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Tobacco use is the leading preventable cause of premature disease and death in the United States. Approximately, 34 million U.S. adults currently smoke cigarettes. We developed a method for automated sample preparation and liquid chromatography-tandem mass spectrometry quantitation of 14 tobacco-related analytes: nicotine (NICF), cotinine (COTF), trans-3′-hydroxycotinine (HCTF), menthol glucuronide (MEG), anabasine (ANBF), anatabine (ANTF), isonicoteine (ISNT), myosmine (MYOS), beta-nicotyrine (BNTR), bupropion (BUPR), cytisine (CYTI), varenicline (VARE), arecaidine (ARD), and arecoline (ARL). The method includes automated solid-phase extraction using customized positive-pressure functions. The preparation scheme has the capacity to process a batch of 96 samples within 4 h with greater than 88% recovery for all analytes. The 14 analytes, separated within 4.15 min using reversed-phase liquid chromatography, were determined using a triple-quadrupole mass spectrometer with atmospheric-pressure chemical ionization and multiple reaction monitoring in negative and positive ionization modes. Wide quantitation ranges, within 1.2–72,000 ng/mL, were established especially for COTF, HCTF, MEG, and NICF to quantify the broad range of biomarker concentrations found in the U.S. population. The method accuracy is above 90% while the overall imprecision is below 7%. Finally, we tested urine samples from 90 smokers and observed detection rates of over 98% for six analytes with urinary HCTF and MEG concentrations ranging from 200–14,100 and 60–57,100 ng/mL, respectively. This high throughput analytical process can prepare and analyze a sample in 9 min and along with the 14-compound analyte panel can be useful for tobacco-exposure studies, in smoking-cessation programs, and for detecting changes in exposure related to tobacco products and their use.

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.

Urinary cotinine is one of the most commonly measured biomarkers reflecting recent exposure to nicotine. In some cases a simple qualitative dichotomization of smokers and non-smokers is all that is required. NicAlert® test strips have been evaluated for this purpose, but other recently introduced, inexpensive single-line test strips have not. In this study we evaluated two such strips with nominal cutoffs of 200 and 10 ng/mL. A total of 800 urine samples with known cotinine concentrations determined by an LC–MS-MS method were examined, including 400 urine samples ranging from 0.23 to more than 24,000 ng/mL by the 200 ng/mL strip, and 400 samples with concentrations 98%. Our results suggest that these simple and inexpensive lateral flow immunoassay test strips can provide useful qualitative estimates of nicotine exposures for appropriate applications within the inherent limitations of sensitivity and precision of the immunoassay test strip format.

Background: Biomarkers of tobacco exposure have a central role in studies of tobacco use and nicotine intake. The most significant exposure markers are nicotine itself and its metabolites in urine. Therefore, it is important to evaluate the performance of laboratories conducting these biomarker measurements. Methods: This report presents the results from a method performance study involving 11 laboratories from 6 countries that are currently active in this area. Each laboratory assayed blind replicates of seven human urine pools at various concentrations on three separate days. The samples included five pools blended from smoker and nonsmoker urine sources, and two additional blank urine samples fortified with pure nicotine, cotinine, and hydroxycotinine standards. All laboratories used their own methods, and all were based on some form of liquid chromatography/tandem mass spectrometry. Results: Overall, good agreement was found among the laboratories in this study. Intralaboratory precision was good, and in the fortified pools, the mean bias observed was < + 3.5% for nicotine, approximately 1.2% for hydroxycotinine, and less than 1% for cotinine (1 outlier excluded in each case). Both indirect and direct methods for analyzing the glucuronides gave comparable results. Conclusions: This evaluation indicates that the experienced laboratories participating in this study can produce reliable and comparable human urinary nicotine metabolic profiles in samples from people with significant recent exposure to nicotine. Impact: This work supports the reliability and agreement of an international group of established laboratories measuring nicotine and its metabolites in urine in support of nicotine exposure studies. Cancer Epidemiol Biomarkers Prev; 27(9); 1083–90. ©2018 AACR.

Background:Given the rapid increase in the popularity of e-cigarettes and the paucity of associated longitudinal health-related data, the need to assess the potential risks of long-term use is essential.Objective:To compare exposure to nicotine, tobacco-related carcinogens, and toxins among smokers of combustible cigarettes only, former smokers with long-term e-cigarette use only, former smokers with long-term nicotine replacement therapy (NRT) use only, long-term dual users of both combustible cigarettes and e-cigarettes, and long-term users of both combustible cigarettes and NRT.Design:Cross-sectional study.Setting:United Kingdom.Participants:The following 5 groups were purposively recruited: combustible cigarette–only users, former smokers with long-term (≥6 months) e-cigarette–only or NRT-only use, and long-term dual combustible cigarette–e-cigarette or combustible cigarette–NRT users (n = 36 to 37 per group; total n = 181).Measurements:Sociodemographic and smoking characteristics were assessed. Participants provided urine and saliva samples and were analyzed for biomarkers of nicotine, tobacco-specific N-nitrosamines (TSNAs), and volatile organic compounds (VOCs).Results:After confounders were controlled for, no clear between-group differences in salivary or urinary biomarkers of nicotine intake were found. The e-cigarette–only and NRT-only users had significantly lower metabolite levels for TSNAs (including the carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL]) and VOCs (including metabolites of the toxins acrolein; acrylamide; acrylonitrile; 1,3-butadiene; and ethylene oxide) than combustible cigarette–only, dual combustible cigarette–e-cigarette, or dual combustible cigarette–NRT users. The e-cigarette–only users had significantly lower NNAL levels than all other groups. Combustible cigarette–only, dual combustible cigarette–NRT, and dual combustible cigarette–e-cigarette users had largely similar levels of TSNA and VOC metabolites.Limitation:Cross-sectional design with self-selected sample.Conclusion:Former smokers with long-term e-cigarette–only or NRT-only use may obtain roughly similar levels of nicotine compared with smokers of combustible cigarettes only, but results varied. Long-term NRT-only and e-cigarette–only use, but not dual use of NRTs or e-cigarettes with combustible cigarettes, is associated with substantially reduced levels of measured carcinogens and toxins relative to smoking only combustible cigarettes.Primary Funding Source:Cancer Research UK

A microchip has been developed on the basis of immno-precipitation approach for fast and sensitive enrichment of low abundant carbonylated proteins. This microfluidic method could enrich molecular biomarkers, which could be further analyzed in the proteomic study of age-related diseases and therapeutic development. In this study, an immunoaffinity-based PDMS micro-device was designed, fabricated, and chemically modified to specifically trap DNP-labeled PTM proteins of low abundance from a complex protein mixture. Carbonylated protein is selected as a representative PTM protein to illustrate the wide application of this immuno-based microchip for other PTMs which could be readily labeled by different antibody groups. Surface characterization methods such as atomic force microscopy and fluorescence microscopy were used to evaluate the construction of glutaraldehyde- and antibody- terminated PDMS substrates in the device fabrication. Quantitative study was also applied to study the target protein capture and elution efficiency of the device. In a testing mixture consisting of smaller amount of test model-In Vitro oxidized cytochrome c and large blocking protein BSA, a high sensitivity and specificity for only carbonylated protein biomarkers was demonstrated using this on-chip immnuoaffinity based extraction/enrichment. For this highly dense 193-post arrays μ-chip, a low abundance of 159 ng of standard in vitro test model- cytochrome c was enriched at flow speed of 5 μL/min within 110 min. We demonstrated that this nascent micro-immunoprecipitation (μ-IP) method is capable for enrichment of biomarkers in protein post-translation modification related diseases and promise great advance in early disease detection.

Ammonia in mainstream smoke is present in both the particulate and vapor phases. The presence of ammonia in the cigarette filler material and smoke is of significance because of the potential role ammonia could have in raising the “smoke pH.” An increased smoke pH could shift a fraction of total nicotine to free-base nicotine, which is reportedly more rapidly absorbed by the smoker. Methods measuring ammonia in smoke typically employ acid filled impingers to trap the smoke. We developed a fast, reliable method to measure ammonia in mainstream smoke without the use of costly and time consuming impingers to examine differences in ammonia delivery. The method uses both a Cambridge filter pad and a Tedlar bag to capture particulate and vapor phases of the smoke. We quantified ammonia levels in the mainstream smoke of 50 cigarette brands from 5 manufacturers. Ammonia levels ranged from approximately 1μg to 23μg per cigarette for ISO smoking conditions and 38μg to 67μg per cigarette for Canadian intense smoking conditions and statistically significance differences were observed between brands and manufacturers. Our findings suggest that ammonia levels vary by brand and are higher under Canadian intense smoking conditions.

Glutathione plays an essential role in the intracellular antioxidant defense against oxidant radicals, especially the •OH radical. To understand the early and progressive cellular changes in the development of Alzheimer's disease (AD), we investigated reduced glutathione/oxidized glutathione (GSH/GSSG) status in a double mutated AD transgenic mouse model (B6.Cg-Tg), which carries Swedish amyloid-β protein precursor mutation (AβPPswe) and exon 9 deletion of the PSEN1 gene. In this study, we quantified and compared both GSH/GSSG and mixed-disulfide (Pr-SSG) levels in blood samples and three anatomic positions in brain (cerebrum, cerebellum, and hippocampus) at 3 age stages (1, 5, and 11 months) of AD transgenic (Tg)/wild type mice. The present study was designed to characterize and provide insight into the glutathione redox state of both brain tissues and blood samples at different disease stages of this Tg model. The level of Pr-SSG increased in all AD brain tissues and blood compared with controls regardless of age. The GSH/GSSG ratio in AD-Tg brain tissue started at a higher value at 1 month, fell at the transitional period of 5 months, right before the onset of amyloid plaques, followed by an increase in GSSG and associated decrease of GSH/GSSG at 11 months. These results suggest that formation of Pr-SSG may be an early event, preceding amyloid plaque appearance, and the data further implies that tissue thiol redox is tightly regulated. Notably, the high basal levels of mixed-disulfides in hippocampus suggest a potential for increased oxidative damage under oxidizing conditions and increased GSSG in this vulnerable region.

Post-translational modifications (PTM) of proteins play essential roles in cellular physiology and disease. The identification of protein substrates and detection of modification site helps understand PTM-mediated regulation in essential biological pathways and functions in various diseases. However, PTM proteins are typically present only at trace levels, making them difficult to identify in mass spectrometry based proteomics. In this paper, we report a novel and sensitive affinity chromatography on the avidin-functionalized poly(methyl methacrylate) (PMMA) microchip for enrichment of nanogram (ng) amount of PTMs. The chemical modification of poly(methyl methacrylate) (PMMA) surfaces yield avidin-terminated PMMA surfaces after UV radiation and consecutive EDC mediated coupling (amide reaction). This functionalized PMMA micro-device was developed to identify and specifically trap biotinylated PTM proteins of low abundance from complex protein mixture. Here we selected carbonylated protein as a representative PTM to illustrate the wide application of this affinity microchip for any PTMs converted into a tractable tag after derivatization. The surface topography, surface functional group mapping and elemental composition changes after each modification step of the treatment process were systematically measured qualitatively and quantitatively by atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. Quantitative study of biotinlated carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this subproteome enrichment micro-device can be assembled with other lab-on-a-chip components for follow-up protein analysis.

A stable, protein C activating membrane-mimetic film was produced on a polyelectrolyte multilayer (PEM) by in-situ photopolymerization of a phospholipid assembly containing thrombomodulin (TM). The monoacrylated phospholipid monomer was initially synthesized and prepared as unilamellar vesicles with varying molar concentrations of TM. Notably, in mixed-lipid systems, K m values for protein C activation increased in direct proportion to the mole fraction of polymerizable lipid, which was likely due to reduced membrane mobility after photopolymerization. Membrane-mimetic films were also constructed on planar substrates with predictable surface concentrations of catalytically active TM. Significantly, at a TM surface concentration of 170 fmol/cm 2 , the rate of protein C activation was comparable to that measured for a variety of confluent endothelial cell monolayers. Serial measurements of contact angles and protein C activation confirmed short-term film stability under a variety of in vitro conditions. Moreover, 1 2 5 I labeling of TM demonstrated little change in TM surface concentration over periods of up to 28 days. Significantly, polymeric lipid membranes functionalized with thrombomodulin efficiently inhibited thrombin generation. We believe that the design of membrane-mimetic films that have the capacity to activate the protein C pathway will provide a useful strategy for generating "actively" antithrombogenic surfaces.

A stabilized, membrane-mimetic film was produced on a polyelectrolyte multiplayer (PEM) by in-situ photopolymerization of an acrylate functonalized phospholipid assembly at a solid-liquid interface. The phospholipid monomer was synthesized, prepared as unilamellar vesicles, and fused onto close-packed octadecyl chains as part of an amphiphilic terpolymer anchored onto the PEM by electrostatic interactions. The lipid film displayed an advancing contact angle of ∼ 60°, elemental composition, as determined by X-ray photoelectron spectroscopy, was in agreement with that anticipated for a lipid membrane. Data obtained from both high-resolution scanning electron microscopy and ellipsometry were consistent with the formation of a supported lipid monolayer. In addition, polarized external reflection infrared spectroscopy revealed significant acyl chain ordering induced on lipid fusion and polymerization. Doping the lipid assembly with a fluorescein terminated polymerizable lipid provided visible confirmation of film formation and its stability under a variety of conditions, including shear rates of 2000 sec - 1 . Transport studies demonstrated that the addition of a lipid film significantly reduced barrier permeability for compounds in excess of 70 kD. The ability to coat microbeads (d ∼ 300 μm) with a robust membrane-mimetic film, while preserving encapsulated cell viability is illustrated, thereby establishing a new strategy for modulating the physiochemical and biological properties of immunoisolation barriers for cell transplantation.

Most sample preparation methods characteristically involve intensive and repetitive labor, which is inefficient when preparing large numbers of samples from population-scale studies.This study presents a robotic system designed to meet the sampling requirements for large population-scale studies. Using this robotic system, we developed and validated a method to simultaneously measure urinary anatabine, anabasine, nicotine and seven major nicotine metabolites: 4-Hydroxy-4-(3-pyridyl)butanoic acid, cotinine-N-oxide, nicotine-N-oxide, trans-3'-hydroxycotinine, norcotinine, cotinine and nornicotine. We analyzed robotically prepared samples using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry in positive electrospray ionization mode using scheduled multiple reaction monitoring (sMRM) with a total runtime of 8.5 min.The optimized procedure was able to deliver linear analyte responses over a broad range of concentrations. Responses of urine-based calibrators delivered coefficients of determination (R(2)) of0.995. Sample preparation recovery was generally higher than 80%. The robotic system was able to prepare four 96-well plate (384 urine samples) per day, and the overall method afforded an accuracy range of 92-115%, and an imprecision of15.0% on average.The validation results demonstrate that the method is accurate, precise, sensitive, robust, and most significantly labor-saving for sample preparation, making it efficient and practical for routine measurements in large population-scale studies such as the National Health and Nutrition Examination Survey (NHANES) and the Population Assessment of Tobacco and Health (PATH) study.

8-Hydroxy-2′-deoxyguanosine (8-OHdG) is one of the major forms of oxidative deoxyribonucleic acid (DNA) damage, and is commonly analyzed as an excellent marker of DNA lesions. The purpose of this study was to develop a sensitive method to accurately and rapidly quantify the 8-OHdG by using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The method involved the use of specific antibody to detect DNA lesions (8-OHdG) and consecutive fluorescence labeling. Next, the urine sample with 8-OHdG fluorescently labeled along with other constituents was resolved by capillary electrophoretic system and the lesion of interest was detected using fluorescence detector. The limit of detection was 0.18 fmol, which is sufficient sensitivity for detection and quantification of 8-OHdG in untreated urine samples. The relative standard deviation (RSD) was found to be 11.32 % for migration time, and 5.52 % for peak area. To demonstrate the utility of this method, the urinary concentration of 8-OHdG in an Alzheimer’s transgenic mouse model was determined. Collectively, our results indicate that this methodology offers great advantages such as high separation efficiency, good selectivity, low limit of detection (LOD), simplicity and low cost of analysis.

We here report an antibody functionalized microimmunoprecipitation (μIP) method used for enrich lowabundant post-translational modified (PTM) proteins. The device is a fabricated by inert, nontoxic and disposable polydimethylsiloxane (PDMS) using a silane-based chemical modification protocol, which yield an antibody-terminated PDMS surfaces. In this study, the μIP device is specifically designed for purification of carbonylated protein, a representative example here to illustrate the potential applications for any other PTMs, which could be immuno-tagged by specific antibodies. The test model- in vitro oxidized Bovine Serum Albumin (BSA) was first derivitized by dinitrophenylhydrazide (DNPH) and then captured by the anti-DNP immobilized on this μIP device. The surface functional group mapping was systematically analyzed and validated by fluorescence microscopy. Quantitative study of DNP-derivatized carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this proteome enrichment μIP device can be assembled with other lab-on-a-chip components, such as microelectrophoresis or micro-chromatographic devices for follow-up protein analysis. This selective enrichment of modified proteins greatly facilitates the study of low abundant protein biomarkers discovery.

S-nitrosylation (also referred to as nitrosation), a reversible post translational modification (PTM) of cysteine, plays an important role in cellular functions and cell signalling pathways. Nitrosylated proteins are considered as biomarkers of aging and Alzheimer's disease (AD). Microfluidics has been widely used for development of novel tools for separation of protein mixtures. Here we demonstrate two-dimensional micro-electrophoresis (2D μ-CE) separations of nitrosylated proteins from the human colon epithelial adenocarcinoma cells (HT-29) and AD transgenic mice brain tissues. Sodium dodecyl sulphate micro-capillary gel electrophoresis (SDS μ-CGE) and microemulsion electrokinetic chromatography (MEEKC) were used for the first and second dimensional separations, respectively. The effective separation lengths for both dimensions were 10 mm, and electrokinetic injection was used with field strength at 200 V cm(-1). After 80 s separation in the first CGE dimension, fractions were successfully transferred to a second MEEKC dimension for a short 10 s separation. We first demonstrate this 2D μ-CE separation by resolving five standard proteins with molecular weight (MW) ranging from 20 to 64 kDa. We also present a high peak capacity 3D landscape image of nitrosylated proteins from HT-29 cells before and following menadione (MQ) treatment to induce oxidative stress. Additionally, to illustrate the potential of the 2D μ-CE separation method for rapid profiling of oxidative stress-induced biomarkers implicated in AD disease, the nitrosylated protein fingerprints from 11-month-old AD transgenic mice brain and their age matched controls were also generated. To our knowledge, this is the first report on 2D profiling of nitrosylated proteins in biological samples on a microchip. The characteristics of this biomarker profiling will potentially serve as the screening for early detection of AD.

S-glutathionylation (Pr–SSG) is a specific post-translational modification of cysteine residues by the addition of glutathione. S-Glutathionylated proteins induced by oxidative or nitrosative stress play an essential role in understanding the pathogenesis of the aging and age-related disorder, such as Alzheimer’s disease (AD). The purpose of this research is to develop a novel and ultrasensitive method to accurately and rapidly quantify the Pr–SSG by using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). The derivatization method is based on the specific reduction of protein-bound S-glutathionylation with glutaredoxin (Grx) and labeling with thiol-reactive fluorescent dye (Dylight 488 maleimide). The experiments were performed by coupling the derivatization method with CGE-LIF to study electrophoretic profiling in in vitro oxidative stress model–S-glutathionylated bovine serum albumin (BSA-SSG), oxidant-induced human colon adenocarcinoma (HT-29) cells, brain tissues, and whole blood samples from an AD transgenic (Tg) mouse model. The results showed almost an eightfold increase in S-glutathionyl abundance when subjecting HT-29 cells in an oxidant environment, resulting in Pr–SSG at 232 ± 10.64 (average ±SD; n = 3) nmol/mg. In the AD–Tg mouse model, an initial quantitative measurement demonstrated the extent of protein S-glutathionylation in three brain regions (hippocampus, cerebellum, and cerebrum), ranging from 1 to 10 nmol/mg. Additionally, we described our developed method to potentially serve as a highly desirable diagnostic tool for monitoring S-glutathionylated protein profile in minuscule amount of whole blood. The whole blood samples for S-glutathionyl expression of 5-month-old AD–Tg mice are quantified as 16.3 μmol/L (=7.2 nmol/mg protein). Altogether, this is a fast, easy, and accurate method, reaching the lowest limit of Pr–SSG detection at 1.8 attomole (amol) level, reported to date.

We report a proof of principle study for the use of oxalyldihydrazide as a crosslinker for enrichment of carbonylated proteins within a microfluidic chip. Surface modification steps are characterized and analyzed using analytical techniques. We use oxidized cytochrome c as our model protein and demonstrate the chip's ability to capture carbonylated targets. After 100 min of continuous loading, the chip is capable of capturing 7.5 μg of carbonylated protein. All the proteins captured are eluted out of the chip using the elution protocol. Finally, we demonstrate the chip's specificity for oxidized targets by mixing oxidized cytochrome c and TRITC-BSA, with cytochrome c in low abundance. The results show that the chip is efficient at finding its target when unoxidized proteins are present. This is the first report to suggest the use of immobilized oxalyldihydrazide on a microchip as an enrichment methodology for low abundance proteins in a sample.