A review of the use of saliva cotinine as a marker of tobacco smoke exposure

Cotinine, the major metabolite of nicotine, is a useful marker of exposure to tobacco smoke. It can be measured in plasma, urine, or saliva. However, distinguishing between active and passive smoking on the basis of a cotinine measurement may be difficult. In order to evaluate the relationship between saliva cotinine concentration and self-reported tobacco smoke exposure in both active and passive smokers, an English-language literature search using MEDLINE was conducted (1973-1989), and the bibliographies of identified articles were reviewed. Of 43 originally identified articles, only 22 met the criteria for inclusion. Specific information regarding population studied, reported tobacco smoke exposure, method of measurement, and cotinine concentrations was assessed. Passive smokers usually have cotinine concentrations in saliva below 5 ng/ml, but heavy passive exposure can result in levels greater than or equal to 10 ng/ml. Levels between 10 and 100 ng/ml may result from infrequent active smoking or regular active smoking with low nicotine intake. Levels greater than 100 ng/ml are probably the result of regular active smoking. Four categorizations of tobacco smoke exposure are suggested on the basis of saliva cotinine concentrations.     

Minor tobacco alkaloids as biomarkers for tobacco use: comparison of users of cigarettes, smokeless tobacco, cigars, and pipes

OBJECTIVES: This study (1) determined levels of various tobacco alkaloids in commercial tobacco products. (2) determined urinary concentrations, urinary excretion, and half-lives of the alkaloids in humans; and (3) examined the possibility that urine concentrations of nicotine-related alkaloids can be used as biomarkers of tobacco use. METHODS: Nicotine intake from various tobacco products was determined through pharmacokinetic techniques. Correlations of nicotine intake with urinary excretion and concentrations of anabasine, anatabine, nornicotine, nicotine, and cotinine were examined. By using urinary excretion data, elimination half-lives of the alkaloids were calculated. RESULTS: Alkaloid levels in commercial tobacco products, in milligrams per gram, were as follows: nicotine, 6.5 to 17.5; nornicotine, 0.14 to 0.66; anabasine, 0.008 to 0.030; and anatabine, 0.065 to 0.27. Measurable concentrations of all alkaloids were excreted in the urine of most subjects smoking cigarettes, cigars, and pipes and using smokeless tobacco. Correlations between nicotine intake and alkaloid concentrations were good to excellent. CONCLUSIONS: Anabasine and anatabine, which are present in tobacco but not in nicotine medications, can be used to assess tobacco use in persons undergoing nicotine replacement therapy.     

Simultaneous measurement of urinary total nicotine and cotinine as biomarkers of active and passive smoking among Japanese individuals

Objectives

Measuring urinary cotinine is a popular and established method of biologically monitoring exposure to tobacco smoke. However, the lower detection limit of cotinine often impedes the evaluation of passive (second-hand) smoking and this, together with unconverted nicotine, does not reflect actual levels of exposure. Furthermore, a portion of the Japanese population might have decreased ability to metabolize nicotine. The present study was therefore carried out to validate the simultaneous analysis of total concentrations of free nicotine and cotinine and their glucuronides to determine actual levels of voluntary and involuntary exposure to cigarette smoke.

Methods

Urine samples from 118 Japanese smokers and 117 non-smokers were analyzed using gas chromatography–mass spectrometry. Voluntary and involuntary smoking status was self-reported and workplace smoking restrictions were objectively evaluated.

Results

The integrated sum of all concentrations showed 2.2- and 2.4-fold higher total levels (free and glucuronide) of nicotine and cotinine relative to the free levels. Median (quartiles) of total nicotine and cotinine were 1635 (2222) and 3948 (3512) ng/mL in smokers, and 3.5 (5.3) and 2.8 (4.2) ng/mL in non-smokers. Concentrations of urinary nicotine were higher than those of cotinine in 21 % of smokers and in 54 % of non-smokers. Nicotine and cotinine levels were significantly associated with a smoking habit, as well as being significantly associated with the workplace and home environments of non-smokers.

Conclusions

The present method can monitor voluntary and involuntary exposure to tobacco smoke. Measuring total urinary nicotine levels might be useful for analyzing exposure to cigarette smoke among non-smokers.     

Relationship between environmental tobacco smoke and urinary cotinine levels in passive smokers at their residence

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 >19 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 significant 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.     

Application of in vitro biotransformation data and pharmacokinetic modeling to risk assessment

The adverse biological effects of toxic substances are dependent upon the exposure concentration and the duration of exposure. Pharmacokinetic models can quantitatively relate the external concentration of a toxicant in the environment to the internal dose of the toxicant in the target tissues of an exposed organism. The exposure concentration of a toxic substance is usually not the same as the concentration of the active form of the toxicant that reaches the target tissues following absorption, distribution, and biotransformation of the parent toxicant. Biotransformation modulates the biological activity of chemicals through bioactivation and detoxication pathways. Many toxicants require biotransformation to exert their adverse biological effects. Considerable species differences in biotransformation and other pharmacokinetic processes can make extrapolation of toxicity data from laboratory animals to humans problematic. Additionally, interindividual differences in biotransformation among human populations with diverse genetics and lifestyles can lead to considerable variability in the bioactivation of toxic chemicals. Compartmental pharmacokinetic models of animals and humans are needed to understand the quantitative relationships between chemical exposure and target tissue dose as well as animal to human differences and interindividual differences in human populations. The data-based compartmental pharmacokinetic models widely used in clinical pharmacology have little utility for human health risk assessment because they cannot extrapolate across dose route or species. Physiologically based pharmacokinetic (PBPK) models allow such extrapolations because they are based on anatomy, physiology, and biochemistry. In PBPK models, the compartments represent organs or groups of organs and the flows between compartments are actual blood flows. The concentration of a toxicant in a target tissue is a function of the solubility of the toxicant in blood and tissues (partition coefficients), blood flow into the tissue, metabolism of the toxicant in the tissue, and blood flow out of the tissue. The appropriate degree of biochemical detail can be added to the PBPK models as needed. Comparison of model simulations with experimental data provides a means of hypothesis testing and model refinement. In vitro biotransformation data from studies with isolated liver cells or subcellular fractions from animals or humans can be extrapolated to the intact organism based upon protein content or cell number. In vitro biotransformation studies with human liver preparations can provide quantitative data on human interindividual differences in chemical bioactivation. These in vitro data must be integrated into physiological models to understand the true impact of interindividual differences in chemical biotransformation on the target organ bioactivation of chemical contaminants in air and drinking water.     

Correlating atmospheric and biological markers in studies of secondhand tobacco smoke exposure and dose in children and adults

OBJECTIVE: We sought to directly compare secondhand smoke (SHS) atmospheric markers to each other and to SHS dosimetric biomarkers, permitting intercomparison of clinical and atmospheric studies. METHODS: We used atmospheric and pharmacokinetic (PK) models for the quantitative estimation of SHS exposure and dose for infants, children, and adults, based on building smoker density and air exchange rate, and from exposure duration, default PK parameters, and respiration rates. RESULTS: We estimate the SHS serum cotinine doses for the typical and most-exposed individuals in the U.S. population; predictions compare well to measurements on a national probability sample. Using default respiration rates, we estimate serum cotinine dose from SHS nicotine exposure for 40 adults exposed to SHS in an environmental chamber; predictions agreed with observations. We correlate urine cotinine and hair nicotine levels for 127 infants exposed to parental smoking, and estimate corresponding atmospheric nicotine exposure via PK modeling. CONCLUSIONS: Our "Rosetta Stone" Equations allow the SHS atmospheric markers, respirable particles, nicotine, and carbon monoxide, to be related to the SHS biomarkers, cotinine in blood, urine, and saliva and nicotine in hair, permitting intercomparison of clinical and atmospheric studies of SHS for the first time.     

Personal exposure to environmental tobacco smoke: salivary cotinine, airborne nicotine, and nonsmoker misclassification.

A large study was conducted to assess exposure to environmental tobacco smoke (ETS) in a geographically dispersed study population using personal breathing zone air sampling and salivary cotinine levels. Approximately 100 self-reported nonsmoking subjects in each of 16 metropolitan areas were recruited for this investigation. Cumulative distributions of salivary cotinine levels for subjects in smoking and nonsmoking homes and workplaces exhibited a general trend of decreasing salivary cotinine levels with decreasing time spent in smoking environments. Median salivary cotinine levels for the four experimental cells in the study (product of smoking and nonsmoking home and workplaces) were comparable to those reported for a large national study of serum levels of cotinine (Third National Health and Nutrition Examination Survey, NHANES III), when the latter was corrected for expected differences between serum and saliva concentrations. However, the most highly exposed group in this study had a median salivary cotinine concentration approximately a factor of 2 greater than that of the comparable group in the NHANES III study. Misclassification rates, both simple (for self-reported nonsmokers) and complex (self-reported lifetime never smokers), were near the median of those reported for other studies. Estimated misclassification rates for self-reported lifetime never-smoking females are sufficiently high (2.95% using a discrimination level of 106 ng/ml) that, if used in the Environmental Protection Agency (EPA) risk assessment related to ETS and lung cancer, would place the lower 90% confidence interval (CI) for relative risk at nearly 1.00, i.e., no statistically significant increased risk. For the 263 most highly exposed subjects in the study whose self-reported nonsmoking status was accurate, the correlation between airborne exposure to nicotine and average salivary cotinine is so small, on an individual basis, that it makes the relationship useless for estimating exposure on a quantitative basis. When subjects are grouped according to likely categories of nicotine exposure, correlation between group median airborne nicotine exposure and salivary cotinine level increases dramatically. The comparison improves for the most highly exposed subjects, suggesting that such quantitative comparisons are useful for only those subjects who are exposed to the higher levels of ETS. However, airborne nicotine exposure for most of the subjects does not account for estimated systemic levels of nicotine, based on salivary cotinine levels.     

Stages of change, smoking characteristics, and cotinine concentrations in smokers: setting priorities for smoking cessation

ObjectiveWe assessed whether the salivary cotinine content of daily smokers varied with the readiness to quit and smoking characteristics.MethodsThis cross-sectional study was conducted in Barcelona, Spain (n = 1245) in 2004–2005. We administered a questionnaire to assess smoking behaviour and collected saliva to determine the cotinine content. We determined the distribution of 278 adult daily smokers across different stages of change and categorised them by individual and smoking characteristics. We used medians and interquartile ranges (IQR) to relate cotinine concentrations to different stages of change, tobacco consumption, and nicotine dependence based on the Fagerström Test for Nicotine Dependence (FTND).ResultsAround 68%, 22%, and 11% of smokers were in precontemplation, contemplation, and preparation stages, respectively. A mean of 17.0 cigarettes was smoked daily, with no differences among stages of change. The median cotinine concentration was 151.3 ng/ml (IQR: 83.2–227.8 ng/ml), with no differences among stages of change. The cigarette consumption scores, FTND, and time to first cigarette of the day were positively associated with cotinine concentration.ConclusionsThe cotinine concentration was similar among the stages of change, but varied within each stage according to the number of cigarettes smoked, time to first cigarette of the day, and nicotine dependence.     

Tea drinking, passive smoking, smoking deception and serum cotinine in the Scottish heart health study

Following a recent claim that the use of cotinine in body fluids, to assess passive smoking and smoking “deception”, was confounded by metabolic individuality, and by non-tobacco sources of dietary nicotine, particularly tea, data were examined from a large cross-sectional survey in a tea-drinking population. In 3383 men and women aged 44–59 years from the Scottish Heart Health Study, defined as non-smokers, both by self-report and by low thiocyanate and expired air carbon monoxide levels, serum cotinine showed minimal association with self-reported daily average tea consumption. However, there was a strong correlation between degree of self-reported passive tobacco smoke exposure and median serum cotinine level. In the same survey, serum cotinine in 4144 self-reported non-smokers and in 3326 smokers showed entirely different distributions, but the same range, suggesting heavy nicotine intake in some “non-smokers”. These analyses confirm that cotinine levels in true non-smokers reflect far more the nicotine in inhaled ambient tobacco smoke than they do nicotine in tea. Some smoking “deceivers” have the same degree of exposure to nicotine as heavy smokers. Despite individual variability, the claim of confounding is poorly supported, and cotinine is confirmed as an indicator both of passive smoking and of smoking deception.     

Cotinine in an ultrafiltrate of saliva.

BACKGROUND. We have developed a device for the simplified collection of a prepurified sample of saliva in the mouth. METHOD. The device is based on the principle of an osmotic pump and accumulated about 1.2 ml of an ultrafiltrate of saliva within 8 min. We have investigated the ultrafiltrate for its utility as a biological medium in the evaluation of cigarette smoking status. RESULTS. (a) In 58 matched samples from 13 subjects, the correlation coefficient for the cotinine concentration in the saliva and the ultrafiltrate was 0.95; (b) in matched plasma and ultrafiltrate samples from 27 smokers, the correlation coefficient for the cotinine concentrations was 0.96 with plasma containing 1.2 times the ultrafiltrate mean; (c) in a nonsmoker, elevated cotinine levels could be detected in the ultrafiltrate more than 24 hr after smoking 2 cigarettes, and the pattern of rise and decrease reflected that in whole saliva; and (d) in a habitual smoker; the mean cotinine concentration in the ultrafiltrate was 157 ng/ml (SD +/- 25.7 ng/ml) during a period of smoking 15 cigarettes per day and dropped to a mean of 47 ng/ml (SD +/- 10.5) when smoking was reduced to 5 cigarettes per day; after cessation of smoking, detectable concentrations of cotinine persisted for up to 5 days. CONCLUSION. The device facilitated the aesthetic, noninvasive collection of a biological sample useful in the validation of smoking status.     

Maternal tobacco smoke exposure and persistent pulmonary hypertension of the newborn.

We propose that in utero exposure to tobacco smoke products places a newborn at risk for persistent pulmonary hypertension of the newborn (PPHN). To test this hypothesis, infants with PPHN were identified. Healthy newborns of similar ethnicity were identified as a comparison group. Cord blood cotinine concentrations and maternal questionnaires were obtained. The number of women exposed to tobacco smoke in each group ascertained by questionnaire was borderline significantly different (38.7% vs. 20.5%; p = 0.080). However, more PPHN infants had detectable cotinine in their cord blood (64.5% vs. 28.2%; p = 0.002), and the median cotinine concentrations were significantly higher (5.2 ng/ml vs. 2 ng/ml; p = 0.051) than the comparison infants. Among infants delivered to nonsmoking women, more PPHN infants had detectable cotinine (50% vs. 19%; p = 0.015), and the cotinine concentrations were higher (3.5 ng/ml vs. 1.65 ng/ml; p = 0.022) than the comparison group. We conclude that active and passive smoking during pregnancy is a risk factor for PPHN. Therefore, we recommend that pregnant women cease smoking and avoid environmental tobacco smoke. Key words. cotinine, newborns, passive, persistent pulmonary hypertension, smoking, tobacco smoke pollution.     

A micromethod for determination of nicotine and cotinine in blood and urine by gas chromatography. Results obtained from various smokers]

The determination of nicotine and cotinine in plasma and urines of smokers is carried out by gas-chromatography with an Ucon-Polar 50 HB 2000 alkalinized column and a specific N detector, using diphenylamine as internal standard. The technique for extraction and chromatographic analysis is developed. The limit values for a correct determination are : 0,2 ng for nicotine and 1 ng for cotinine. The results show that the transit of nicotine and cotinine into the blood is very fast. They confirm the rapidity of the urinary excretion of nicotine, the slowness of that of cotinine, and the relation between the eliminated amounts and the urinary flow.     

Behavioral factors predicting serum cotinine concentrations of male smokers in a Japanese community.

Cigarette-derived toxic substances are inhaled along with the nicotine that is absorbed to satisfy the smoker's physical demand. Therefore, serum cotinine, a metabolite of nicotine, may be considered to be an indirect marker of absorption of the other toxic substances from smoking. However, few studies have examined factors related to serum cotinine concentrations in natural settings. The authors, therefore, have studied relations among patterns of smoking behavior and serum cotinine concentrations of community residents. Subjects were 60 smoking men living in the town of Yamasaki, Hyogo Prefecture, Japan. Number of daily cigarettes, depth of inhalation, hours from the last cigarette smoked and the total nicotine tolerance score were significantly, while neither nicotine-yields nor butt length of a discarded cigarette was correlated with serum cotinine concentrations. Multiple regression analysis confirmed that depth of inhalation and hours from the last cigarette smoked were independently significant. This finding suggests that attention only to self-reported daily number of cigarettes smoked may not be sufficient to detect smokers who are actually at high risk. Healthcare workers should also pay attention to smokers' patterns of smoking, particularly depth of inhalation.     

Racial differences in serum cotinine levels among smokers in the Coronary Artery Risk Development in (Young) Adults study.

Cotinine was measured in the serum of nearly all 5,115 18-30 year old, Black and White, men and women participating in the Coronary Artery Risk Development in (Young) Adults Study, 30 percent of whom reported current cigarette smoking. Ninety-five percent of the reported smokers had serum cotinine levels indicative of smoking (greater than 13 ng/ml). The median cotinine level was higher in Black than White smokers (221 ng/ml versus 170 ng/ml; 95 percent CI for difference: 34, 65) in spite of the fact that estimated daily nicotine exposure and serum thiocyanate were higher in Whites. The difference persisted after controlling for number of cigarettes, nicotine content, frequency of inhalation, weekly sidestream smoke exposure, age, gender, and education. A reporting bias and nicotine intake were ruled out as explanations for the racial difference suggesting that the metabolism of nicotine or the excretion of cotinine may differ by race. Racial differences in cotinine levels may provide clues to the reasons for the observed lower cessation rates and higher rates of some smoking-related cancers in Blacks.     

Development of an anti-cotinine vaccine to potentiate nicotine-based smoking cessation strategies

Nicotine replacement therapies (NRT) have limited success in smoking cessation. The efficacy of nicotine may be compromised by its main metabolite, cotinine. An anti-cotinine vaccine to remove this antagonism could enhance the efficacy of NRT. We show that cotinine is a weak nicotinic agonist and decreases responses to nicotine, consistent with antagonism through receptor desensitisation. trans-4-Thiol cotinine was coupled to tetanus toxoid, and rats immunised repeatedly. Vaccination raised antibodies specific for cotinine that do not recognise other metabolites or nicotine. Increased serum cotinine concentrations following nicotine administration indicate sequestration of cotinine by antibodies, encouraging further evaluation of this vaccine in behavioural models of nicotine addiction and relapse.     

In silico toxicology: simulating interaction thresholds for human exposure to mixtures of trichloroethylene,tetrachloroethylene, and 1,1,1-trichloroethane

In this study, we integrated our understanding of biochemistry, physiology, and metabolism of three commonly used organic solvents with computer simulation to present a new approach that we call "in silico" toxicology. Thus, we developed an interactive physiologically based pharmacokinetic (PBPK) model to predict the individual kinetics of trichloroethylene (TCE), perchloroethylene (PERC), and methylchloroform (MC) in humans exposed to differently constituted chemical mixtures of the three solvents. Model structure and parameterization originate from the literature. We calibrated the single-compound PBPK models using published data and described metabolic interactions within the chemical mixture using kinetic constants estimated in rats. The mixture model was used to explore the general pharmacokinetic profile of two common biomarkers of exposure, peak TCE blood levels and total amount of TCE metabolites generated, in rats and humans. Assuming that a 10% change in the biomarkers corresponds to a significant health effect, we calculated interaction thresholds for binary and ternary mixtures of TCE, PERC, and MC. Increases in the TCE blood levels led to higher availability of the parent compound for glutathione conjugation, a metabolic pathway associated with kidney toxicity/carcinogenicity. The simulated change in production rates of toxic conjugative metabolites exceeded 17% for a corresponding 10% increase in TCE blood concentration, indicating a nonlinear risk increase due to combined exposures to TCE. Evaluation of metabolic interactions and their thresholds illustrates a unique application of PBPK modeling in risk assessment of occupational exposures to chemical mixtures.     

Breast feeding and smoking hygiene: major influences on cotinine in urine of smokers'' infants.

The determinants of urine cotinine levels were studied in a group of 101 infants aged 3 months, including 79 infants whose mothers were current smokers. At a pre-arranged home visit the infants' mothers completed an interviewer-administered questionnaire, and samples of maternal urine and breast milk and infants' urine were collected. Cotinine and nicotine levels were determined by gas-liquid chromatography. Infant urine cotinine levels ranged from 0 to 140 micrograms/l (0-1120 ng cotinine/mg creatinine). A linear dose response relation between mother's smoking rate and infant urine cotinine level was observed among breast-fed infants (r = 0.79, p less than 0.001). The relation was weaker among infants fed by both breast and bottle (r = 0.56, p = 0.01) and was not apparent among bottle-fed infants (r = 0.15, p = 0.16). In addition to mode of feeding and mother's smoking rate, mother's smoking "hygiene" (assessed by the reported frequency of smoking while feeding and with infant in same room) was independently associated with infant urine cotinine level. Father's smoking pattern and exposure to smoke outside the household did not relate significantly to infant cotinine levels. We conclude that when mothers smoke, breast feeding is the principal determinant of cotinine in infants' urine. It is likely that most of this cotinine comes from cotinine in mothers' breast milk, but further research is needed to establish how much nicotine is ingested by breast-fed infants of mothers who smoke, and to investigate possible health effects.     

Importance of exposure to gaseous and particulate phase components of tobacco smoke in active and passive smokers

Summary The uptake of tobacco smoke constituents from gaseous and particulate phases of mainstream smoke (MS), inhaled by smokers, and of environmental tobacco smoke (ETS), breathed in by non-smokers, was investigated in two experimental studies. Tobacco smoke uptake was quantified by measuring carboxyhemoglobin (COHb), nicotine and cotinine in plasma and urine and the data obtained were correlated with urinary excretion of thioethers and of mutagenic activity. An increase in all biochemical parameters was observed in smokers inhaling the complete MS of 24 cigarettes during 8 h, whereas only an increase in COHb and, to a minor degree, in urinary thioethers was found after smoking the gas phase of MS under similar conditions. Exposure of non-smokers to the gaseous phase of ETS or to whole ETS at similar high concentrations for 8 h led to identical increases in COM, plasma nicotine and cotinine as well as urinary excretion of nicotine and thioethers which were much lower than in smokers. Urinary mutagenicity was not found to be elevated under either ETS exposure condition. As shown by our results, the biomarkers most frequently used for uptake of tobacco smoke (nicotine and cotinine) indicate on the one hand the exposure to particulate phase constituents in smoking but on the other hand the exposure to gaseous phase constituents in passive smoking. Particle exposure during passive smoking seems to be low and a biomarker which indicates ETS particle exposure is as yet not available. These findings emphasize that risk extrapolations from active smoking to passive smoking which are based on cigarette equivalents or the use of one biomarker (e.g. cotinine) might be misleading.     

Occupational exposure of non-smoking restaurant personnel to environmental tobacco smoke in Finland

BACKGROUND: Environmental tobacco smoke (ETS) exposure levels in different restaurant types in Finland were assessed before the National Tobacco Act restricting smoking in restaurants was activated. METHODS: Exposure to ETS was determined by measuring nicotine in the breathing zone of non-smoking restaurant workers and by quantification of the nicotine metabolites cotinine and 3-hydroxycotinine in the urine of these workers during one whole work week. Altogether 23 workers from 15 restaurants were included in the study. RESULTS: The geometric mean (GM) breathing-zone nicotine level was 3.9 microg/m(3) (3.7 microg/m(3) in pubs, 1.4 microg/m(3) in dining restaurants, and 10.2 microg/m(3) in nightclubs). The GM cotinine and trans-3'-hydroxycotinine level in urine were 3.3 ng/mg((creatinine)) and 15.3 ng/mg((creatinine)), respectively. The exposure to ETS of restaurant workers in dining restaurants was clearly lower than that of workers in pubs and nightclubs as indicated by all ETS-markers used in the present study. During the work week, the cotinine and 3'-hydroxycotinine levels in urine of the study subjects increased. The correlation between breathing zone nicotine and urine cotinine and hydroxycotinine was 0.66 for both compounds. Post-shift cotinine and hydroxycotinine levels were not significantly higher than the pre-shift levels. CONCLUSIONS: If 9 ng cotinine/mg((creatinine)) is considered as the level above which heavy exposure has occurred, then this level was exceeded by 14 (approximately 60%) subjects at least once during the work week. Nicotine metabolite concentrations in the urine increased during the work week in 80% of the subjects, and the increase was especially noticeable for subjects working in both pubs and nightclubs. The study indicates that measures to restrict ETS exposure in restaurants are needed.