thiocyanate (Butts et al., 1974), and carbon monoxide (Ashton et al., 1981) concentrations in blood have been used as biomarkers to quantify exposure to ETS in direct and passive smokers. Nicotine concentrations in air have been used as an

indicator of ETS exposure in many studies (Rothberg et al., 1998). Unlike with direct smoking, it is very difcult to quantify a passive smokers exposure to ETS. Therefore, a method for quantifying ETS exposure in passive smokers is needed. It is believed that the most accurate method is to measure the level of cotinine, a biomarker of nicotine metabolism, which has a long biological half-life in the blood, saliva, and urine. Conitine is present in higher concentrations in blood than other biomarkers and is chemically stable (Etzel, 1990). This study investigated the relationship between indoor nicotine concentrations in homes and the passive smoking residents urinary cotinine to creatinine ratio (CCR).

Methods
Sample Collection Morning urine samples were collected from 57 non-smoking women between 28 and 69 years of age who spent more than 19 h per day at their respective residences in Seoul, Korea. On the same day that urine specimens were taken, indoor air samples were collected from each of their 57 homes and analyzed for nicotine. All samples were collected between September and November 1999. Urine samples were

1. Address all correspondence to: Dr. Dongchun Shin, C.P.O. Box 8044, Seoul, Republic of Korea. Tel.: 82-2-361-5361. Fax: 82-2-2-3920239. E-mail: dshin5@yumc.yonsei.ac.kr

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collected in sterile bottles, retrieved at the end of the sampling day, and stored at 701C until analysis. Nicotine was collected for 24 h using a personal air sampler (Gillian Co., USA) that drew 1.5 l/min of air through a glass ber lter coated with a 4% sodium bisulfate solution. The sampling equipment was placed in the center of the living room at a height of approximately 1.5 m to simulate the breathing zone of a seated person. The families of participated homes completed a questionnaire, detailing the time spent at home per day, whether family members smoked at home and where in the home they smoked, the number of cigarettes smoked at home per day, and what type of home the participants lived in (detached house, tenement house or apartment). Smoking locations were classied as either indoors or outdoors (on a veranda, or near a window or door to a garden or hall). Most tenement houses in Korea are two- or three-story buildings (smaller than apartment buildings), and each at is typically owned by one household.

Table 1. Instrumental analysis conditions of nicotine using GC/MSD.

Parameters Port temp. Volume Mode Inlet Flow (Pressure) Oven: Initial GC/MSD conditions 2501C 1 ml Splitless 1.51C/min, constant 801C for 0.5 min 201C/min to 2001C 2001C held for 3 min J&W 1235063 (non-polar) i.d. 0.2 mm, 20 mm, 0.33 mm 9.5 min 70 eV

Column Run time Ionization energy

Table 2. Instrumental analysis conditions of cotinine using HPLC/ UV.

Parameters Column Temp. HPLC conditions LC-C18 (4.6 mm 25 cm, ID 5 mm, Supelcosil) Column: 201C Sample: 201C Acetonitrile: water (9: 91) Add triethylamine 5 ml, sodium Heptanesulfonate 600 mg, potassium Phosphate 30 mmol/l, citric acid 30 mmol/l, pH 4.4 35 min 1.2 ml/min 10 ml Absorbance at 260 nm

Sample Analysis The glass ber lters from nicotine samples were extracted with 0.1 ml ethanol. To produce a free base, 2 ml of 10 N NaOH was added and the nicotine was extracted into 0.25 ml heptane. In all, 50 ml of a quinoline solution containing 0.005 mg/ml of nicotine-methyl-d3 as an internal standard were added (Odgen et al., 1989). Nicotine concentrations in the resulting samples were determined using a Hewlett-Packard 5890II gas chromatograph with mass selective detector (GC/MSD, HewlettPackard, Avondale, USA). Table 1 gives the GC/MSD parameters used for nicotine quantication. A 2 ml sample of urine was mixed with 50 ml of 0.01 mg/ml 2-phenylimidazole in methanol (as an internal standard) and 0.15 ml of 0.5 M sodium hydroxide. Dichloromethane was used for the liquidliquid extraction of the subjects urine sample. The mixture was stirred and 3 ml of dichloromethane was added. The mixture was shaken for 15 min and centrifuged at 5000 rpm for 15 min. The organic layer was separated and evaporated to dryness at 401C under a nitrogen stream. The aqueous layer was mixed with 1.5 ml of 0.5 M HCl, 0.15 ml of 0.5 M sodium hydroxide and 3 ml of dichloromethane, and recentrifuged to separate the organic layer. A reversed-phase column was used for the analysis using a high-performance liquid chromatography-ultraviolet and visible detector (HPLC/UV, Waters Assoc., Milford, MA, USA). Table 2 shows the HPLC/UV parameters for cotinine analysis. The Jaffe reaction method was used to measure the creatinine level in each urine specimen (Tietz, 1986). The urinary creatinine concentration, a standard urine-specic reference, was used to correct for dilution differences among individuals urine. The urinary cotinine concentration divided by the urinary creatinine concentration gives the urinary
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Mobile phase

Run time Flow rate Injection volume Detection

CCR. When normalized by creatinine measurements, urinary cotinine measurements performed as well as or better than reported exposures in assessments of lung function (Haddow et al., 1994). The recovery rate of a known amount of nicotine in a glass ber lter coated with 4% sodium bisulfate was measured as 98.772.3%. A urine sample that had not been exposed to nicotine was dosed with a known amount of cotinine, and the conitine concentration measured to determine the conitine recovery rate: 92.673.7%. Measured concentrations were corrected using these recovery rates. The limit of detection (LOD) is dened as three standard deviations above the average measured difference between the sample and the blank signals (US EPA, 1984). The LOD values were 0.0005 mg/m3 for airborne nicotine and 0.007 mg/ ml for cotinine in urine.

Statistical Analysis As the data did not follow a Gaussian distribution, the KruskalWallis one-way test and the Wilcoxon rank sum test were used to analyze the nicotine levels in indoor air and the passive smokers CCR levels according to the smoking habits
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of their households. SAS (release 8.01 for Windows) was used to calculate the nonparametric Spearmans correlation coefcient and to conduct simple regression analysis.

Results
Concentration of Nicotine in Indoor Air In this study, indoor nicotine levels ranged from nondetectable (ND) to 17.39 mg/m3 (Figure 1). The median nicotine concentrations in houses where smoking occurred indoors or outdoors (1.24 mg/m3) were signicantly higher than those in houses where no smoking occurred (0.25 mg/ m3) (po0.001). In houses where smoking occurred indoors, the median nicotine level (5.90 mg/m3) was signicantly higher than in houses where smoking occurred only on the veranda or outdoors (0.86 mg/m3) (po0.001) (Table 3). The number of cigarettes smoked per day in the home was also signicantly correlated with nicotine concentration (po0.05). The median nicotine concentration in tenement houses (1.15 mg/m3) was higher than in detached houses (0.58 mg/m3) or apartments (0.34 mg/m3), though not signicantly so (p40.05). Nonsmoking homes were included in each of these house categories. For two homes, the classication was not known. For each house category, we compared homes where

Figure 1. Distribution of nicotine concentrations in indoor air at home.

smoking occurred with homes where no smoking occurred. Indoor and outdoor smoking locations could not be statistically compared within each house category because there were too few subjects in each subcategory. The median nicotine concentration in tenement houses where indoor or outdoor smoking occurred was higher than in other house categories where smoking occurred (Table 3).

Concentration of Urinary Cotinine for Passive Smokers The CCR was between ND and 384.68 mg/g creatinine (Figure 2). The median CCR level in passive smokers who

Table 3. Nicotine concentration in indoor air and urinary cotinine to creatinine ratio (CCR) of passive smokers.
Classes Variables Nicotine (mg/m3) Median Smoking habits Home smoker No (n 31) Yes (n 26) Smoking location No smoking (n 31) Veranda or outdoors (n 18) Indoors (n 8) Number of daily cigarettes at home 15 (n 16) 6 or more (n 10) House type A detached house (n 20) No (n 12) Home smoker Yes (n 8) Tenement house (n 11) No (n 5) Home smoker Yes (n 6) Apartment (n 24) No (n 13) Home smoker Yes (n 11) n: number of subjects. 1.14 15.29 0.34 0.24 0.507 14.16 8.23 1.15 0.18 0.023 10.39 6.81 3.83 0.346 p-value CCR (mg/g creatinine) Median p-value

0.25 1.24 0.25 0.86 5.90 0.97 3.23

o0.001

7.53 9.96 7.53 4.27 49.45 4.11 26.38

0.133

o0.001

0.008

0.048

0.014

Environment characteristics

0.683 0.58 0.32 0.177 19.84 10.49 1.93 9.36 0.89

0.886

0.291

0.855

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Figure 2. Distribution of urinary cotinine concentrations for the subjects.

lived with smokers (9.96 mg/g creatinine) was not signicantly different from that of passive smokers who lived with nonsmokers (7.53 mg/g creatinine). However, the median CCR of passive smokers who lived with indoor smokers (49.45 mg/ g creatinine) was higher than that of passive smokers who lived with smokers who only smoked on the veranda or outdoors (4.27 mg/g creatinine) and of those who lived with non-smokers (7.53 mg/g creatinine) (Table 3). This study found that the CCR of passive smokers was signicantly correlated to the smoking location (po0.01) and the number of cigarettes consumed daily in the home (po0.05). However, the type of house was not correlated to passive smokers CCR (po0.05). Among the passive smokers who lived in homes with indoor or outdoor smoking, the median cotinine level was 19.84 mg/g creatinine for women living in detached houses, 10.31 mg/g creatinine for women living in tenement houses, and 14.16 mg/g creatinine for women living in apartments (Table 3).

Figure 3. Correlation between indoor nicotine and CCR levels of passive smokers. (a) All subjects, (b) subjects exposed to ETS at home, (c) subjects with no ETS exposure at home.

Relationship Between Indoor Nicotine Levels and Passive Smokers CCR There was no signicant correlation between nicotine levels in the indoor air of non-smoking homes and the CCRs of passive smokers who lived in a non-smoking home. However, a Spearmans correlation coefcient of 0.805 (po0.001) indicates a statistically signicant relationship between the indoor air nicotine levels in homes with members who smoke at home and the CCR of the passive smokers who live there (Figure 3). The following regression equation was obtained using simple regression analysis of these data: Y (urinary cotinine) 21.03 X (nicotine in indoor air) 14.20.

Discussion
Several approaches have been used to study the effect of ETS exposure on passive smokers. Studies on smoking and health
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have estimated exposure on the basis of the reported number of cigarettes smoked daily (Kannel, 1981), which is subject to bias and accuracy limitations (Oddoze et al., 1999). Lee (1995) assessed childrens ETS exposure in investigations are based on parents self-reports; hence, they could be biased. For example, there is a possibility of deceptive underreporting of exposure in children by smoking parents. Further, several factors are of importance for the exposure, such as the amount of tobacco smoked, room size, ventilation, and proximity to smokers. It may be difcult to estimate these factors in a questionnaire (Willers et al., 2000). In all, 75% or more of the nicotine that is emitted from a cigarette is emitted into the air as sidestream smoke, which contributes substantially to ETS (Committee on Passive Smoking, 1986). Nicotine in ETS is breathed into the nose and throat and is inhaled into the lungs by nonsmokers (Iwase et al., 1991). When nicotine is taken into the body through the lungs, it enters the bloodstream and is circulated to various body organs. On average, 7080% of nicotine is converted to cotinine (Jarvis et al., 1984). Measurements of plasma or saliva thiocyanate levels, expired carbon monoxide levels, and carboxyhemoglobin levels have been used to measure subjects total cigarette smoke intake (Luepker et al., 1981). However, these methods lack sensitivity and specicity. Cotinine measurement is more accurate because cotinine is chemically stable. Cotinine measurement is superior to nicotine measurement, because urinary pH has less inuence on cotinine excretion than on nicotine excretion (Benowitz, 1988), and because cotinine has a longer half-life (1940 h) in the body than nicotine (2 h). Thus, cotinine reects longterm, rather than recent, ETS exposure (Rosenberg et al., 1980). Benowitz et al. (1991) reported that cotinine has
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limited use as a biomarker because the metabolism of cotinine differs among individuals and because there is no standard analysis method. However, cotinine has been used as a biomarker for direct and passive smokers nicotine intake because of its consistently high sensitivity and specicity (Baranowski et al., 1988). Furthermore, it was reported that it is the best means available to measure the extent of ETS exposure (Etzel, 1990). Urinary cotinine was measured using a simple, reversedphase, high-performance liquid chromatographic method (Hariharan et al., 1991). This method has proved reliable for assessing non-smokers exposure to ETS, and tractable for routine analysis, with no derivatization (Georg and Gerhard, 1987; Parviainen and Barlow, 1988), no long liquidliquid extraction over several steps (Hariharan et al., 1991) and no need for special detectors such as a photodiode array detector (Grimaldi et al., 1993). In this study, the detection limit (dened as signal-to-noise ratio 3) for cotinine was 0.007 mg/l, which agrees well with the values of o1 mg/l reported by Hariharan et al. (1991) and 0.5 mg/l by Oddoze et al. (1998). In this study, the home nicotine levels were signicantly different (po0.001) among the following categories: no smoking in the home (median 0.25 mg/m3), smoking on the veranda or outdoors (median 0.86 mg/m3), and smoking indoors (median 5.90 mg/m3). The corresponding median urinary CCRs were 7.53 mg/g creatinine, 4.27 mg/g creatinine, and 49.45 mg/g creatinine, respectively. Yoon (1995) reported home nicotine concentrations of 3.1773.02 and 1.6072.40 mg/m3, in summer and winter, respectively, in Seoul, Korea. Hansen et al. (2001) reported that urinary excretion of the nicotine metabolites in smokers was approximately 100 times higher than that observed in passive and non-smokers. In addition, Oddoze et al. (1999) reported that urinary cotinine concentrations in children were signicantly correlated (po0.001) with the number of cigarettes the parents smoked. Wakeeld et al. (2000) reported that the mothers smoking behavior was correlated with urinary CCRs of the child, and that a total absence of cigarette smoking by parents was associated with signicantly lower urinary CCRs in children after adjusting for age. If both parents reported that they were smoking in their home (with or without other smokers), their childrens median urinary cotinine levels were approximately 17 times higher than those of children from homes with no indoor smoking (Willers et al., 2000). The urinary cotinine level of nonsmokers living with smokers was 11.4 mg/l, while that of nonsmokers living alone or with other non-smokers was 4.4 mg/l (Thompsom et al., 1990). In this study, the correlation coefcient between home air nicotine concentrations and urinary CCRs of passive smokers in homes where smoking occurred was 0.8319. Three recent studies have reported the relation between ambient air nicotine concentrations and cotinine concentraJournal of Exposure Analysis and Environmental Epidemiology (2004) 14(S1)

tions in the urine or saliva of non-smokers. Studies by Marbury et al. (1993) and Henderson et al. (1989) of children in the home, and by Coultas et al. (1990) of adults in the workplace, found correlations between ambient air nicotine concentrations and urinary cotinine concentrations (correlation coefcients: r 0.81, r 0.68, and r 0.60, respectively). These correlations are likely as high as can be expected given the sources of variability. These results support urinary cotinine concentration as a biomarker of ETS-derived nicotine exposure. The median CCR of passive smokers living in non-smoking homes was higher than that of passive smokers living with smokers who smoke on the veranda or outdoors, as shown in Table 3. Similarly, relatively high CCRs in non-smoking homes and relatively low nicotine levels in homes where smoking occurred were measured in some cases (Figure 3). Figure 3(c) shows the relationship between nicotine concentrations in nonsmokers homes and urinary CCRs (correlation coefcient 0.1879). One house may have been misclassied as non-smoking because of a guest who smoked in the home or because of smoke introduced through ventilation. That single nicotine concentration dominates the correlation. When that data point was removed, the correlation coefcient was 0.0312. These results may be explained by house ventilation, gas stove usage, smoking of guests, etc. This study has limited utility as a cross-sectional study. However, the signicant correlation found in this study between indoor nicotine concentrations and urinary cotinine concentrations supports the hypothesis that ETS exposure through passive smoking causes nicotine intake. Future studies of other factors that affect indoor nicotine concentrations, such as ventilation rates, and their effects on CCRs, are needed, as are studies of the health of passive smokers.

Conclusions and implications

Urinary cotinine concentration was used in this study as a biomarker for nicotine intake in passive smokers exposed to ETS. A statistically signicant, simple regression was calculated relating urinary cotinine concentrations in nonsmoking women to nicotine concentrations in their home indoor air. A doseresponse relationship was established between the magnitude of passive exposure to tobacco smoke and the urinary excretion of cotinine. In addition, the location of smoking within the home and the number of cigarettes smoked per day in the home affected both the nicotine concentrations in indoor air and the passive smokers CCRs. Urinary cotinine, which is easy to quantify by HPLC, is presently the best biochemical marker for measuring the level of exposure to tobacco smoke, and it can be measured in any epidemiological survey of respiratory and lung function.
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Acknowledgements
This study was supported by a grant (HMP-99-M-09-0006) from the 1999 Good Health R&D Project, at the Ministry of Health & Welfare, Korea.

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