Comprehensive metabolic study of nicotine in equine plasma and urine using liquid chromatography/high-resolution mass spectrometry for the identification of unique biomarkers for doping control.

Nicotine is classified as a stimulant, and its use is banned in horse racing and equestrian sports by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. Because nicotine is a major alkaloid of tobacco leaves, there is a potential risk that doping control samples may be contaminated by tobacco cigarettes or smoke during sample collection. In order to differentiate the genuine doping and sample contamination with tobacco leaves, it is necessary to monitor unique metabolites as biomarkers for nicotine administration and intake. However, little is known about the metabolic fate of nicotine in horses. This is the first report of comprehensive metabolism study of nicotine in horses. Using liquid chromatography/electrospray ionization high-resolution mass spectrometry, we identified a total of 17 metabolites, including one novel horse-specific metabolite (i.e., 4-hydroxy-4-(3-pyridyl)-N-methylbutanamide), in post-administration urine samples after nasoesophageal administration of nicotine to three thoroughbred mares; eight of these compounds were confirmed based on reference standards. Among these metabolites, N-hydroxymethylnorcotinine was the major urinary metabolite in equine, but it could only be tentatively identified by mass spectral interpretation due to the lack of reference material. In addition, we developed simultaneous quantification methods for the eight target analytes in plasma and urine, and applied them to post-administration samples to establish elimination profiles of nicotine and its metabolites. The quantification results revealed that trans-3'-hydroxycotinine could be quantified for the longest period in both plasma (72 h post-administration) and urine (96 h post-administration). Therefore, this metabolite is the most appropriate monitoring target for nicotine exposure for the purpose of doping control due to its long detection times and the availability of its reference material. Further, we identified trans-3'-hydroxycotinine as a unique biomarker allowing differentiation between nicotine administration and sample contamination with tobacco leaves.

Relations between toxicity and altered tissue distribution and urinary excretion of nicotine, cotinine, and hydroxycotinine after chronic oral administration of nicotine in rats.

Tissue distribution and urinary excretion of nicotine, cotinine, and hydroxycotinine after multiple oral administration of nicotine to rats for 4 weeks were studied. Physiological change and serum biochemical parameters were also measured to check dysfunction of organs. Significant change of glutathione S-transferase, aspartate aminotransferase, blood urea nitrogen, and physiological parameters indicated the toxicity in liver and kidney, at the dose of 5 and 10 mg/kg/day. Only the concentration and total amount of cotinine, not nicotine or hydroxycotinine, in the liver and the kidney showed a proportional dose-dependent increase and were highly correlated with toxicity. Saturation of metabolizing enzymes for nicotine was estimated by the change of urinary excreted amount ratio between nicotine and its metabolites. Metabolizing enzyme to produce cotinine from nicotine was saturated after multiple oral dosing for 4 weeks in a low dose (1 mg/kg/day), but within 1 week in the dose of 5 and 10 mg/kg/day.

Relationship of human toenail nicotine, cotinine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol to levels of these biomarkers in plasma and urine.

Recently, we developed sensitive and quantitative methods for analysis of the biomarkers of tobacco smoke exposure nicotine, cotinine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human toenails. In this study, we further evaluated the newly developed toenail biomarkers by investigating their relationship to demographic factors, reported exposure, plasma nicotine, cotinine, and trans-3'-hydroxycotinine, and urinary NNAL. Toenails of 105 smokers, mean age 38.9 years (range, 19-68), were analyzed. Fifty-five (53.4%) were male, with approximately equal numbers of Whites and African-Americans. The average number of cigarettes smoked per day was 18 (range, 5-50). There was no effect of age or gender on the toenail biomarkers. Toenail NNAL was higher in White than in African-American participants (P = 0.019). Toenail nicotine and toenail cotinine correlated significantly with cigarettes smoked per day (r = 0.24; P = 0.015 and r = 0.26; P = 0.009, respectively). Toenail nicotine correlated with plasma nicotine (r = 0.39; P < 0.001); toenail cotinine correlated with plasma cotinine (r = 0.45; P < 0.001) and plasma trans-3'-hydroxycotinine (r = 0.30; P = 0.008); and toenail NNAL correlated with urine NNAL (r = 0.53; P = 0.005). The results of this study provide essential validation data for the use of toenail biomarkers in investigations of the role of chronic tobacco smoke exposure in human cancer.

Effect of delivered dosage of cigarette smoke toxins on the levels of urinary biomarkers of exposure.

Urinary metabolites of tobacco smoke toxins are often used as biomarkers for the evaluation of active and passive exposure to cigarette smoke toxins. In a study of healthy smokers, we investigated concentrations of urinary biomarkers in relation to concentrations of selected toxins in mainstream cigarette smoke as determined by machine smoking of cigarettes in a manner that mimics an individual's smoking behavior (topography). Concentrations of nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and benzo(a)pyrene, in mainstream smoke determined under human smoking conditions, and their urinary metabolites cotinine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and 1-hydroxypyrene were established for 257 individuals who smoked low-yield (0.1-0.8 mg Federal Trade Commission nicotine/cigarette; mean, 0.66; n = 87), medium-yield (0.9-1.2 mg nicotine/cigarette; mean, 1.1; n = 109), and high-yield cigarettes (nicotine, >1.3 mg nicotine/cigarette; mean, 1.41; n = 61). Levels of urinary metabolites expressed per unit of delivered parent compounds decreased with increased smoke emissions. In smokers of low-, medium-, and high-yield cigarettes, the respective cotinine (ng/mg creatinine)-to-nicotine (mg/d) ratios were 89.4, 77.8, and 57.1 (low versus high; P = 0.06); the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (pmol/mg creatinine)-to-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (ng/d) ratios were 0.81, 0.55, and 0.57 (low versus high; P = 0.05); and the 1-hydroxypyrene (pg/mg creatinine)-to-benzo(a)pyrene (ng/d) ratios were 1.55, 1.13, and 0.97 (low versus high; P = 0.008). Similarly, means of cotinine per unit of delivered nicotine in smokers who consumed <20 cigarettes per day was 3.5-fold higher than in those who smoked >20 cigarettes per day. Likewise, a negative correlation was observed between cotinine-to-nicotine ratios and delivered doses of nicotine in subgroups of smokers who used the identical brand of cigarette, namely a filter tip-vented Marlboro (r = -0.59), which is a popular brand among Euro-Americans, and Newport (r = -0.37), a menthol-flavored cigarette without filter tip vents that is preferred by African-Americans. Thus, the intensity of the exposures significantly affects the levels of urinary biomarkers of exposure and should be taken into account in the evaluation of human exposure to cigarette smoke toxins.

Environmental tobacco smoke and risk of spontaneous abortion.

BACKGROUND: Studies of exposure to environmental tobacco smoke (ETS) and risk of spontaneous abortion are limited to a few studies of self-reported exposure, and the results have been inconsistent. The aim of this study was to investigate risk of early spontaneous abortion related to ETS and active smoking as defined by plasma cotinine levels. METHODS: We conducted a population-based case-control study in Uppsala County, Sweden, between January 1996 and December 1998. Cases were 463 women with spontaneous abortion at 6 to 12 completed weeks of gestation, and controls were 864 pregnant women matched to cases according to the week of gestation. Exposure status was defined by plasma cotinine concentrations: nonexposed, <0.1 ng/mL; ETS-exposed, 0.1-15 ng/mL; and exposed to active smoking, >15 ng/mL. Multivariable analysis was used to estimate the relative risk of spontaneous abortion associated with exposure to ETS and active smoking. RESULTS: Nineteen percent of controls and 24% of cases were classified as having been exposed to ETS. Compared with nonexposed women, risk of spontaneous abortion was increased among both ETS-exposed women (adjusted odds ratio = 1.67; 95% confidence interval = 1.17-2.38) and active smokers (2.11; 1.36-3.27). We could not show a differential effect of exposure to ETS or active smoking between normal and abnormal fetal karyotype abortions. CONCLUSIONS: Nonsmoking pregnant women exposed to ETS may be at increased risk of spontaneous abortion. Given the high prevalence of ETS exposure, the public health consequences of passive smoking regarding early fetal loss may be substantial.

A high-performance thin-layer chromatographic technique to screen cocaine in urine samples.

Qualitative identification of cocaine and its metabolites in urine samples is generally carried out by an immunoassay technique followed by a gas chromatographic/mass spectrometric confirmation of presumptive positives. Nevertheless, other chromatographic techniques such as thin-layer chromatography or gas chromatography could also be used to screen several types of drugs of abuse especially for forensic and legal purposes. In the present work, the capability of high performance thin-layer chromatography (HPTLC) to detect cocaine in urine samples and discriminate it from interfering substances (local anaesthetic, caffeine and nicotine) was studied. Twenty urine samples of drug addicts were submitted to the HPTLC method. Unaltered cocaine present in the urine samples and cocaine obtained after methylation of benzoylecgonine (main cocaine metabolite) with diazomethane were detected in all tested samples. In conclusion, the proposed HPTLC method showed to be useful to detect cocaine in biological matrices.

Nicotine and cotinine in adults' urine: The German Environmental Survey 1998.

In 1998, the German Environmental Survey (GerES III) recruited approximately 5000 adults between the ages of 18 and 69 years. The study population for these analyses consisted of 1580 smokers (34% of the total population) and 3126 nonsmokers. Nicotine and cotinine concentrations in urine were determined by HPLC methods with UV-detection and corrected for creatinine. Nicotine and cotinine concentrations differed between smokers and nonsmokers by factors of 10-100. The multiple linear regression models used for the analyses of nicotine detection in the urine of smokers explained 43.2% and 42.3% of the total volume-specific and creatinine-specific variances, respectively. Cigarette smoking was the major factor responsible for 41% of the total variance. The explained variances of the cotinine results were larger, 51.0% and 49.3% of the total variance were volume-specific and creatinine-specific, respectively. More than 20% of nonsmokers in GerES III were exposed to environmental tobacco smoke at home, at work or in other places. The logistic regression analysis approach used for the group of nonsmokers showed the greatest effects for those exposed to tobacco smoke at home (adjusted OR varied between 4 and 6). These results were seen for nicotine as well as for cotinine excretion. Exposure to tobacco smoke in the workplace doubled the risk for the detection of nicotine and cotinine in urine. When other risk factors such as age, sex, social status, community size, season of urine collection, and the consumption of food containing nicotine such as potatoes, cabbage, tea were included, the effect estimates for tobacco smoke exposure remained unchanged. A new federal bill to diminish environmental tobacco smoke (ETS) exposure in the workplace was recently passed in Germany, but protection of nonsmokers from smoking family members at home needs more attention.

A comparison of urinary biomarkers of tobacco and carcinogen exposure in smokers.

Recently, several potential harm reduction strategies, such as reduction in the number of cigarettes smoked and the use of modified cigarette products, have been discussed as possible means by which to reduce tobacco-related disease. To assess any potential reduction in harm by either of these approaches requires an accurate assessment of tobacco toxin exposure. We have recently completed a cigarette reduction study in which smokers were required to reduce the number of cigarettes smoked by 75%. This reduction took place over a 6-week period. We report here the comparison of urinary concentrations of tobacco alkaloid and tobacco carcinogen biomarkers in a subset of these same smokers during a 7-week period prior to any reduction in cigarette consumption. Urine samples were collected at four time points and analyzed for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and its glucuronide, 1-hydroxypyrene, anatabine, free nicotine, total nicotine (free plus glucuronidated), free cotinine, total cotinine (free plus glucuronidated), and total trans-3'-hydroxycotinine (free plus glucuronidated). Anatabine is a minor alkaloid that may be useful in assessing tobacco exposure in individuals using nicotine replacement therapies. Urinary anatabine levels were well correlated (P < 0.0001) with both free and total nicotine (r = 0.753 and 0.773, respectively). Anatabine levels were also correlated with free cotinine (r = 0.465; P < 0.001), total cotinine (r = 0.514; P < 0.001), and total NNAL (r = 0.633; P < 0.001). These data support the role of anatabine as a biomarker of tobacco exposure. 1-Hydroxypyrene is a biomarker of polycyclic aromatic hydrocarbon exposure, but unlike NNAL it is not tobacco specific. Whereas urinary concentrations of 1-hydroxypyrene were consistent across the four visits, the levels were not correlated with NNAL, anatabine, nicotine, or any nicotine metabolites.