RELATIONSHIP BETWEEN ENVIRONMENTAL TOBACCO SMOKE AND URINARY COTININE LEVELS IN PASSIVE SMOKERS AT THEIR RESIDENCE

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1 RELATIONSHIP BETWEEN ENVIRONMENTAL TOBACCO SMOKE AND URINARY COTININE LEVELS IN PASSIVE SMOKERS AT THEIR RESIDENCE YW Lim 1, HJ Kim 2, DC Shin 3, SJ Lee 2, SE Park 4, JY Yang 2 and CS Hong 5 l Dept. of Environmental Health, Seonam University, Korea 2 The Institute for Environmental Research, Yonsei University, Seoul, Korea 3 Dept. of Preventive Medicine, College of Medicine, Yonsei University, Seoul, Korea 4 Envioneer Co., Ltd, Seoul, Korea 5 Dept. of Internal Medicine, Institute of Allergy, Yonsei University College of Medicine, Seoul, Korea ABSTRACT Studies on the effects of environmental tobacco smoke (ETS), using recorded air concentrations, may be subject to bias. Cotinine, a metabolite of nicotine, is recommended as a valid quantitative measure of exposure to ETS. The purpose of this study is to correlate environmental tobacco smoke with urinary cotinine levels in passive smokers at home. We measured urinary cotinine concentrations in 57 housewives and the nicotine level in the indoor air of their living rooms over a period of 24 hours. Levels of nicotine and urinary cotinine were analyzed using GC/MS and HPLC/UV. At the same time, we gave a spirometric test to the subjects and questionnaires about smoking to the subjects families. We found significant correlation between nicotine levels in the indoor air and the passive smokers' urinary cotinine to creatinine ratio (CCR). On the other hand, age and urinary cotinine levels were not significantly correlated. The smoking habits of the smokers were also a major determinant factor in the urinary cotinine levels of the passive smokers. INDEX TERMS ETS, Cotinine, Passive smoker, Indoor air, Biomarker INTRODUCTION Recently not only direct smoking, but also the possibility of the noxious effect on the human body caused by passive smoking has become of increasing importance as one of the public health problems. ETS contains large amount of noxious substances such as nicotine, acrolein, formaldehyde, polynuclear aromatic hydrocarbons(pahs), dimethylnitrosamine and also harms the human body with carcinogens, cocarcinogens, ciliotoxins, irritants, etc(hew, 1993; John HB, 1991). Among these substances, nicotine is known as a substance which causes an acute toxic effect even at a low concentrations (Stephanie M et al., 2). Accordingly, ETS, a major source of indoor air pollution, has a high likelihood of being noxious to the health of passive smokers. In 1997, Brownson reported that passive smoking increases the risk of pulmonary cancer (Brownson RC, Eriksen MP et al., 1997). An increased incidence of chronic coughs, chronic wheezing, and respiratory infections are also observed in children exposed to parental smoking Zmirou D, Blatier JF et al., 199). Nicotine, carboxyhemogloblin(sillett et al.,1978; Pojer et al., 1984), thiocyanate (Butts et al., 1974) and carbonmonoxide(ashton et al., 1981) are used as biomarkers to form a valid Contact author dshin5@yumc.yonsei.ac.kr 526

2 quantitative estimate of the exposure to ETS in direct and passive smokers. Unlike direct smoking, it is very hard to evaluate a passive smoker s individual exposure dose, thus a method of objective evaluation is required. It is thought that the most ideal way is to analyze cotinine, a biomarker of ETS exposure, which has a long biological half-life in the blood, saliva and urine. It exists in higher concentrations in a fluid form than other markers and remains chemically stable (Etzel, 199; Stellman, 1998; Dwaine and Jiang, 1986). In this study, we derived the relationship between indoor nicotine levels in homes and passive smokers' urinary CCR levels. METHODS From Sept. to Nov. 1999, we placed personal air samplers at the height of 1.5m in the living rooms of 57 housewives. To these, we connected a filter cassette fitted with a glass fiber filter coated with a 4% sodium bisulfate solution. From these, we extracted the nicotine from the air using a flow rate of 1.5L/min for 2 24hours. We also collected their urine samples in sterilized bottles held at -7 until analysis. The Jaffe reaction method was used to measure the creatinine level in each urine specimen. We put the ETS filter in a centrifuge tube with distilled water and ethanol for voltex mixing. To produce a free base, 1N NaOH was added, and heptane was used for elution and the sample was subsequently analyzed using GC/MS. Dichloromethane was used for the liquid-liquid extraction of the subject's urine sample, and a reversed phase column was used for analysis using HPLC/UV. We also surveyed the smoking habits of their families, and their house structure. For the spirometric test, nearly all the housewives underwent an FEV 1 test using a spectrometer. To evaluate the nicotine levels in the indoor air and the passive smokers' CCR levels according to smoking habits, a Kruskal-Wallis one-way test and Wilcoxon rank sum test were used since the data didn't follow a gaussian distribution. We used SAS version 8.1 to calculate the nonparametric spearman correlation coefficient and to do a simple regression analysis. RESULTS Analysis of nicotine and cotinine In this study, we were not able to measure the total amount of nicotine in the filters or cotinine in the urine samples. To determine what percentage of each we were able to measure, we measured the recovery of a known amount of nicotine in a glass fiber filter coated with 4% sodium bisulfate; in this case, we were able to measure % of the total amount in the filter. In the case of cotinine, we used a urine sample which hadn't been exposed to ETS and dosed it with a known amount of cotinine; we were able to measure % of the total amount. The concentration of nicotine in indoor air In these homes, the concentration of indoor nicotine was measured at ND /. Classifying the families into smoking families and non-smoking families, the results indicated that the nicotine was of a much higher concentration in smoking families than in non-smoking families with a statistically significant difference(p<.1). In the families whose members smoked in the living room (or rooms), the nicotine level was much higher than for the families whose members smoked only on the veranda or outdoors(p<.1). The number of cigarettes consumed daily in the home was also significantly associated with a high concentration of nicotine.(p<.5). By classification of house type, we found that the 527

3 nicotine level in tenement houses was higher than that of detached houses and apartments. (Table 1). The concentration of urinary cotinine for passive smokers We compared the CCR in passive smokers from smoking families with that of the subjects from non-smoking families; there was no great difference between the two cases. We then divided the families into three groups: families with indoor smokers, families with outdoor smokers who smoke only on the veranda or outdoors, and non-smoking families. Measuring the CCR in passive smokers for each case, we found that the smoking location was a significant variable (p<.1). The CCR of passive smokers was significantly associated with the number of cigarettes consumed daily in the home (p<.5). The house type, however didn't affect passive smokers' CCR. The relationship between nicotine levels indoor and the passive smokers CCR The average age of all the subjects was about 47(28 69), and there was no relationship between age and CCR level. The Spearmen correlation coefficient of.85(p<.1) meant that the relation between indoor air nicotine levels in the homes of smokers and the passive smokers' CCR was statistically significant. Using this data and a simple regression analysis, it yielded the significant regression equation of Y(urinary cotinine)=21.3 X(nicotine in indoor) Table 1. Concentration of nicotine indoors and urinary cotinine concentrations for passive smokers Class Variables Nicotine (/m 3 ) CCR (/g crea) Median p-value Median p- value Indoor smoker Smoking habits Environment characteristics No (n=31) Yes (n=26) Smoking location No smoking (n=31) Veranda or outdoor (n=18) Indoor at home (n=8) Number of daily cigarettes at home 1 ~ 5 (n=16) or more (n=1) House type A detached house (n=2) Te nement house (n=11) Apartment (n=24) n : the number of subjects Clinical test We gave a FEV 1 test to 48 subjects. The average level was % (63~1%). DISCUSSION There have been studies about the effect of ETS expos ure on passive smokers using several different approaches. Cotinine, which is generally used as a biomarker, is produced by one of the cigarettes' special substances, nicotine, and is absorbed in the body and metabolized. 528

4 Because the metabolism of cotinine differs from individual to individual and there is no standard analysis method, it is reported that cotinine has limited use as a biomarker (Benowitz, 1991). Not withstanding this, it is used as a biomarker for direct and passive smokers because of its consistently high sensitivity and specificity(baranowski, 1998; Dimch- Ward, 1997). Further, it is reported that it is the best means available to measure ETS.(Etzel et al, 199). In this study, to allow for variance between individuals, nicotine-methyl-d 3 and 2- phenylimidazole were used as internal standard references for measuring the accuracy of the analysis of nicotine and urinary cotinine. Creatinine, a urine specific standard reference, was measured and used for individual urinary correction. This study has limited usefulness as a cross sectional study, however the significant correlation between nicotine indoors and urinary cotinine, investigated in this study, could be a basis for presuming the harmfulness of passive smoking through ETS exposure. Future parallel studies concerning several elements affecting the indoor nicotine concentrations and its effect, such as daily ventilation and studies related to the health symptoms of passive smokers, are expected to be needed r 2 = r 2 = (a) Total subjects (n=57) (b) ETS exposure at home (n=26) r 2 = (c) ETS non-exposure at home (n=31) Figure 1. Correlation between indoor nicotine and CCR of passive smokers. (a) Total subjects, (b) ETS exposure at home, (c) ETS non-exposure at home 529

5 CONCLUSIONS AND IMPLICATIONS In this study, urinary cotinine was chosen as a biomarker for passive smokers exposed to ETS, and a quantitative analysis was done. After analyzing the correlation between this and nicotine concentrations in indoor air, a statistically significant simple regression equation was produced. From the results of FEV1 tests, we could not establish whether or not ETS exposure had an influence on the subjects. However, through measur ements of urinary cotinine in passive smokers, it is reasonable to assume that ETS exposure does have an effect. In addition, it was found that the smoking location and the number of cigarettes consumed daily at home affected nicotine levels in indoor air and affected passive smokers CCR levels. Therefore, passive smoking, even in children, should be avoided. ACKNOWLEDGEMENTS This study was supported by a grant (HMP-99-M-9-6) of the 1999 Good Health R&D Project. Ministry of Health & Welfare, Korea. REFERENCES Ashton H, Stepney R, Thompson JW Should intake of carbon monoxide be used as a guide to intake of other smoke constituents? Br Med J. Vol.282, pp Baranowski J, Pochopieri G et al Determination of nicotine, cotinine and caffeine in meconium using high-performance liquid chromatography. Journal of chromatography B. Vol.49, pp Benowitz NL, Jacob P, Denaro C Stable isotope studies of nicotine kinetics and bioavailability. Clin Pharmacol Ther. Vol.49, pp Brownson RC, Eriksen MP, Davis RM, Warner KE Environmental tobacco smoke: health effect and policies to reduce exposure. Annu. Rev. Public Health. Vol. 18, pp Butts WC, Kuehneman M, Widdowson GM automated method for determining serum thiocyanate to distinguish smokers from nonsmokers. Clin Chem. Vol.2, pp Etzel RA A review of the use of saliva cotinine as a marker of tobacco smoke exposure. In Benowitz NL, Jacob P, Denaro C. Stable isotope studies of nicotine kinetics and bioavailability. Clin Pharmacol Ther Vol.49, pp Hew Smoking and Health- A report of the sugeon general. Department of health, education and welfare. Pub. No. (PHS) Vol.79 (66). John HB Tobacco; Wilson TD(eds); Harrison s principles of internal medicine. New York: MC Graw-Hill, pp Pojer R, Whitfield JB, Poulos V et al Carboxyhemoglobin, cotinine, and thiocyanate assay compared for distinguishing smokers from non-smokers. Clin Chem. Vol.3, pp Sillett RW, Wilson MB, Malcolm RE, Ball KP deception among smokers. Br Med J. Vol.2, pp Stephanie M, Pendergrass, Larry B, Jacox. 2. Development of versatile method for the detection of nicotine in air. AIHAJ. Vol.61, pp Zmirou D, Blatier JF, Andre E, Ferley JP, Balducci F, Rossum F, Delormas P Tabagisme passif et risque respiratoire. Une synthese quantitative de la literature. Rev Mal Resp. Vol.7, pp