Characterization of cotinine degradation in a newly isolated Gram-negative strain Pseudomonas sp. JH-2.

Cotinine, the primary metabolite of nicotine in the human body, is an emerging pollutant in aquatic environments. It causes environmental problems and is harmful to the health of humans and other mammals; however, the mechanisms of its biodegradation have been elucidated incompletely. In this study, a novel Gram-negative strain that could degrade and utilize cotinine as a sole carbon source was isolated from municipal wastewater samples, and its cotinine degradation characteristics and kinetics were determined. Pseudomonas sp. JH-2 was able to degrade 100 mg/L (0.56 mM) of cotinine with high efficiency within 5 days at 30 ℃, pH 7.0, and 1% NaCl. Two intermediates, 6-hydroxycotinine and 6-hydroxy-3-succinoylpyridine (HSP), were identified by high-performance liquid chromatography and liquid chromatograph mass spectrometer. The draft whole genome sequence of strain JH-2 was obtained and analyzed to determine genomic structure and function. No homologs of proteins predicted in Nocardioides sp. JQ2195 and reported in nicotine degradation Pyrrolidine pathway were found in strain JH-2, suggesting new enzymes that responsible for cotinine catabolism. These findings provide meaningful insights into the biodegradation of cotinine by Gram-negative bacteria.

Serum hydroxycotinine was associated with chronic kidney disease (CKD): a cross-sectional study based on NHANES.

OBJECTIVE: Smoking has been suggested as a modifiable and cardiovascular risk factor for chronic kidney disease (CKD). Although long-term smoking has been associated with CKD, the potential relationship between its metabolite hydroxycotinine and CKD has not been clarified. METHODS: A total of 8,544 participants aged 20 years and above from the National Health and Nutrition Examination Survey (NHANES) 2017 - March 2020 were enrolled in our study. CKD was defined by estimated glomerular filtration rate (eGFR) < 60 mL/(min*1.73 m2). Serum hydroxycotinine was measured by an isotope-dilution high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric (ID HPLC-APCI MS/MS) method with a lower limit of detections (LLOD) at 0.015 ng/mL. The non-linear relationship was explored with restricted cubic splines (RCS). Pearson's correlation coefficient and a multivariate logistic regression model were used for correlation analysis. RESULTS: Serum hydroxycotinine and eGFR were negatively correlated in both non-CKD group (r= -0.05, p < 0.001) and CKD group (r= -0.04, p < 0.001). After serum hydoxycotinine dichotominzed with LLOD, serum hydroxycotinine ≥ 0.015 ng/mL was negatively correlated with eGFR not only in non-CKD group (r = -0.05, p < 0.001) but also in CKD group (r = -0.09, p < 0.001). After adjusting for comprehensive confounders, results from the multivariate logistic regression analysis showed that participants with serum hydroxycotinine ≥ 0.015 ng/mL had increased odds of CKD (OR = 1.505, p < 0.001). CONCLUSIONS: Serum hydroxycotinine might be positively associated with CKD. Further study is warranted to find the right concentration of hydroxycotinine to measure the CKD.

Mass spectrometry-based metabolomics study of nicotine exposure in THP-1 monocytes.

The tobacco alkaloid nicotine is known for its activation of neuronal nicotinic acetylcholine receptors. Nicotine is consumed in different ways such as through conventional smoking, e-cigarettes, snuff or nicotine pouches. The use of snuff has been associated with several adverse health effects, such as inflammatory reactions of the oral mucosa and oral cavity cancer. We performed a metabolomic analysis of nicotine-exposed THP-1 human monocytes. Cells were exposed to 5 mM of the alkaloid for up to 4 h, and cell extracts and medium subjected to untargeted liquid chromatography high-resolution mass spectrometry. Raw data processing revealed 17 nicotine biotransformation products. Among these, cotinine and nornicotine were identified as the two major cellular biotransformation products. The application of multi- and univariate statistical analyses resulted in the annotation, up to a certain level of identification, of 12 compounds in the cell extracts and 13 compounds in the medium that were altered by nicotine exposure. Of these, four were verified as methylthioadenosine, cytosine, uric acid, and L-glutamate. Methylthioadenosine levels were affected in both cells and the medium, while cytosine, uric acid, and L-glutamate levels were affected in the medium only. The effects of smoking on the pathways involving these metabolites have been previously demonstrated in humans. Most of the other discriminating compounds, which were merely tentatively or not fully identified, were amino acids or amino acid derivatives. In conclusion, our preliminary data suggest that some of the potentially adverse effects related to smoking may also be expected when nicotine is consumed via snuff or nicotine pouches.

Determination of cotinine and 3-hydroxynicotinine in human serum by liquid chromatography-tandem mass spectrometry and its application.

In this study, we developed a method for determining cotinine and 3-hydroxycotinine in human serum and established a methodology for an in-depth study of tobacco exposure and health. After the proteins in the human serum samples were precipitated with acetonitrile, they were separated on a ZORBAX SB-Phenyl column with a mobile phase of methanol encompassing 0.3% formic acid-water encompassing 0.15% formic acid. The measurement was performed on an API5500 triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Cotinine, 3-hydroxycotinine, and cotinine-d3 isotope internal standards were held for 2.56 minutes, 1.58 minutes, and 2.56 minutes, respectively. In serum, the linear range was 0.05 to 500 ng·mL-1 for cotinine and 0.50 to 1250 ng·mL-1 for 3-hydroxycotinine. The lower limit of quantification (LLOQ) was 0.05 ng·mL-1 and 0.5 ng·mL-1 for cotinine and 3-hydroxycotinine, respectively. The intra-day and inter-day relative standard deviations were <11%, and the relative errors were within ±â€…7%. Moreover, the mean extraction recoveries of cotinine and 3-hydroxycotinine were 98.54% and 100.24%, respectively. This method is suitable for the rapid determination of cotinine and 3-hydroxycotinine in human serum because of its rapidity, sensitivity, strong specificity, and high reproducibility. The detection of cotinine levels in human serum allows for the identification of the cutoff value, providing a basis for differentiation between smoking and nonsmoking populations.

Affinity of Nicotinoids to a Model Nicotinic Acetylcholine Receptor (nAChR) Binding Pocket in the Human Brain.

The binding affinity of nicotinoids to the binding residues of the α4ß2 variant of the nicotinic acetylcholine receptor (nAChR) was identified as a strong predictor of the nicotinoid's addictive character. Using ab initio calculations for model binding pockets of increasing size composed of 3, 6, and 14 amino acids (3AA, 6AA, and 14AA) that are derived from the crystal structure, the differences in binding affinity of 6 nicotinoids, namely, nicotine (NIC), nornicotine (NOR), anabasine (ANB), anatabine (ANT), myosmine (MYO), and cotinine (COT) were correlated to their previously reported doses required for increases in intracranial self-stimulation (ICSS) thresholds, a metric for their addictive function. By employing the many-body decomposition, the differences in the binding affinities of the various nicotinoids could be attributed mainly to the proton exchange energy between the pyridine and non-pyridine rings of the nicotinoids and the interactions between them and a handful of proximal amino acids, namely Trp156, Trpß57, Tyr100, and Tyr204. Interactions between the guest nicotinoid and the amino acids of the binding pocket were found to be mainly classical in nature, except for those between the nicotinoid and Trp156. The larger pockets were found to model binding structures more accurately and predicted the addictive character of all nicotinoids, while smaller models, which are more computationally feasible, would only predict the addictive character of nicotinoids that are similar to nicotine. The present study identifies the binding affinity of the guest nicotinoid to the host binding pocket as a strong descriptor of the nicotinoid's addiction potential, and as such it can be employed as a fast-screening technique for the potential addiction of nicotine analogs.

Application of HPLC-QQQ-MS/MS and New RP-HPLC-DAD System Utilizing the Chaotropic Effect for Determination of Nicotine and Its Major Metabolites Cotinine, and trans-3'-Hydroxycotinine in Human Plasma Samples.

The routine techniques currently applied for the determination of nicotine and its major metabolites, cotinine, and trans-3'-hydroxycotinine, in biological fluids, include spectrophotometric, immunoassays, and chromatographic techniques. The aim of this study was to develop, and compare two new chromatographic methods high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS), and RP-HPLC enriched with chaotropic additives, which would allow reliable confirmation of tobacco smoke exposure in toxicological and epidemiological studies. The concentrations of analytes were determined in human plasma as the sample matrix. The methods were compared in terms of the linearity, accuracy, repeatability, detection and quantification limits (LOD and LOQ), and recovery. The obtained validation parameters met the ICH requirements for both proposed procedures. However, the limits of detection (LOD) were much better for HPLC-QQQ-MS/MS (0.07 ng mL-1 for trans-3'-hydroxcotinine; 0.02 ng mL-1 for cotinine; 0.04 ng mL-1 for nicotine) in comparison to the RP-HPLC-DAD enriched with chaotropic additives (1.47 ng mL-1 for trans-3'-hydroxcotinine; 1.59 ng mL-1 for cotinine; 1.50 ng mL-1 for nicotine). The extraction efficiency (%) was concentration-dependent and ranged between 96.66% and 99.39% for RP-HPLC-DAD and 76.8% to 96.4% for HPLC-QQQ-MS/MS. The usefulness of the elaborated analytical methods was checked on the example of the analysis of a blood sample taken from a tobacco smoker. The nicotine, cotinine, and trans-3'-hydroxycotinine contents in the smoker's plasma quantified by the RP-HPLC-DAD method differed from the values measured by the HPLC-QQQ-MS/MS. However, the relative errors of measurements were smaller than 10% (6.80%, 6.72%, 2.04% respectively).

A Simple and High-Throughput LC-MS-MS Method for Simultaneous Measurement of Nicotine, Cotinine, 3-OH Cotinine, Nornicotine and Anabasine in Urine and Its Application in the General Korean Population.

Measuring nicotine metabolites is the most objective method for identifying smoke exposure. Liquid chromatography--tandem mass spectrometry (LC-MS-MS) can measure multiple metabolites and is sensitive enough to detect low concentrations of metabolites. Therefore, we developed a simple and high-throughput method for measuring nicotine, cotinine, trans-3'-hydroxycotinine (3-OH cotinine), nornicotine and anabasine for population-based studies using LC-MS-MS. Each 30 µL of urine sample was diluted with 90 µL of acetonitrile containing five deuterated internal standards. Chromatographic separation used a C18 column, and LC-MS-MS analysis was performed with a multiple reaction monitoring mode. The chromatographic run time for each sample was 6.5 min. The method was validated by evaluating selectivity, interference, limit of detection, lower limit of quantification, precision, accuracy, linearity, extraction recovery, matrix effect and carryover according to guidelines. Our methods required a short preparation time (∼20 min) while simultaneously measuring five markers for smoking status. No endogenous or exogenous interference was found. Our method showed excellent precision and accuracy: within-run coefficient of variation (CV) 2.9-9.4%, between-run CV 4.8-8.7% and bias -10.1 to 5.3%. Linear dynamic ranges were 1-10,000 ng/mL for nicotine, nornicotine and anabasine; 2-5,000 ng/mL for cotinine and 5-15,000 ng/mL for 3-OH cotinine. Extraction recovery was consistent (87-109%) across concentrations. No significant matrix effect or carryover was observed. The validated method was applied to 849 urine samples. In samples from the 125 current smokers, nicotine, cotinine, 3-OH cotinine, nornicotine and anabasine were detected in 97.6, 99.2, 98.4, 96.8 and 87.2%, respectively. No markers were detected in 93.9% of 609 nonsmokers. The overlapping detection of multiple markers made it possible to identify the smoking status even in current smokers with a low concentration of cotinine. Our LC-MS-MS method using a simple sample preparation technique is sensitive and effective for screening of smoking status in the general population.

Parahydrogen hyperpolarization of minimally altered urine samples for sensitivity enhanced NMR metabolomics.

Parahydrogen hyperpolarization has been shown to enhance NMR sensitivity in urine analysis by several orders of magnitude if urine samples are prepared by solid phase extraction (SPE). We present a different approach, developed for minimal sample alteration before analysis. Removing SPE from the workflow allows to retain a wider range of metabolites and paves the way towards more universal hyperpolarized NMR metabolomics of low abundance metabolites.

Serum Concentrations of Cotinine and Trans-3'-Hydroxycotinine in US Adults: Results From Wave 1 (2013-2014) of the Population Assessment of Tobacco and Health Study.

INTRODUCTION: The Population Assessment of Tobacco and Health (PATH) Study is a nationally representative cohort of tobacco product users and nonusers. The study's main purpose is to obtain longitudinal epidemiologic data on tobacco use and exposure among the US population. AIMS AND METHODS: Nicotine biomarkers-cotinine (COT) and trans-3'-hydroxycotinine (HCT)-were measured in blood samples collected from adult daily tobacco users and nonusers during Wave 1 of the PATH Study (2013-2014; n = 5012; one sample per participant). Participants' tobacco product use and exposure to secondhand smoke were categorized based on questionnaire responses. Nonusers were subdivided into never users and recent former users. Daily tobacco users were classified into seven tobacco product use categories: exclusive users of cigarette, smokeless tobacco, electronic cigarette, cigar, pipe, and hookah, as well as polyusers. We calculated sample-weighted geometric mean (GM) concentrations of cotinine, HCT, and the nicotine metabolite ratio (NMR) and evaluated their associations with tobacco use with adjustment for potential confounders. RESULTS: The GMs (95% confidence intervals) of COT and HCT concentrations for daily tobacco users were 196 (184 to 208) and 72.5 (67.8 to 77.4) ng/mL, and for nonusers they were 0.033 (0.028 to 0.037) and 0.021 (0.018 to 0.023) ng/mL. Exclusive smokeless tobacco users had the highest COT concentrations of all user groups examined. The GM NMR in daily users was 0.339 (95% confidence interval: 0.330 to 0.350). CONCLUSIONS: These nationally representative estimates of serum nicotine biomarkers could be the basis for reference ranges characterizing nicotine exposure for daily tobacco users and nonusers in the US adult population. IMPLICATIONS: This report summarizes the serum nicotine biomarker measurements in Wave 1 of the PATH Study. We are reporting the first estimates of HCT in serum for daily tobacco users and nonusers in the noninstitutionalized, civilian US adult population; the first nationally representative serum COT estimates for daily exclusive users of different tobacco products and daily polyusers; and the first nationally representative estimate of the serum NMR in daily tobacco users by age, race/ethnicity, and sex.

Study on the daily Ad Libitum smoking habits of active Korean smokers and their effect on urinary smoking exposure and impact biomarkers.

INTRODUCTION: Understanding interactions of smoking topography with biomarkers of exposure to tobacco is essential for accurate smoking risk assessments. METHODS: In this study, the smoking topography and the levels of tobacco smoke exposure urinary biomarkers of a sample of active Korean smokers were quantified and measured. The results were used to investigate the effect of daily activities and smoking time on the smoking topography. Moreover, correlations between the smoking topography parameters and biomarkers were assessed. RESULTS: No significant effect of either the daily activities or time on the smoking topography of the subjects were observed. Synchronic correlations of the cigarette consumption per day (CPD) and the average flow per puff with both urinary cotinine and trans-3'-hydroxycotinine were significant. For the urinary nicotine metabolites, the peak levels appeared when the CPD was over 19 cigarettes per day and the average puff velocity was between 35 and 45 ml/s. Nevertheless, when the average flow was over 60 ml/s, the levels of cotinine and trans-3'-hydroxycotinine significantly dropped. CONCLUSIONS: The findings of this study may be beneficial for further smoking risk assessments with contributions of both the smoking topography and biomarkers to provide current smokers with applicable cession programs.Clinical significanceSmoking habits and levels of urinary biomarkers of Korean smokers are investigated.People with a higher dependency on nicotine smoke cigarettes with slower puffs.Effects of daily activities or time on smoking topography were not significant.Correlations between smoking topography and urinary biomarkers were significant.Peak biomarker levels were observed under certain smoking topography conditions.

Cotinine Hydroxylase CotA Initiates Biodegradation of Wastewater Micropollutant Cotinine in Nocardioides sp. Strain JQ2195.

Cotinine is a stable toxic contaminant, produced as a by-product of smoking. It is of emerging concern due to its global distribution in aquatic environments. Microorganisms have the potential to degrade cotinine; however, the genetic mechanisms of this process are unknown. Nocardioides sp. strain JQ2195 is a pure-culture strain that has been reported to degrade cotinine at micropollutant concentrations. This strain utilizes cotinine as its sole carbon and nitrogen source. In this study, a 50-kb gene cluster (designated cot), involved in cotinine degradation, was predicted based on genomic and transcriptomic analyses. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3), which initiated cotinine catabolism, was identified and characterized. CotA from Shinella sp. strain HZN7 was heterologously expressed and purified and was shown to convert cotinine into 6-hydroxycotinine. H218O-labeling and electrospray ionization-mass spectrometry (ESI-MS) analysis confirmed that the hydroxyl group incorporated into 6-hydroxycotinine was derived from water. This study provides new molecular insights into the microbial metabolism of heterocyclic chemical pollutants. IMPORTANCE In the human body, cotinine is the major metabolite of nicotine, and 10 to 15% of generated cotinine is excreted in urine. Cotinine is a structural analogue of nicotine and is much more stable than nicotine. Increased tobacco consumption has led to high environmental concentrations of cotinine, which may have detrimental effects on aquatic ecosystems and human health. Nocardioides sp. strain JQ2195 is a unique cotinine-degrading bacterium. However, the underlying genetic and biochemical foundations of cotinine degradation are still unknown. In this study, a 50-kb gene cluster (designated cot) was identified by genomic and transcriptomic analyses as being involved in the degradation of cotinine. A novel three-component cotinine hydroxylase gene (designated cotA1A2A3) catalyzed cotinine to 6-hydroxy-cotinine. This study provides new molecular insights into the microbial degradation and enzymatic transformation of cotinine.

Rapid, sensitive, and reliable quantitation of nicotine and its main metabolites cotinine and trans-3'-hydroxycotinine by LC-MS/MS: Method development and validation for human plasma.

New nicotine delivery products are gaining market share. For evaluation of their characteristics, toxicokinetic investigations are in current research focus. For reliable determination of blood plasma levels of nicotine and its main metabolites cotinine and trans-3'-hydroxycotinine, a quantitation method based on LC-ESI-MS/MS was developed and validated. Addition of isotope labeled internal standards prior to rapid sample preparation using protein precipitation with methanol was chosen for sample preparation. Different stationary phases were tested and phenyl-hexyl separation was found to be superior to HILIC, C18, and C8 stationary phases. Ion suppression effects caused by hydrophilic early eluting matrix were eliminated by the adjustment of an adequate retention utilizing a phenyl-hexyl separation stationary phase. Exchange of acetonitrile as organic mobile phase by methanol and elevation of pH value of aqueous mobile phase containing 5 mM NH4Ac to 4.50 improved the chromatographic resolution. The limits of quantitation for nicotine, cotinine, and hydroxycotinine were 0.15, 0.30, and 0.40 ng/mL, respectively. Linearity was proven by matrix matched calibration for the whole working range from 0.50 ng/mL to 35.0 ng/mL for nicotine and from 6.00 to 420 ng/mL for cotinine and hydroxycotinine (Mandel's fitting test with R2 > 0.995). Quality control samples at four different levels (0.50, 1.50, 17.5, 28.0 ng/mL for nicotine and 6.00, 18.0, 210, 336 ng/mL for cotinine and hydroxycotinine) in plasma were analyzed six times on three days. Mean accuracies ranged from 87.7% to 105.8% for nicotine, from 90.3% to 102.9% for cotinine, and from 99.9% to 109.9% for hydroxycotinine. Intra- and inter-day precisions (RSD %) were below 15% for all analytes (<20% for LLOQ). As proof of concept, the method was successfully applied to a real plasma sample from a cigarette smoking volunteer.

Urinary Cotinine and Cotinine + Trans-3'-Hydroxycotinine (TNE-2) Cut-points for Distinguishing Tobacco Use from Nonuse in the United States: PATH Study (2013-2014).

BACKGROUND: Determine the overall, sex-, and racially/ethnically-appropriate population-level cotinine and total nicotine equivalents (TNE-2, the molar sum of the two major nicotine metabolites) cut-points to distinguish tobacco users from nonusers across multiple definitions of use (e.g., exclusive vs. polytobacco, and daily vs. non-daily). METHODS: Using Wave 1 (2013-2014) of the U.S. Population Assessment of Tobacco and Health (PATH) Study, we conducted weighted Receiver Operating Characteristic (ROC) analysis to determine the optimal urinary cotinine and TNE-2 cut-points, stratified by sex and race/ethnicity. RESULTS: For past 30-day exclusive cigarette users, the cotinine cut-point that distinguished them from nonusers was 40.5 ng/mL, with considerable variation by sex (male: 22.2 ng/mL; female: 43.1 ng/mL) and between racial/ethnic groups (non-Hispanic other: 5.2 ng/mL; non-Hispanic black: 297.0 ng/mL). A similar, but attenuated, pattern emerged when assessing polytobacco cigarette users (overall cut-point = 39.1 ng/mL, range = 5.5 ng/mL-80.4 ng/mL) and any tobacco users (overall cut-point = 39.1 ng/mL, range = 4.8 ng/mL-40.0 ng/mL). Using TNE-2, which is less impacted by racial differences in nicotine metabolism, produced a comparable pattern of results although reduced the range magnitude. CONCLUSIONS: Because of similar frequency of cigarette use among polytobacco users, overall cut-points for exclusive cigarette use were not substantially different from cut-points that included polytobacco cigarette use or any tobacco use. Results revealed important differences in sex and race/ethnicity appropriate cut-points when evaluating tobacco use status and established novel urinary TNE-2 cut-points. IMPACT: These cut-points may be used for biochemical verification of self-reported tobacco use in epidemiologic studies and clinical trials.

Concentrations of serum hydroxycotinine for US adult smokers aged ≥ 20 years by type of smoker.

Cross-sectional survey data (N = 3264) from the National Health and Nutrition Examination Survey for 2013-2018 were used to investigate how serum hydroxycotinine concentrations vary among US adult smokers aged ≥ 20 years by smoker type. Those reporting using tobacco products during the last 5 days were classified as smokers. Smokers were classified as being cigarette only smokers, cigar only smokers, cigar and cigarette smokers, dual cigarette and e-cigarette smokers, e-cigarette only smokers, smokeless tobacco only users, and all other smokers. Regression models stratified by smoker type with log10 transformed values of serum hydroxycotinine as dependent variable were fitted to compute adjusted geometric means (AGM) for each type of smoker. The order in which various types of smokers were found to have AGMs for serum hydroxycotinine was cigarette and e-cigarette users (64.61 ng/mL), cigarette only smokers (53.17 ng/mL), smokeless tobacco only users (44.89 ng/mL), cigar and cigarette smokers (36.99 ng/mL), e-cigarette only users (32.52 ng/mL), smokers of miscellaneous tobacco products (20.32 ng/mL), and cigar smokers only (10.75 ng/mL). Compared to this as presented in a recent study, the order in which serum cotinine AGMs were: smokeless tobacco only users (272 ng/mL), cigarette only smokers (152.5 ng/mL), cigarette-e-cigarette or e-cigarette only users (146.3 ng/mL), smokers of miscellaneous tobacco products (105.5 ng/mL), cigar and cigarette smokers (92.5 ng/mL), cigar smokers only (65.1 ng/mL). Among cigarette only smokers, males had lower AGM than females (47.18 vs. 59.91 ng/mL, p < 0.01), but the reverse was true for smokeless tobacco only and miscellaneous smokers. In general, differences for hydroxycotinine levels did not exist among non-Hispanic white and non-Hispanic black smokers. Among US adults, cigarette only and dual cigarette-e-cigarette smokers had the highest, and cigar smokers had the lowest concentrations of serum hydroxycotinine.

Comparative analysis of the concentrations of serum cotinine and hydroxycotinine for US children, adolescents, and adults: impact of exposure to environmental tobacco smoke at home and other indoor environments.

This study was carried out to investigate how serum cotinine and hydroxycotinine concentrations compare and vary by age, gender, race/ethnicity, smoking, and exposure to environmental tobacco smoke (ETS) at home and other indoor environments. Data from NHANES for 2013-2018 for US children aged 3-11 years (N = 3834), nonsmoker (N = 1963) and smoker (N = 247) adolescents aged 12-19 years, and nonsmoker (N = 10,334) and smoker (N = 3264) adults aged ≥ 20 years were analyzed by fitting regression models with log10 transformed values of serum cotinine and hydroxycotinine as dependent variables. Models stratified by age and smoking status were fitted. Those reporting using tobacco products during the last 5 days were classified as smokers. For cotinine, males had higher cotinine concentrations than females for children, adolescent smokers, and nonsmoker adults. Non-Hispanic Blacks were found to have lower concentrations of both cotinine and hydroxycotinine than non-Hispanic Whites for adult smokers (p < 0.01) only. The ratio of concentrations of those exposed to ETS at home to those not exposed to ETS at home for hydroxycotinine was 6.3 for nonsmoker adults and as low as 1.39 for adult smokers.

Exposure to volatile organic compounds may be associated with oxidative DNA damage-mediated childhood asthma.

Volatile organic compounds (VOCs) are important and ubiquitous air pollutants, which may lead to a significant increase in the prevalence of respiratory diseases. To investigate the relationships between VOCs exposure and childhood asthma, 252 asthmatic children and 69 healthy children were recruited. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG, a biomarker of oxidative DNA damage), trans-3'-hydroxycotinine (OH-Cot, a biomarker of passive smoking) and 27 VOC metabolites were simultaneously determined by an ultra-high-performance liquid chromatography-tandem mass spectrometer. Results showed that levels of 8-OHdG and most VOC metabolites in asthmatic children were significantly higher than those in healthy children. More than half of the VOC metabolites were significantly and positively associated with OH-Cot with maximal ß coefficient of 0.169, suggesting that second-hand smoking is one important source of VOCs exposure for children in Guangzhou. Significant dose-response relationships between most VOC metabolites and 8-OHdG were observed. Each unit increase in ln-transformed VOC metabolite levels was significantly associated with 5.5-32% increase in ln-transformed 8-OHdG level. Moreover, each unit increase in ln-transformed 8-OHdG level was associated with an 896% increased odd ratios (OR) of asthma in children (OR = 9.96, 95% confidence intervals (CI): 4.75, 20.9), indicating that oxidative stress induced by VOCs exposure may have a significant impact on childhood asthma. Urinary 3-&4-Methylhippuric acid (3-&4-MHA, OR: 5.78, 95% CI: 3.50, 9.54), rac 2-Aminothiazoline-4-carboxylic acid (ATCA, OR: 2.90, 95% CI: 1.69, 4.99) and N-Acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA, OR: 2.76, 95% CI: 1.73, 4.43) which may derive from m/p-xylene, cyanide and 1,3-butadiene exposure, respectively, could significantly and maximally increase the odds of asthma. Interestingly, they also had the strongest associations with 8-OHdG among all investigated VOC metabolites. Moreover, DHBMA strongly correlated with most VOC metabolites. Hence, DHBMA is a suitable biomarker to indicate not only VOCs exposure profile, but also the DNA damage-mediated asthma induced by VOCs.

Transferability of Ancestry-Specific and Cross-Ancestry CYP2A6 Activity Genetic Risk Scores in African and European Populations.

The Nicotine Metabolite Ratio (NMR; 3-hydroxycotinine/cotinine), a highly heritable index of nicotine metabolic inactivation by the CYP2A6 enzyme, is associated with numerous smoking behaviors and diseases, as well as unique cessation outcomes. However, the NMR cannot be measured in nonsmokers, former smokers, or intermittent smokers, for example, in evaluating tobacco-related disease risk. Traditional pharmacogenetic groupings based on CYP2A6 * alleles capture a modest portion of NMR variation. We previously created a CYP2A6 weighted genetic risk score (wGRS) for European (EUR)-ancestry populations by incorporating independent signals from genome-wide association studies to capture a larger proportion of NMR variation. However, CYP2A6 genetic architecture is unique to ancestral populations. In this study, we developed and replicated an African-ancestry (AFR) wGRS, which captured 30-35% of the variation in NMR. We demonstrated model robustness against known environmental sources of NMR variation. Furthermore, despite the vast diversity within AFR populations, we showed that the AFR wGRS was consistent between different US geographical regions and unaltered by fine AFR population substructure. The AFR and EUR wGRSs can distinguish slow from normal metabolizers in their respective populations, and were able to reflect unique smoking cessation pharmacotherapy outcomes previously observed for the NMR. Additionally, we evaluated the utility of a cross-ancestry wGRS, and the capacity of EUR, AFR, and cross-ancestry wGRSs to predict the NMR within stratified or admixed AFR-EUR populations. Overall, our findings establish the clinical benefit of applying ancestry-specific wGRSs, demonstrating superiority of the AFR wGRS in AFRs.

Biocompatible SPME fibers for direct monitoring of nicotine and its metabolites at ultra trace concentration in rabbit plasma following the application of smoking cessation formulations.

The ultra-trace determination of nicotine and its 4 major metabolites (cotinine, nornicotine, norcotinine and anabasine) from rabbit plasma was achieved by a newly developed solid phase microextraction-liquid chromatography-tandem mass spectrometry method. Extraction of the target analytes was performed with hydrophilic/lipophilic balance-polyacrylonitrile SPME fibers. Dual fiber extraction was necessary to guarantee improved recovery at parts-per-trillion levels. Liquid chromatographic analysis was achieved in a 6-min run using a C18 (1.9 µm C18, 50 mm x 2.1 mm) column with a mobile phase flow rate of 0.4 mL/min. Tandem mass spectrometry was used for detection and quantification in positive electrospray ionization (ESI+) mode for all the targeted analytes. Two stable isotope-labeled internal standards were used for signal correction and accurate quantification. The mass spectrometer with laminar flow ion flux transport, guaranteed improved signal stability, minimal contamination of the ion guide and reproducibility into the first quadrupole analyzer. The method was validated in line with the Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The results met the acceptance criteria as proposed by the FDA: accuracy was tested at 0.35, 10 and 75 µg L - 1 and ranged between 98.3-112.2% for nicotine, 94.1-101.9% for cotinine, 94.7-107.0% for nornicotine, 81.1-107.2% for norcotinine and 94.3-115.2% for anabasine, with precision up to 14.2%. Stability tests indicated that all the targeted analytes were stable in the desorption solution for at least 1 week. LOQs ranged from 0.05 to 1 µg L-1. The method was successfully applied to analyze plasma samples obtained from rabbits following transdermal application of a smoking cessation formulation loaded with solid lipid nanoparticles containing a nicotine-stearic acid conjugate.

A prospective study of the association between rate of nicotine metabolism and alcohol use in tobacco users in the United States.

BACKGROUND: Rate of nicotine metabolism has been identified as a biochemical risk factor for nicotine use and dependence; however, its role in alcohol consumption and related outcomes is not well understood. The current research examined nicotine metabolism rate as a risk factor for alcohol use among current tobacco users. We also examined sex differences in these associations. METHOD: Data were taken from Waves 1 and 2 of the Population Assessment of Tobacco and Health (PATH) study, a national longitudinal study of tobacco use and associated health outcomes. The nicotine metabolite ratio (NMR) was calculated as the ratio of trans-3' hydroxycotinine to cotinine in urine samples provided at wave 1. Alcohol use outcomes included past 30-day NIAAA-defined hazardous drinking status, total drinks, and alcohol-related consequences. All analyses controlled for alcohol use at Wave 1. RESULTS: NMR at Wave 1 predicted increased odds of meeting hazardous drinking criteria, adjusted odds ratio (aOR) = 1.14, 95 % CI = 1.06; 1.23, p = 0.001, greater total alcohol consumption amount, adjusted rate ratio (aRR) = 1.21, 95 % CI = 1.12; 1.30, p < 0.001, and more alcohol consequences, aRR = 1.07, 95 % CI = 1.01; 1.13, p = 0.018, at wave 2. No significant sex differences were identified. NMR remained a significant predictor of alcohol use in models controlling for severity of nicotine exposure in cigarette smokers. CONCLUSIONS: NMR may be a shared risk factor for harmful nicotine and alcohol use that contributes to their co-occurrence.

Anabasine-based measurement of cigarette consumption using wastewater analysis.

Community tobacco use can be monitored over time using wastewater-based epidemiological approaches by estimating the mass loads of nicotine and its metabolites, cotinine, or hydroxycotinine, in wastewater. However, due to the use of nicotine in smoking cessation products, other sources of nicotine contribute to cotinine and hydroxycotinine loads. The use of nicotine replacement therapies could vary in space and time and mask the true rates of tobacco consumption. Therefore, this work evaluated the content of tobacco specific markers, anatabine and anabasine, in cigarettes, in urine of smokers, and in wastewater. The results indicated that the anabasine content in both licit and illicit cigarettes in Australia is less variable than anatabine and is therefore considered a better measure of tobacco consumption. A study determining the excretion of tobacco-specific alkaloids of smoking and non-smoking volunteers gave an average urinary mass load of anabasine of 4.38 µg/L/person and a daily mass load of 1.13 µg/day/person. Finally, this was compared with the mass loads of anabasine from wastewater-based epidemiology data of 3 µg/day/person to estimate cigarette rates in a South Australian city: equivalent to 2.6 cigarettes/person/day. The rate of decline of cigarette use was greater when using anabasine as a measure of consumption compared with cotinine. This is the first study to estimate the rate of anabasine excretion, which can be used to estimate tobacco use independent of therapeutically prescribed nicotine.