Continuous exposure of nicotine and cotinine retards human primary pterygium cell proliferation and migration

Pterygium is a triangular-shaped hyperplastic growth, characterized by conjunctivalization, inflammation, and connective tissue remodeling. Our previous meta-analysis found that cigarette smoking is associated with a reduced risk of pterygium. Yet, the biological effect of cigarette smoke components on pterygium has not been studied. Here we reported the proliferation and migration properties of human primary pterygium cells with continuous exposure to nicotine and cotinine. Human primary pterygium cells predominantly expressed the α5, β1, and γ subunits of the nicotinic acetylcholine receptor. Continuous exposure to the mixture of 0.15 μM nicotine and 2 μM cotinine retarded pterygium cell proliferation by 16.04% (P = 0.009) and hindered their migration by 11.93% ( P = 0.039), without affecting cell apoptosis. SNAIL and α-smooth muscle actin protein expression was significantly downregulated in pterygium cells treated with 0.15 μM nicotine-2 μM cotinine mixture by 1.33- ( P = 0.036) and 1.31-fold ( P = 0.001), respectively. Besides, the 0.15 μM nicotine-2 μM cotinine mixture also reduced matrix metalloproteinase (MMP)-1 and MMP-9 expressions in pterygium cells by 1.56- ( P = 0.043) and 1.27-fold ( P = 0.012), respectively. In summary, this study revealed that continuous exposure of nicotine and cotinine inhibited human primary pterygium cell proliferation and migration in vitro by reducing epithelial-to-mesenchymal transition and MMP protein expression, partially explaining the lower incidence of pterygium in cigarette smokers.     

In vitro effect of nicotine and cotinine on the susceptibility of LDL oxidation and hemoglobin glycosylation

Nicotine, a major component of cigarette smoke, plays an important role in the development of cardiovascular disease and lung cancer in smokers. This study was designed to determine the in vitro effects of nicotine and its metabolite cotinine on the susceptibility of LDL to oxidation and hemoglobin glycosylation. Three different concentrations of each one (10, 15, 25 g/ml) were used. Our data show that nicotine and cotinine are inhibitors for Cu²⁺-induced LDL oxidation but also they increase the glycosylation degree of hemoglobin. Nicotine at final concentrations of (10, 15, 25 g/ml) increases the rate of hemoglobin glycosylation 25, 32 and 47%, respectively, and cotinine at final concentrations increase the rate of glycosylation 8, 10 and 12%, respectively. Therefore promoting hemoglobin glycosylation is one of the alternations caused by smoking that increase risk of cardiovascular disease.     

Simultaneous estimation of nicotine and cotinine levels in biological fluids using high-resolution capillary-column gas chromatography combined with solid phase extraction work-up

A rapid and sensitive capillary gas-chromatographic method with nitrogen-sensitive detection is reported for the simultaneous analysis of nicotine and cotinine levels occurring in the plasma, saliva, and urine of regular tobacco smokers. The proposed assay has a linear output, has satisfactory accuracy over the range of concentrations of both amines encountered in active smokers, and has also been successful in the analysis of the urine samples of passive smokers. Its lower limit of sensitivity is 0.2 ng of nicotine and 0.5 ng of cotinine per ml of plasma or saliva or per 100 l of urine.The beneficial characteristics of the presented method were achieved by the combination of solid phase extraction of 0.1–1.0 ml of fluid specimens, capillary column gas chromatography with splitless injection and nitrogen sensitive detection, and the use of separate, structurally analogous compounds as internal standards for nicotine. The suitability of the assay is shown by plasma concentration-time curves of nicotine and cotinine in a steady smoker during a 24 hours period.     

Separation of pH,dilution, lonic strength and chemical matrix effects for biological monitoring of urines with the Microtox® test using nicotine,cotinine and reference urines

The aim was to investigate the factors influencing light emission from Photobacterium phosphoreum in the Microtox® test to interpret bioassay results for urine. Four reference urines were assessed as reference materials for the bioassay. Nicotine and cotinine were investigated as urinary markers for tobacco exposure. The optimum luminescence conditions were: 1.85%–3.25% NaCl, 0.33–0.58 mol/L ionic strength, and pH 5.8–6.7. Low pH values and high concentration of toxic trace metals were important factors in this study. Unexpacted toxicity for a Standard Reference Material was attributed to zinc contamination. Nicotine and cotinine together exhibited antagonistic effects in 2% saline but this could not be observed in the urines because of substantial urine toxicity. Thus practical urinary biological monitoring with the Microtox® test necessitates excretion of metabolites and compounds that are much more toxic than the urine components. Also, separation of the effects of physical factors like pH, ionic strength and dilution is essential before chemical toxicity effects can be assigned. This is the first report of Microtox® EC₅₀ values for nicotine and cotinine. The results have application to environmental samples since analyses are often uncontrolled relative to pH, ionic strength and dilution.     

Relationship of advanced oxidative protein products in human saliva and plasma: age- and gender-related changes and stability during storage

Abstract

The blood levels of advanced oxidative protein products (AOPP) elevate in aging and age-related diseases. However, saliva AOPP in healthy humans have been unexplored. Thus, we investigated 143 Chinese healthy adults to assay age- and gender-related changes in saliva and plasma AOPP levels and the stability of saliva AOPP stored both at ??20°C and ??80°C. We found the mean AOPP levels in saliva and plasma of 119 subjects were 7.51?±?3.20 and 28.31?±?5.53 μmol/L (μM). An age-dependent increase was observed in both saliva and plasma AOPP levels. This increase was particularly significant in the elderly subjects compared with that in the young and middle-aged ones. A significant positive correlation among age, saliva and plasma AOPP levels was observed. No gender-dependent difference was observed in either saliva or plasma AOPP levels during the aging process. Furthermore, AOPP levels in the 24 saliva samples showed no significant change at every successive determination during 4 weeks at ??80°C, whereas those levels significantly increased after 7 days of storage at ??20°C. These results indicate the feasibility to screen aging biochemical indicators using saliva AOPP as an alternative to blood AOPP. Saliva AOPP samples are suitable to be stored at ??80°C.     

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

Isolation and microsequencing of a novel cotinine receptor

SUMMARY 1. Nicotine and its main derivative, cotinine, are reported to have distinct central activities in mammals. In this study, the cotinine receptor was separated by biochemical procedures including radio receptor, affinity-chromatography, SDS–PAGE, and N-terminal sequencing assays.2. Consistently, the results showed that distinctive cotinine receptors exist in different tissues of mammals. In rat brain, the affinity chromatography and [¹²⁵I]cotinine receptor essays were used to isolate a 40-kDa protein (p40) with higher affinity for cotinine than alpha-bungarotoxin and nicotine. The N-terminus amino acid sequences of the p40 and its internal tryptic peptides showed no identity to recently described protein sequences, with the exception of homology to the human p205 synovial fluid protein.3. These results, in agreement with other behavioral studies, are the first molecular evidence for distinctive nicotine and cotinine receptors in mammals.     

Determination of nicotine,anabasine, and cotinine in urine and saliva samples using single-drop microextraction

A simple, sensitive, and inexpensive singe-drop microextraction (SDME) followed by gas chromatography and flame-ionization detection (GC-FID) was developed for determination of nicotine, anabasine, and cotinine in human urine and saliva samples. The target compounds were extracted from alkaline aqueous sample solution into an organic acceptor drop suspended on the tip of a 25-μL GC microsyringe in the aqueous sample solution. This microsyringe was also used for direct injection after extraction. Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.5–25.0, 0.5–65.0, and 0.5–45.0 mg L^?1 for nicotine, anabasines and cotinine, respectively. The method detection limit values were in the range of 0.33–0.45 mg L^?1. Intra-day and inter-day precisions for peak area ratios were in the range of 1.3–9.2% and 2.0–7.0%, respectively. The proposed procedure was successfully applied to the determination of analytes in spiked urine and saliva samples with satisfactory results. The mean relative recoveries of spiked water samples ranged over 71.2–111.0%, with relative standard deviations varying from 2.3% to 10.0%.     

Effect of nicotine on basal and bombesin stimulated canine plasma levels of gastrin, cholecystokinin and pancreatic polypeptide

The influence of nicotine on the basal and bombesin (BBS) stimulated plasma levels of gastrin, cholecystokinin (CCK) and pancreatic polypeptide (PP) was investigated in conscious dogs. Plasma levels of nicotine and gastrointestinal (GI) hormones were measured by employing gas liquid chromatography and specific radioimmunoassay (RIA). The basal levels of gastrin, CCK and PP were found to be in pg/ml (pmol/l) (mean +/- S.E.), 28 +/- 5 (13 +/- 3), 252 +/- 32 (66 +/- 8) and 347 +/- 136 (83 +/- 32), respectively and these values remained unchanged with nicotine. Significant increases in levels of gastrin, CCK and PP were, however, found with infusions of BBS alone or with BBS in combination with nicotine. Gastrin levels were higher whereas CCK and PP levels were lower with BBS alone than with BBS plus nicotine. The peak values for CCK and PP, but not gastrin, were less during second BBS infusion. These results indicate that nicotine, in presence of bombesin, has an inhibitory effect on the release of gastrin and a stimulatory effect on the release of PP and CCK.     

Origin, structure, and biological activities of peroxidases in human saliva

Human whole saliva contains two peroxidases, salivary peroxidase (hSPO) and myeloperoxidase (hMPO), which are part of the innate host defence in oral cavity. Both hSPO as well as human milk lactoperoxidase (hLPO) are coded by the same gene, but to what extent the different producing glands, salivary and mammary glands, affect the final conformation of the enzymes is not known. In human saliva the major function of hSPO and hMPO is to catalyze the oxidation of thiocyanate (SCN(-)) in the presence of hydrogen peroxide (H(2)O(2)) resulting in end products of wide antimicrobial potential. In addition cytotoxic H(2)O(2) is degraded. Similar peroxidation reactions inactivate some mutagenic and carcinogenic compounds, which suggests another protective mechanism of peroxidases in human saliva. Although being target of an active antimicrobial research, the structure-function relationships of hSPO are poorly known. However, recently published method for recombinant hSPO production offers new tools for those investigations.     

Analysis of the human saliva proteome

Interest in the characterization of the salivary proteome has increased in the last few years. This review discusses the different techniques and methodologies applied to the separation and identification of salivary proteins. Nowadays, proteomic techniques are the state of the art for the analysis of biologic materials and saliva is no exception. 2D electrophoresis and tryptic digest analysis by mass spectrometry are the typical methodology, but new approaches using 2D liquid chromatography/mass spectrometry methods have already been introduced for saliva analysis. Due to their important physiologic role in the oral cavity, low-molecular-weight proteins and peptides are also included in this article and the methodologies discussed.     

Protein content of human saliva in various psycho-emotional states

The protein composition of human saliva depends on psycho-emotional state of individuals. Depression was accompanied by decrease of proteins of molecular masses ranging from 20 to 200 kD, whereas emotionally positive intellectual activity caused the opposite effect. It is suggested that human saliva may be used as an experimental model for the development of diagnostics of various psycho-physiological states.     

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C₁₈ column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.     

A comparison of native and non-urate Total Antioxidant Capacity of fasting plasma and saliva among middle-aged and older subjects

Objectives: As plasma and salivary total antioxidant capacity (TAC) is mainly contributed by uric acid (UA), the present study measures non-urate TAC (Nu-TAC). The aim of the study was to correlate plasma native TAC, Nu-TAC and UA with their salivary analogues, and compare the UA contribution in both body fluids using two different methods.

Methods: The study involved 55 middle-aged and older subjects (66.7?±?4.5 years). TAC was determined simultaneously with two methods (ferric reducing ability of plasma – FRAP, 2.2-diphenyl-1-picryl-hydrazyl – DPPH and countertypes for saliva – FRAS and DPPHS test), with and without UA (native TAC and Nu-TAC, respectively). Plasma UA and salivary UA (SUA) were assessed.

Results: Subjects with increased FRAP, DPPH and UA had higher FRAS, DPPHS and SUA, respectively (P?P?Discussion: Our findings suggest that saliva is a good predictor for native plasma TAC but not for Nu-TAC. UA level is comparably dominant in saliva and in plasma according to DPPH, but lower in plasma according to FRAP.     

Analysis of risperidone and 9-hydroxyrisperidone in human plasma, urine and saliva by MEPS-LC-UV

Risperidone is currently one of the most frequently prescribed atypical antipsychotic drugs; its main active metabolite 9-hydroxyrisperidone contributes significantly to the therapeutic effects observed. An original analytical method is presented for the simultaneous analysis of risperidone and the metabolite in plasma, urine and saliva by high-performance liquid chromatography coupled to an original sample pre-treatment procedure based on micro-extraction by packed sorbent (MEPS). The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 73% (v/v) acidic phosphate buffer (30 mM, pH 3.0) containing 0.23% triethylamine and 27% (v/v) acetonitrile. The UV detector was set at 238 nm and diphenhydramine was used as the internal standard. The sample pre-treatment by MEPS was carried out on a C8 sorbent. The extraction yields values were higher than 92% for risperidone and 90% for 9-hydroxyrisperidone, with RSD for precision always lower than 7.9% for both analytes. Limit of quantification values in the different matrices were 4 ng/mL or lower for risperidone and 6 ng/mL or lower for the metabolite. The method was successfully applied to plasma, urine and saliva samples from psychotic patients undergoing therapy with risperidone, with satisfactory accuracy results (recovery>89%) and no interference from other drugs. Thus, the method seems to be suitable for the therapeutic drug monitoring of schizophrenic patients using the three different biological matrices plasma, urine and saliva.     

Determination of nicotine and cotinine in human serum by means of LC/MS

As part of a joint clinical research project to study the effects of nicotine on the brain, a HPLC electrospray ionisation mass spectrometry method with a solid-phase extraction sample preparation was developed for the quantitative determination of nicotine and cotinine in human serum in volunteers. The measured concentrations of nicotine and cotinine were used as control for smoking behaviour. A X-Bridge-column from Waters, and a SSQ 7000 single quadropole mass spectrometer with a TSP liquid chromatographic system were used. The method includes a simple and robust sample preparation and this assay has been shown to be of a sufficient sensitivity for this application. The limits of quantification were 5 and 2 ng/ml for cotinine and nicotine, respectively. A simultaneous study was conducted to measure nicotine receptor availability and the vigilance in the same group of volunteers.     

High-performance liquid chromatographic assay for N-glucuronidation of nicotine and cotinine in human liver microsomes

A method for the determination of N-glucuronidation of nicotine and cotinine in human liver microsomes by high-performance liquid chromatography was developed. Nicotine or cotinine was incubated with human liver microsomes and UDP-glucuronic acid in a 200-microl incubation mixture. The nicotine N-glucuronide (Nic-glu) and cotinine N-glucuronide (Cot-glu) formed were analyzed by ion-pair chromatography with a C-18 column. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3, and the limit of quantification was 10 pmol/200 microl mixture for both Nic-glu and Cot-glu. Linear standard curves were obtained within the concentration ranges 25-1000 pmol/200 microl mixture for Nic-glu and 100-5000 pmol/200 microl mixture for Cot-glu. The intraassay precision and accuracy were < or =11.1% coefficient of variation (CV) and 97.5-106.6% for Nic-glu and < or =4.6% CV and 96.7-100.4% for Cot-glu. The interassay precision and accuracy were < or =7.2% CV and 98.2-106.1% for Nic-glu and < or =4.6% CV and 96.8-99.3% for Cot-glu. This is the first report of the in vitro determination of Nic-glu and Cot-glu in human liver microsomes. Furthermore, this highly sensitive HPLC method can be used for the determination of Nic-glu and Cot-glu in biological specimens in vivo.     

Quantification of manganese and mercury in toenail in vivo using portable X-ray fluorescence (XRF)

Background and objective: Toenail is an advantageous biomarker to assess exposure to metals such as manganese and mercury. Toenail Mn and Hg are in general analyzed by chemical methods such as inductively coupled plasma mass spectrometry and atomic absorption spectrophotometry. In this project, a practical and convenient technology—portable X-ray florescence (XRF)—is studied for the noninvasive in vivo quantification of manganese and mercury in toenail.

Material and methods: The portable XRF method has advantages in that it does not require toenail clipping and it can be done in 3?min, which will greatly benefit human studies involving the assessment of manganese and mercury exposures. This study mainly focused on the methodology development and validation which includes spectral analysis, system calibration, the effect of toenail thickness, and the detection limit of the system. Manganese- and mercury-doped toenail phantoms were made. Calibration lines were established for these measurements.

Results: The results show that the detection limit for manganese is 3.65?μg/g (ppm) and for mercury is 0.55?μg/g (ppm) using 1?mm thick nail phantoms with 10?mm soft tissue underneath.

Discussion and conclusion: We conclude that portable XRF is a valuable and sensitive technology to quantify toenail manganese and mercury in vivo.     

Simultaneous determination of nicotine and cotinine in plasma using capillary column gas chromatography with nitrogen-sensitive detection

A rapid and sensitive method is described for the simultaneous determination of nicotine and its principal metabolite, cotinine, in plasma. A one-step extraction procedure is employed and the quantitative analyses are performed by capillary column gas chromatography using a thermionic specific detector. Other special measures to avoid contamination from external sources such as atmosphere, solvents and laboratory equipment, which constitutes the major limiting factor of nicotine assay, were also undertaken. The structural analogues of nicotine and cotinine, N-methylanabasine and N-ethylnorcotinine, are used as internal standards. Moreover, a micromethod, which requires only 0.1 ml of plasma and found to be suitable for analysis of cotinine in finger-tip samples of blood, is described. Linearity over the concentration ranges 5–100 ng of nicotine per ml of plasma and 5–500 ng of cotinine per ml of plasma is demonstrated. The precision of the method has been investigated by determining the reproducibility at different levels of nicotine and cotinine within the working ranges, for both 1-ml and 0.1-ml samples of plasma.