Determination of Cotinine, 3'-Hydroxycotinine and Nicotine 1'-Oxide in Urine of Passive and Active Young Smokers by LC-Orbitrap-MS/MS Technique.

Tobacco smoke is probably the most significant factor conducing to toxic xenobiotics exposure to humans. The aim of the study was to develop a rapid and sensitive method for the determination of selected nicotine metabolites in urine of tobacco smokers and passive smokers. The method for removing protein and extracting the metabolites involved the centrifugation of urine with acetonitrile. Cotinine, trans-3'-hydroxycotinine, and (2'S)-nicotine 1'-oxide in the supernatant were determined using the LC-Orbitrap-MS/MS technique, with the selected ion monitoring (SIM) and parallel reaction monitoring (PRM) modes used. The recovery of these analytes added to the urine samples ranged from 72% to 101%. Repeatability and reproducibility were less than 3.1% and 10.1%, respectively. The study was carried out among medical students. The group was selected as representatives of young people and who as future physicians should be more aware of the effects of nicotine use. Concentration levels of cotinine and trans-3'-hydroxycotinine determined in ng/mL in the urine of cigarette smokers were 70- and 58-fold higher, respectively, compared to passive smokers. Higher concentrations were recorded in the urine of those passively exposed to tobacco smoke than in non-smokers, confirming that passive exposure to tobacco smoke is not harmless to the human body. However, no significant differences were observed in the concentration of (1'S,2'S)-nicotine 1'-oxide in the samples of individuals from various groups.

Women suffering from systemic lupus erythematosus are characterized by low blood levels of α-dicarbonyl compounds.

Introduction: Systemic lupus erythematosus (SLE) is a chronic, autoimmune disease, often characterised by severe course and unclear etiopathogenesis. The reaction of protein glycoxidation, also known as glycation, may be linked to etiopathogenesis of SLE. Advanced glycation end-products (AGEs) exhibit cytotoxic properties, affect cellular signalling, impair functions of extracellular proteins, and may act as neoepitopes. Glucosone (GS), glyoxal (GO), and methylglyoxal (MGO) are examples of α-dicarbonyl compounds (α-DCs) partaking in glycoxidation. The study aimed to evaluate concentrations of these three compounds in blood serum of SLE patients, and to compare the results with healthy individuals. Material and methods: 31 women suffering from SLE and 26 healthy individuals were included in the study. High-performance liquid chromatography with fluorescence detection was applied to evaluate concentrations of α-DCs in their serum samples. Correlations between the results and parameters such as disease duration time, age, glomerular filtration rate (GFR), Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K), and creatinine were analysed. Results: The SLE patients exhibited lower concentrations of glucosone, glyoxal, and methylglyoxal than the control group. Analysis of correlations showed a difference between the examined groups. Conclusions: In women suffering from SLE the course of α-DCs metabolism is altered. SLE patients are characterised by low serum levels of α-DCs. We hypothesise that either hindered proteasomal degradation or fast consumption of α-DCs in oxidative conditions may cause the observed low concentration of these compounds.

Polycyclic Aromatic Hydrocarbons (PAHs) in Grilled Marshmallows.

The aim of this study was to assess potential health risks among children and adolescents consuming various grilled marshmallows using a survey and to determine polycyclic aromatic hydrocarbons (PAHs) in these food products. PAH analysis in grilled marshmallows included a dilution stage with deionized water and liquid-liquid extraction with cyclohexane and solid-phase extraction (SPE). PAH fractions were initially analyzed via high-performance thin-layer chromatography, and PAH concentrations were determined via gas chromatography with a tandem mass detector using the selective reaction monitoring (SRM) mode. This study on the consumption of grilled marshmallows was conducted among approximately 300 children and adolescents. The preliminary results indicated that "raw" marshmallows did not contain PAHs. However, the obtained data suggested the exposure of young people to carcinogenic PAHs from grilled marshmallows (63.5% of them consumed marshmallows). Carcinogenic benzo(a)pyrene (BaP) was determined in all samples. The profile of PAH concentrations in the extracts isolated from various grilled types of marshmallows was similar (r2 > 0.8000), regardless of the grilling method. Compared to the white sugar confection, higher concentrations of PAHs were determined in multicolored marshmallows. The lack of social awareness about exposure to carcinogenic substances is alarming.

Application of High-Performance Liquid Chromatography with Fluorescence Detection for Non-Polar Heterocyclic Aromatic Amines and Acridine Derivatives Determination in Pork Loin Roasted in a Roasting Bag.

Heat treatment of meat can lead to the formation of carcinogenic organic compounds. The influence of dried fruits on the formation of non-polar heterocyclic aromatic amines (carbolines) and nitrogen derivatives of polycyclic aromatic hydrocarbons (azaarenes) in roasted pork loin was elucidated. Two hundred grams of fruit per 1 kg of meat were used as stuffing. Carbolines, derivatives of pyridoimidazole and pyridoindole, and azaarenes (benzoacridines and dibenzoacridines) were determined by means of high-performance liquid chromatography with fluorescence detection. The total concentration of six δ-, γ- and α-carbolines in roasted pork loin was 1.3 ng/g. This content decreased by 64%, 58%, and 54% in pork loin stuffed with prunes, apricots, and cranberries, respectively. Concentrations of ß-carbolines (harmane and norharmane) increased under the influence of added fruits. The norharmane content increased the most, from 2.2 ng/g in the control sample to 12.3 ng/g in meat prepared with cranberries. The harmane content increased from 1.0 ng/g to 3.6 ng/g in meat with prunes. The total concentration of azaarenes (two benzoacridines and dibenzo[a,c]acridine), which was close to 0.1 ng/g, decreased in dishes with prunes and apricots by 54% and 12%, respectively. Azaarenes were not found in samples of meat stuffed with cranberries.

The effect of onion and garlic on non-polar heterocyclic aromatic amines (α-, ß-, γ- and δ-carbolines) formation in pan-fried meat and gravy.

Thermal treatment of protein-rich food can lead to the formation of biologically active heterocyclic aromatic amines (HAAs). One of the methods to learn how to reduce the content as well as the influence of these compounds on heath is the study of factors inhibiting their synthesis. In the current investigation, the effect of onion and garlic on the formation of six possibly carcinogenic non-polar HAAs (α-, γ- and δ-carbolines) and two co-mutagenic ß-carbolines (harmane and norharmane) was evaluated by comparing their contents in meat and gravy samples obtained from pan-fried pork dishes prepared in the presence and absence of these vegetables. Carbolines were isolated from food samples by solid phase extraction. The quantitative analysis was performed by high-performance liquid chromatography with fluorescence detection. The concentrations of individual compounds in dishes prepared without added vegetables ranged from 0.02 ng g-1 (3-amino-1,4-dimethyl-5 H-pyrido(4,3-b)indole; Trp-P-1) to 10.1 ng g-1 of meat (2-amino-9 H-pyrido[2,3-b]indole; AαC). Onion (30 g/100 g of meat) and garlic (15 g/100 g of meat) lowered the total content (in meat and gravy) of the α-, δ- and γ-carbolines in the range from 52% to 87%. In contrast, onion caused an increase in the norharmane concentration both in meat and gravy. The percentage of carbolines in the gravies (assuming that their total content in meat and gravy is 100%) was higher in dishes prepared with onion and garlic than in dishes without these seasonings.

Antibodies to Heat Shock Proteins 90α and 90ß in Psoriasis.

One of many hypotheses of psoriasis pathogenesis supposes an overexpression of heat shock proteins (Hsps) in different skin layers and systemic immunologic response to them. Hsp90 is one of the most abundant chaperone in eukaryotic cells. The number of studies concerning the role of Hsp90 and anti-Hsp90 antibodies in etiopathogenesis of various diseases is also constantly expanding. Still, there are not many reports concerning potential involvement of this Hsp family or anti-Hsp90 immunization in pathomechanism of psoriasis. The aim of the study was the estimation of anti-Hsp90α and anti-Hsp90ß IgG antibodies in the sera of the psoriatic patients at different phases of disease activity in comparison to the sera of healthy individuals. The study material consisted of sera from psoriasis patients (n = 80) in active phase and in the remission phase and healthy individuals (n = 80). Concentrations of anti-Hsp90α and anti-Hsp90ß IgG antibodies were determined using ELISA technique. In the patients with psoriasis (both in the active phase of the disease and in the remission phase) concentrations of anti-Hsp90α antibodies were significantly higher than in healthy individuals and they correlated positively with psoriasis area severity index values. The mean concentrations of anti-Hsp90ß antibodies in the psoriatic patients and healthy controls were comparable. The obtained results indicate an existence of increased immunological response to Hsp90α in psoriasis. It may suggest the role of the extracellular form of this chaperone and/or anti-Hsp90α antibodies in etiopathogenesis of this dermatosis. The inhibition of Hsp90α may represent a novel therapeutic approach to treat psoriasis.

Pregnancy-Associated Plasma Protein A (PAPP-A) Concentration in Population of Healthy Young People: Interactions with Tobacco Smoke and Anti-oxidative Status.

Pregnancy-associated plasma protein A (PAPP-A) is a high-molecular zinc-binding metalloproteinase that was first detected in the serum of pregnant women. It can also be detected in men and non-pregnant women. Recently, a growing interest in determining the concentration of PAPP-A as a marker of oxidative stress and atherosclerotic processes has been observed. Among the factors that could potentially influence the PAPP-A formation is the exposure to tobacco smoke. Some components of tobacco smoke have an immediate effect on the body and also direct influence on the cardiovascular system. The aim of this study was to evaluate the relation between PAPP-A concentration and either passive or active exposure to tobacco smoke in the population of medicine students (n = 152). The relation between PAPP-A concentration and chosen markers of inflammatory response and anti-oxidative processes was analyzed. The samples of serum, urine, and saliva were collected and main nicotine metabolites in urine samples were determined using ELISA technique. Comparison of the PAPP-A concentrations in the study group revealed that in the group of active smokers, the concentration of the protein was significantly higher than in the group of passive smokers (p = .04) and the group of not-exposed students (p = .006). PAPP-A concentration showed significant positive correlation with the values of FRAP and main nicotine metabolites. The evident influence of both active and passive tobacco smoke exposure on PAPP-A levels in the studied population of young people who in general are not included in the group of high-risk cardiovascular incidents, shows how important early prevention of anti-health behaviors is.

Human exposure to biologically active heterocyclic aromatic amines arising from thermal processing of protein rich food

Frequent consumption of thermally processed meat was classified by the International Agency for Research on Cancer to directly carcinogenic agents for humans. During the heat treatment of high protein food, mutagenic and carcinogenic, as well as neurotoxic heterocyclic aromatic amines are formed. Epidemiological studies confirm that exposure to some of these compounds may increase the risk of cancer in humans, especially the colon cancer. Most heterocyclic amines contain fried and grilled meat products, and the lowest content of these compounds can be found in boiled and slightly baked dishes. The use of spices and vegetable additives with antioxidant properties allows to obtain dishes with reduced content of these xenobiotics. An effective way to reduce human exposure to cancerogenic amines may be simultaneous consumption, together with meat dishes, products containing fiber which can adsorb molecules of heterocyclic amines in the gastrointestinal tract, as well as enrichment of the diet in the crucifers plants, as isothiocyanates released from them can inhibit the metabolic activation processes of heterocyclic amines. Raising the public awareness of the formation of mutagenic and carcinogenic compounds, including heterocyclic aromatic amines, during the intensive heat treatment of high protein food, as well as the dissemination of knowledge on the conditions regarding the preparation of dishes with reduced content of such compounds could become one of the components of cancer prevention programs in Poland.

Determination of advanced glycation end-products and antibodies against them (anti-CML and anti-CEL) in the serum of Graves' orbitopathy patients before and after methylprednisolone treatment.

INTRODUCTION: The glycation process is a non-enzymatic modification of proteins occurring due to the reactions of reductive carbohydrates. The glycated residues lose their biological functions, and their removal process is ineffective. They accumulate, and as a result they cause an immunological response. The aim of this study was a determination of the concentrations of advanced glycation end-products and antibodies against carboxymethyl lysine (anti-CML) and carboxyethyl lysine (anti-CEL) in the sera of Graves' orbitopathy patients. MATERIAL AND METHODS: The study group were patients from the Division of Endocrinology of the Medical University of Silesia (n = 25) suffering from Graves' orbitopathy. The concentration of AGE-peptides using flow spectrofluorimetry method, and anti-CML and anti-CEL IgG antibodies using immunoenzymatic technique (ELISA), were measured in patients sera before and after methylprednisolone treatment. RESULTS: In sera of the study group the concentrations of AGE-peptides and anti-CML were significantly lower before and after treatment in comparison to the control group (p < 0.05). Mean values of anti-CEL concentrations were comparable (at both phases of treatment) with the value observed in the control group. After treatment the concentrations of AGE-peptides and anti-CEL significantly decreased (p < 0.05); however, the concentration of anti-CML was also lower but the observed change was not significant (p > 0.05). CONCLUSIONS: In the course of Graves' orbitopathy the glycation process is disturbed. The treatment modifies significantly the process by lowering the concentration of advanced glycation end-products and suppressing the immune response to them. (Endokrynol Pol 2016; 67 (4): 383-389).

[Environmental tobacco smoke--assessment of formaldehyde concentration in urine samples of exposed medicine students]. / Srodowiskowy dym tytoniowy--ocena stezen formaldehydu w moczu narazonych studentów medycyny.

Environmental Tobacco Smoke (ETS) is ranked as one of the factors of confirmed carcinogenicity to human. It consists of the mixture of smoke exhaled by the smoker as well as the sidestream smoke and contains many times higher concentrations of some toxic substances in comparison to the amount of toxic compounds inhaled by a smoker. From many years the issue of passive smoking has been the subject of many research and still not all of its aspects of affecting human health have been explored. Apart from the tobacco varieties, also diverse additives added during the process of tobacco manufacturing, including particularly carbohydrates, influence the composition of the environmental tobacco smoke. During smoking they can undergo many complex transformations, as a result of which toxic components of the environmental tobacco smoke are formed, carbonyl compounds in particular, like aldehydes. They are marked by a significant chemical reactivity which enables them to modify amino groups of proteins leading to the changes in their structure, biological functions and often antigenicity. Therefore their influence to the human body is the cause of numerous adverse health effects caused by the increase in free radical processes which can constitute to the source of these compounds. Well known representative of this group of xenobiotics is formaldehyde as a compound that reflects well the environmental exposure to carbonyl compounds. The considerable source of this compound is tobacco smoke. Therefore analysis of formaldehyde in body fluids is a valuable biomonitoring tool of exposure to it. The aim of this study was the evaluation of formaldehyde concentration in urine samples of medicine students exposed to ETS. The study material consisted of 149 urine samples of students from School of Medicine with the Division of Dentistry in Zabrze, Medical University of Silesia. The concentration of formaldehyde in urine samples was determined by a spectrophotometric method using the Purpald reagent. To verify the collected questionnaire data regarding exposure to constituents of tobacco smoke, the immuno-enzymatic method was used to determine main nicotine metabolites in tested urine samples. This enabled dividing the investigated students' group into active smokers, passively exposed to tobacco smoke and not exposed. Analysis of obtained results showed that mean concentration of formaldehyde in urine of active smokers (68.45 ± 58.67 µmol/l) and passive smokers (79.23 ± 53.64 µmol/l) were significantly higher in comparison to not exposed students (42.99 ± 30.29 µmol/l). Mean concentrations of formaldehyde in urine samples of active and passive smokers are comparable. The results of our study allow to conclude that passive exposure to tobacco smoke is an equivalent source of exposure to active smoking regarding formaldehyde adverse influence to human. Applied method enables to quick evaluation of formaldehyde concentration in biological samples.

[Non-smokers about smokers]. / Niepalacy o palaczach.

Although the knowledge that smoking is a health hazard is widely accessible, this addiction remains serious social problem among young generation. It poses a great challenge to therapists, economic consequences impair both national and family budgets and that is a reason why governments and publicly-founded institutions promote anti-nicotine behaviours. But still rate of young smokers is very high. Aim of work was to get a real picture of smokers seen through nonsmoker's eyes. The used questionnaire was prepared exclusively for that survey. There were 97 students of medicine that participated in the poll. The poll showed 70 of them had a negative picture of smokers. Soft personality given in to addiction was the main feature. The knowledge about compounds of tobacco smoke was very poor, much like its influence on the human body. Survey showed general need for further education in that matter during medical studies.

Chronic spontaneous urticaria is characterized by lower serum advanced glycation end-products.

BACKGROUND: Chronic spontaneous urticaria (CSU) is associated with activation of acute phase response. On the other hand, it is known that systemic inflammation may lead to increased formation of advanced glycation end-products (AGEs), associated with pathogenesis of various diseases. AIM: We aim to test whether chronic inflammation manifested by activated acute phase response may provide a mechanism for increased serum AGEs concentration in CSU. METHODS: Concentrations of AGEs were measured spectrofluorimetrically in serum of CSU patients and the healthy subjects. RESULTS: Serum AGEs and albumin concentrations in CSU patients were significantly lower as compared with the healthy subjects. Serum CRP concentration was significantly higher in patients with CSU than in the controls. Significant positive correlation was observed between AGEs and albumin concentrations in the subjects. CONCLUSIONS: CSU is not associated with increased circulating AGEs concentrations, despite the enhanced systemic inflammatory response. Paradoxical decrease of serum AGEs concentrations is probably a reflection of lower concentration of "negative acute phase proteins" such as albumin.

Medicine students and exposure to environmental tobacco smoke.

OBJECTIVE: Although medicine students express positive attitudes toward providing lifestyle counseling, they require more instruction in many areas of health behavior in order to be helpful to their patients. The presented study included the students' questionnaires analysis regarding their lifestyle and exposure to tobacco smoke. The aim of this study was to examine students' exposure to chosen xenobiotics by determination of selected biomarkers in urine samples, which underlay the basis for exposure assessment towards tobacco smoke. MATERIALS AND METHODS: The investigated group consisted of first- and second-year medicine students from the Silesian Medical University (N = 133). Data obtained from a questionnaire survey was compared with the results of chosen biomarkers determined in urine samples. The analyses of the main nicotine metabolites were carried out firstly with use of ELISA, followed by the TLC technique with densitometry. RESULTS: According to questionnaires, every third student examined was exposed to passive smoking. The mean concentration of the main nicotine metabolites determined by ELISA in urine samples of smoking students was 1293.52±396.70 µg/g creatinine. The results of the TLC analysis in the group of smoking students were as follows: for cotinine - 523.10±68.10 µg/g creatinine and for trans-3'-hydroxycotinine - 653.81±62.30 µg/g creatinine. CONCLUSIONS: Medicine students, regardless of their area of study, are a highly-exposed part of the population to tobacco smoke, not only actively but also passively. Tobacco smoke exposure can be assessed by ELISA as a screening method as well as by more specific TLC technique with densitometry.

[Evaluation of the influence of exposure to tobacco smoke on the concentration of the pregnancy-associated plasma protein A in the population of healthy men and non-pregnant women]. / Ocena wplywu dymu tytoniowego na stezenie osoczowego bialka ciazowego typu A w populacji zdrowych mezczyzn i kobiet nieciezarnych.

The chemical variety of tobacco smoke components induces a multidirectional, not yet fully understood effects on living organisms, including both those actively and passively exposed. The chemical compounds present in tobacco smoke, due to its heterogeneous structure and properties undergo different biotransformation processes. Some components of tobacco smoke (for example, carbon monoxide) have an immediate effect on the body, in this case due to the formation of carboxyhemoglobin, and nicotine contained in the tobacco smoke also has direct influence on the cardiovascular system. Additionally, lifestyle imposed by the highly developed society, including a sedentary lifestyle and unhealthy diet cause, that we are exposed to the factors associated with the formation of the so-called lifestyle diseases, like atherosclerosis and cancer. Pregnancy-Associated-PlasmaProtein A (PAPP-A) is a high-molecular zinc-binding metalloproteinase that was first detected in the serum of pregnant women as a protein produced by trophoblasts of properly developing fetus. In the blood of pregnant women PAPP-A exists as heterotetrameric complex (htPAPP-A) consisting of two subunits of PAPP-A. It can also be detected in men and women who are not pregnant but this time labeled PAPP-A is present as a homodimer (dPAPP-A, "free PAPP-A") consisting of two identical subunits devoid of proMBP protein. Recently a growing interest in determining the concentration of PAPP-A as a marker of oxidative stress and atherosclerotic processes is observed, because the increased levels of PAPP-Awere detected in people with a history of cardiovascular incident. PAPP-A is present on the early stages of atherosclerotic plaque instability when it is still possible to prevent a myocardial damage. The influence of smoking and exposure to tobacco smoke on concentrations of PAPP-A is not completely understood. The aim of this study was to determine the concentration of PAPP-A levels in the population of healthy young men and non-pregnant women. In the study author's questionnaire was used to assess exposure to tobacco smoke including knowledge of exposure to selected environmental xenobiotics, unhealthy habits and everyday diet. The study group consisted of 169 students of the first and second year students of the Faculty of Medicine and Department of Medicine and Dentistry, and the second year of Emergency Medicine in Zabrze, Medical University of Silesia. Blood samples were obtained from 153 students. For the determination of protein PAPP-A ELISA method was used. Mean PAPP-A concentration in the study group was 11.52 [ng/ml]. There was a statistically significant difference in the concentration of PAPP-A regarding gender (women 14.6 [ng/ml] vs. men 20.39 [ng/ml]; p = 0.014). In the study group of smoking students average concentration of PAPP-A was lower in comparison with non-smoking students (regardless of gender). Passive exposure to tobacco smoke also affects the levels of PAPP-A in the tested population of young people.

[3-nitrotyrosine determination as nitrosative stress marker and health attitudes of medical students considering exposure to environmental tobacco smoke]. / Oznaczanie 3-nitrotyrozyny jako markera stresu nitrozacyjnego a postawy zdrowotne studentów medycyny z uwzglednieniem narazenia na srodowiskowy dym tytoniowy.

Negative attitudes in health such as cigarette smoking and imbalanced diet play important role in pathogenesis of various diseases. Cigarette smoking constitutes one of the main sources of exposure to cancerogenic and procancerogenic xenobiotics among adults as well as among young people. Many studies have proven that cigarettes smokers more frequently follow less varied diet in comparison to non-smokers. Despite increasing knowledge of Poles regarding harmful effects of cigarettes smoking and numerous antinicotine campaigns, still high number of women and men smoke and the smoking percentage among young people remains high and has not decreased in the recent years. The ongoing research shows that free radicals -the man cause of exposure to oxidative stress- play the seminal role in pathogenesis of civilisation diseases and physiological cell aging processes. Reactive oxygen and nitrogen species present in cigarette smoke due to induced toxic compounds formation, are closely connected with observed increased risk of cancer, Chronic Obstructive Pulmonary Disease (COPD) and arteriosclerosis incidents. Malondialdehyde is one of the most studied product of lipid peroxidation and biomarker of oxidative stress. However, 3-nitrotyrosine is one of the most promising biomarkers regarding changes caused by oxidative stress in living organisms. The presence of 3-nitrotyrosine was observed in many diseases such as coronary artery disease, cancer and diabetes. The aim of the study was the evaluation of free radical processes increase related to tobacco smoke exposure and chosen diet habits by determination of 3-nitrotyrosine in plasma samples collected from the group of medicine students. In our investigation we used an author's questionnaire which served to estimate the exposure to tobacco smoke among medicine students. It took also into account the knowledge of the exposure to other xenobiotics and unhealthy habits/behaviours. The investigated group included 150 students of 1-st and 2-nd year of study at Faculty of Medicine and Dentistry Division in Zabrze, Silesian Medical University. 120 students provided blood samples for further analysis. In the study group 52 students were active smokers and the control group consisted of 68 non-smokers. 3-nitrotyrosine was determined with the use of ELISA technique. The mean concentration of 3-nitrotyrosine was higher in the group of smoking students in comparison to the control group. Diet habits also influenced the concentration of 3-nitrotyrosine. Eating food products of possibly high acrylamide level and drinking alcohol led in particular to observed increased concentrations of 3-nitrotyrosine in both students groups. The correlation between the food habits and the severity of nitrosative stress was also found.

[Oxidative stress biomarkers determination in urine samples in the group of medical students exposed to tobacco smoke]. / Oznaczanie biomarkerów stresu oksydacyjnego w moczu studentów medycyny narazonych na dym tytoniowy.

Cigarette smoking constitutes one of the main sources of exposure to cancerogenic and procancerogenic xenobiotics in the human population. Smoking is not only a bad habit among adults but also among young people. Despite many anti-tobacco campaigns undertaken by European Union countries still the smoking percentage among young people is high and does not decrease in the recent years. Cigarettes smoking induces formation of reactive oxygen species and increases lipids peroxidation, which ultimately results in the production of MDA (malondialdehyde)--one of the oxidative stress biomarkers. Exposure to oxidative stress and the consequences of its influence can be measured by redox parameters monitoring. One of these methods is determination of total antioxidant activity by means of FRAP test (Ferric Reducing Antioxidant Power) in biological samples. The aim of this work was the evaluation of free radical processes increase related to tobacco smoke exposure by measurement of total antioxidant activity FRAP and MDA concentration in urine samples of medical students. In our study the authors'--designed questionnaire regarding the assessment of exposure to tobacco smoke and chosen xenobiotics was used. The investigated group included 134 students of 1-st and 2-nd year of study at Faculty of Medicine and Dentistry Division in Zabrze, Silesian Medical University. 85 students provided urine samples for further analysis. In the study group 40 students were active smokers and the control group consisted of 45 nonsmokers. The mean MDA concentrations in smoking students group were higher in comparison to the control group of non-smoking students. In the group of students exposed to second-hand smoke MDA concentrations were significantly higher in comparison to the group of students non-exposed to second hand smoke. The values of FRAP parameter were significantly higher for students who smoked in comparison with students non-exposed to tobacco smoke. Students exposed to second hand smoke had slightly higher FRAP values in comparison to smoking students. Exposure to tobacco smoke increased the intensity of free radical processes. Our study confirmed the harmful influence of second hand smoke that resulted in observed higher concentrations of determined biomarkers in the group of passive smoking students.

The enzyme-linked immunosorbent assay (ELISA) method for nicotine metabolites determination in biological fluids.

The aim of this study was to present the usefulness of enzyme-linked immunosorbent assay (ELISA) in nicotine metabolites determination in urine and serum samples from active smokers and compare it with the reference to thin layer chromatography (TLC) with densitometry. The specific anti-cotinine antibodies were obtained from rabbit sera after sequential immunization with 4'-carboxycotinine-hemocyanine conjugate. The immunoaffinity chromatography technique with the use of self-prepared cotinine-aninohexyl-sepharose bed enabled the isolation of the specific anti-nicotine metabolites antibodies from the antiserum. Affinity of isolated antibodies to cotinine was passively immobilised on ELISA plates and competition between nicotine metabolites in samples and tracer (horseradish peroxidase-cotinine conjugate) was applied. After the washing stage the enzymatic activity of solid-phase-bound peroxidase was determined. For calibration cotinine perchlorate solutions in appropriate matrix were used. Determination ranges for serum and urine samples were from 3 to 1500 and from 3 to 5000 ng/mL, respectively. Precision within-run and between-run was below 8.7 and 11.3%; mean recovery of cotinine was 100.59% from serum and 88.56% from urine samples. The ELISA method, used in determination of the main nicotine metabolites showed high accuracy and sensitivity. However, this method was less specific than the reference technique (TLC). The high correlation coefficients, r>0.9, between the results of determined nicotine metabolites in urine by means of ELISA and TLC with densitometry confirmed the possibility of the application of ELISA method to practical monitoring of tobacco smoke exposure in large population groups.