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Schütte-Borkovec, Katharina (2008): Vorkommen und Toxikokinetik von Myosmin in Abhängigkeit von Rauchen und Ernährung. Dissertation, LMU München: Faculty of Veterinary Medicine
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Abstract

Occurrence and toxicokinetics of myosmine depending on smoking and nutrition Myosmine, a minor tobacco alkaloid, occurs like the major tobacco alkaloids nicotine, nor-nicotine, anabasine and anatabine in small amounts in tobacco plants and tobacco smoke. Because of its low acute toxicity and the weak affinity to nicotinic receptors, no attention was given to myosmine for a long time. However, this changed with the discovery that myosmine occurs independently of nicotine in a great variety of staple foods, vegetables, fruits, nuts and dairy products and consequently in human milk, plasma and saliva. The possible health im¬pact of myosmine was further demonstrated by its conversion into reactive intermediates with carcinogenic potential. Due to its imine structure, myosmine is readily nitrosated and peroxi-dated. Nitrosation yields the tobacco-specific nitrosamine N'-nitrosonornicotine (NNN), a human carcinogen, which produces tumours in oesophagus, oral cavity and the respiratory tract of rodents. In larger quantities, the same reactive intermediates which are formed by me-tabolic activation of NNN and another tobacco-specific nitrosamine are generated by nitro-sation and peroxidation of myosmine. In biomonitoring studies the resulting DNA and protein adducts did not show the expected correlation with smoking status. Therefore, myosmine has been postulated to be an important additional source of these adducts. In the first chapter, a detailed literature survey is given covering all aspects of occurrence, biosynthesis, toxicokinetics and -dynamics of myosmine in the context of other tobacco alkaloids and tobacco-specific nitrosamines. In the experimental part, an analytical method was developed to determine myosmine, coti-nine and nicotine by gas chromatography/mass spectrometry (GC/MS) in different matrices in order to study the occurrence of myosmine in livestock as well as in humans. First, myosmine was determined in plasma of pigs in dependence of the feeding condition. Second, human sa-liva and toenails from smokers and nonsmokers were analyzed for short and long-term expo-sure to myosmine and cotinine. The nails were also analyzed for nicotine. Finally, myosmine was determined in multiple samples of saliva of eight test persons to study the kinetics of myosmine under controlled dietary conditions. The results can be summarized as follows: • The analytical method has low detection limits for myosmine, nicotine and cotinine in toenails with 0.01, 0.02 and 0.035 ng/mg, respectively, and for myosmine and cotinine in plasma/saliva with 0.0012 and 0.05 ng/ml. The recovery is very high with 91-93% in plasma/saliva and 97–102% in toenails. The intraday precision is ≤ 8% for all analytes in toenails, whereas for plasma/saliva it is 18% with myosmine and 4% with cotinine. The analytical method has a high specificity by the use of deuterated internal standards. • In the plasma of 12 fasting pigs, myosmine was traceable with only one exception. The concentration of myosmine was 0.067 ± 0.049 ng/ml. Within 1 - 2 hours after start of fee-ding the concentration of myosmine in the plasma of 13 pigs was 0.497 ± 0.166 ng/ml, a statistically significant 7-fold difference to the fasting pigs (p < 0.0001). The swill con-tained 124 ng myosmine/g wet weight. • In toenails the concentrations of all analytes were significantly lower in 11 nonsmokers (0.021 ± 0.014 ng/mg) compared to 15 smokers (0.058 ± 0.052 ng/mg, p < 0.01). This 2.8-fold difference in myosmine concentrations between smokers and nonsmokers was clearly less than the 14-fold difference in nicotine concentrations (0.128 ± 0,008 versus 1.789 ± 0.964 ng/mg, p < 0.001). Cotinine was detectable only in toenails of smokers, 1.136 ± 0.843 ng/mg. Significant correlations exists between the concentrations of myosmine and nicotine (Spearman r = 0.67) as well as myosmine and cotinine (r = 0.63). A clearly better correlation (r = 0.83) was found between cotinine and nicotine values. • In saliva samples taken in parallel with toenails, the differences between nonsmokers and smokers were also smaller with myosmine, 0.73 ± 0.65 versus 2.54 ± 2.68 ng/ml than with cotinine, 1.85 ± 4.50 versus 83.14 ± 54.30 ng/ml. Nonetheless, concentrations of myos-mine and cotinine in saliva were highly correlated (p < 0.0001). • In eight volunteers, the kinetics of myosmine in saliva after food intake showed large indi-vidual differences. After four hours of fasting in the morning all subjects took a lunch containing about 2.7 µg of myosmine within half an hour. The basal concentrations of myosmine in saliva at lunch were between 0.05 and 0.28 ng/ml. In one subject only, a rapid rise of myosmine to 3.6 ng/ml was observed within three-quarters of an hour. In two subjects a plateau of 1.1 and 1.5 ng/ml was reached between 2½ and 4½ hours after lunch. The remaining five subjects showed only a weak rise of myosmine concentrations to a maximum of 0.42 ± 0.06 ng/ml within 1½ and 2½ hours. Fitting the data to the Bateman function, an elimination half-life of about 1.6 hours could be estimated which is roughly equivalent to the plasma half-life of nicotine in humans.