The contribution of dietary nicotine and dietary cotinine to salivary cotinine levels as a nicotine biomarker
Introduction
Nicotine is reported to be found in a variety of plants. Leete (1983) reported the presence of nicotine in various plants belonging to 12 families and 24 genera, including the large family of the nightshades (Solanaceae). Besides the tobacco plant (Nicotiana tobaccum), which is cultivated because of its high nicotine content, some vegetables that are common parts of our diet belong to the nightshade family — including tomatoes (Lycopersicon esculentum), potatoes (Solanum tuberosum), eggplants (aubergines, Solanum melonga), and peppers (Capsicum annum and Capsicum frutescens). There has been controversy in the literature concerning the relative amount of nicotine intake from dietary sources in relation to the exposure to environmental tobacco smoke, or even active smoking of a small number of cigarettes (Castro and Monji, 1986, Davis et al., 1991, Domino et al., 1993, Idle, 1990, Jarvis, 1994, Repace, 1994, Sheen, 1988). Osler (1998) recently suggested that non-smokers generally consume a healthier diet than smokers and consequently have a larger consumption of raw and cooked vegetables and teas. If nicotine is present in these foods and beverages, non-smokers may have a larger intake of dietary nicotine.
Recently, Siegmund, Leitner and Pfannhauser (1999b) developed and validated an analytical method based on gas chromatography–mass spectrometry for the determination of nicotine in food substances. Siegmund, Leitner, and Pfannhauser (1999a) have reported detailed results of the determination of nicotine in a variety of vegetables. The matrices analysed include raw and cooked vegetables from the nightshade family that are frequently consumed (i.e. tomatoes, potatoes, and eggplants) and processed foods made from these vegetables such as tomato sauce, ketchup and French fries (Pommes frites). Green peppers and teas were included because nicotine has been reported in these materials with conflicting conclusions (Chappell and Gratt, 1996, Davis et al., 1991, Domino et al., 1993, Idle, 1990, Sheen, 1988). Siegmund et al. (1999a) reported that the nicotine concentrations for various vegetables ranged from 2–7 μg kg−1 for fresh (“as is”) vegetables. The results provided a set of means and standard deviations of the nicotine contents for these food substances. This information was combined with food consumption data from 13 European countries and the USA to perform a Monte Carlo Simulation (Decisioneering, 1996) of the distribution of dietary nicotine intake. The mean and the 95th percentile of dietary nicotine intake based on these data are 1.4 μg day−1 and 2.25 μg day−1, respectively.
In the mammalian system, nicotine is metabolised, whereas the most important metabolic reaction of nicotine is the formation of cotinine by oxidation (Gorrod & Schepers, 1999; Fig. 1). As a consequence of this metabolic pathway, cotinine is generally used as a biomarker of exposure to tobacco smoke and it is typically measured in blood, plasma, saliva or urine (Benowitz, 1996, Lee, 1999). Based on the fact that nicotine was also found in frequently-consumed vegetables (Siegmund et al., 1999a), it was thought that the dietary intake of nicotine would contribute significantly to cotinine levels in biological fluids. Furthermore, a decrease of nicotine concentration was observed during the ripening process of tomatoes, indicating a possible degradation or oxidation of nicotine. Based on these results, investigations were continued to determine whether cotinine is formed by oxidation of nicotine in vegetables. Should this be the case, dietary cotinine would be of potential impact on salivary cotinine levels, due to its direct supply via foods.
The literature contains a number of papers dealing with the determination of cotinine in matrices such as blood, plasma, urine or saliva using gas chromatography or liquid chromatography and a variety of detection systems (Baranowski et al., 1998, Bentley et al., 1999, Bernert et al., 1997, Crooks and Byrd, 1999, Jacob and Byrd, 1999, Moore et al., 1993, Skarping et al., 1988). Nevertheless, from these citations, it is not apparent which of these methods would be most suitable for the determination of cotinine in foods. In addition, no references were found on the determination of cotinine in any food matrix. In this paper we describe methods for the determination of cotinine in various food matrices using liquid chromatographic as well as gas chromatographic techniques, as well as the results from these investigations.
To estimate the influence of dietary factors on cotinine levels in human biological fluids, we used the results of Siegmund et al. (1999a) to calculate a distribution of salivary cotinine concentration that might be expected from dietary intake of nicotine. Furthermore, edible nightshades were analysed for their cotinine concentrations, to determine whether there is a direct cotinine contribution to salivary cotinine via diet.
Section snippets
Chemicals
Dichloromethane, ethyl acetate, sodium sulfate granular (all minimum 99.5% purity) were purchased from Promochem, Wesel, Germany. Butyl acetate (HPLC quality, 99.7%), triethylamine (purum, 99%), cotinine (purum, 98%) and deuterated cotinine ([2H3]methylcotinine, purum 98%; used as internal standard) were purchased from Sigma-Aldrich, Steinheim Germany. Sodium hydroxide (analytical-reagent grade), potassium hydroxide (analytical-reagent grade), hydrochloric acid (analytical-reagent grade),
Dietary cotinine
For the determination of nicotine in food matrices, GC–MS provided a very selective and sensitive method (Siegmund et al., 1999b). Cotinine is a more polar compound than nicotine. In the course of these investigations it was found that cotinine could not be determined using GC–MS with stable performance of the system. The decrease in performance manifested itself as strongly tailing peaks and a decrease of sensitivity, which was mainly dependent on the condition of the injection system and the
Conclusions
In contrast to nicotine that was found in fresh vegetables at the μg kg−1 range, its main human metabolite, cotinine, could not be detected in any edible nightshades or products thereof in concentrations higher than 0.1 μg kg−1. Consequently, the estimation of the influence of diet on the nicotine and/or cotinine level in biological fluids such as saliva is based only on dietary nicotine.
The calculations presented here show that dietary intake of nicotine is not significant in comparison with
Acknowledgements
This work was supported in part by Philip Morris Incorporated. The authors acknowledge the assistance of Helmut Pelzman, of the Landesversuchsanlage für Spezialkulturen, Wies, Austria, for providing the tomato samples.
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