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Recently, the proteomic analysis has become an ideal tool to study the structure and function of platelets. We proposed a nano-liquid chromatography (nano-LC) technique coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) for rat platelet proteome analysis. In this study, we attempted to analyze the rat platelet proteome in two different subcellular fractions: cytosol and membrane. Platelet-rich plasma was collected from healthy rats. The platelet samples were extracted with Subcellular Proteome Extraction Kit to collect subcellular compartments. For further investigations, platelet lysate, cytosol and membrane fractions were used. Enzymatic digestion of proteins was performed using Filter Aided Sample Preparation method with trypsin as a proteolytic enzyme. Tryptic peptides were analyzed using nano-LC-MALDI-TOF/TOF-MS. Platelet proteins identification was performed using the Mascot engine. We identified 238 proteins in the platelet lysate, 210 in the cytosol, and 148 in the membrane fraction. Among them, 45 were unique for platelet lysate, 55 for cytosol, and 34 for the membrane fraction. The gene ontology analysis showed that there were differences in the proteome of cytosol and membrane fractions related to the molecular functions, i.e. coagulative activity. Our results may suggest that the membrane or cytosol location of the proteins with coagulative activity may be responsible for the acute or delayed platelet response to an agonist. The nano-LC-MALDI-TOF/TOF-MS method can be used for identifying proteins of subcellular fraction in rat platelets.

Unlabelled: Pregnant women are exposed to benzodiazepines for therapeutic purposes during gestation. The goal of this study was to evaluate prenatal exposure to benzodiazepines. Time of exposure during course of pregnancy is a significant aspect of fetal exposure to drugs. Benzodiazepine concentration assay in hair of mothers and newborns exposed prenatally to these drugs was performed in the studies. Development, validation and evaluation of benzodiazepine determination method in mothers and their newborns enables assessment of health risks for the child and implementation of adequate therapeutic procedures. We used A LC-ESI-MS/MS method that allowed determination of diazepam (the main benzodiazepine used by pregnant women was diazepam) and its metabolites (nordazepam, oxazepam) in hair of mothers and newborns. LOQ 10 pg/mg of hair was used in the study., Results: concentration of nordazepam was higher than parent drug (diazepam) and higher in newborns' hair when compared to mothers'. The mean concentrations of diazepam in mothers' hair were 31.6±36.0 and 34.1±42.4 pg/mg in the second and third trimester of pregnancy respectively. The mean concentration of diazepam in newborns' hair was higher and reached levels of 53.3±36.5 pg/mg. The mean concentration of nordazepam in the mothers' hair corresponding to the second and third trimester was 52.9±48.1 and 89.9±122.8 pg/mg, respectively. Nordazepam in the newborns' hair was detected at the mean level of 108.1±144.2 pg/mg. It was concluded that diazepam and nordazepam are permanently incorporated into the hair structure. Presence of diazepam and its metabolites in newborn's hair confirms that these benzodiazepines permeate placental barrier. Segmental analysis of mothers' hair enabled the assessment of drug administration time. Diazepam and its metabolites determined in hair of newborns may serve as biomarkers of prenatal exposure to these drugs. The performed LC-MS/MS analysis was accurate enough to determine even low concentrations of benzodiazepines, at the level of few pg/mg of hair. Levels of diazepam detected in hair of newborns were higher than levels determined in mothers. This may confirm the fact, that fetus's ability to metabolize diazepam is scarce. Nordazepam was found in higher concentrations in hair of newborns than in hair of mothers, which may suggest that it is cumulated in child's organism. Other metabolites of diazepam--oxazepam and temazepam--were detected in very few cases, in low concentrations.

The purpose of this study was to evaluate the influence of tobacco smoke on the pharmacokinetics of citalopram (CIT) and desmethylcitalopram (DCIT) and its enantiomers on an animal model. High performance liquid chromatography (HPLC) with a diode array detector (DAD) was used for the identification and quantification of the studied compounds. The HPLC quantification of racemic mixtures of CIT was performed on a C18 column. The limits of detection (LOD) and quantification (LOQ) were: 7 and 10 ng/ml respectively. HPLC separation of citalopram enantiomers (S- and R-CIT) was performed on a Chirobiotic V column. The limits of detection (LOD) and quantification (LOQ) were: 6 and 15 ng/ml for R- and S-CIT respectively. The experiment was carried out on male Wistar rats. The rats were exposed to tobacco smoke for five days (6 hours per day). After the exposure, citalopram was administered in a dose of 10 mg/kg intragastrically. In the control group (non-exposed animals), citalopram was administered in the same way and at an equal dose. The blood of the animals was collected at nine time points. It was found that tobacco smoke exposure inhibits the biotransformation of citalopram. The half-life of the racemic mixture of citalopram after intragastric administration was increased by about 287%. Changes in the pharmacokinetic parameters of S-citalopram (active isomer) show a similar tendency to those of the racemic mixture. The pharmacokinetics of R-citalopram showed no statistically important differences after tobacco smoke exposure. Alterations in the pharmacological parameters of desmethylcitalopram presented an opposite trend to the parent drug. After exposure to tobacco smoke, the induction of metabolism of this compound was observed.

Tobacco smoking during pregnancy is associated with a variety of negative consequences not only for the mother, but also for the developing fetus. Many studies have shown that carcinogens contained in tobacco smoke permeate across the placenta, and are found in fetus. The aim of the study was to determine the prenatal exposure to tobacco-specific carcinogenic N-nitrosamines on the basis of measurements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine of smoking and second-hand smoke (SHS) exposed women and in the first urine of their newborns. A questionnaire documenting demographics and socio-economical data, smoking habits and exposure to SHS was completed by 121 delivering women near or at term. Maternal concentrations of cotinine and NNAL were measured in urine of the mother and the first urine of her newborn infant by liquid chromatography tandem mass spectrometry (LC/MS/MS). The mean concentration of cotinine was 439.2 ng/mg creatinine and NNAL concentration in urine of smoking women was 74.0 pg/mg creatinine, and for her newborn 78.6 pg/mg creatinine. Among mothers exposed to SHS, cotinine and NNAL mean concentration were 23.1 ng/mg creatinine, and 26.4 pg/mg creatinine. In newborns of SHS exposed mothers during pregnancy the mean concentration of NNAL was 34.1 pg/mg creatinine, respectively. Active tobacco smoking as well as passive exposure to smoking during pregnancy is an important source of tobacco specific N-nitrosamines to the fetuses as evidenced by increased concentrations of this carcinogen. Determination of NNAL in maternal urine samples can be a useful biomarker of prenatal exposure of newborn to carcinogenic nitrosamines.