Serum Free Amino Acid Profiling in Differential Diagnosis of Ovarian Tumors-A Comparative Study with Review of the Literature.

Proper preoperative ovarian cancer (OC) diagnosis remains challenging. Serum free amino acid (SFAA) profiles were investigated to identify potential novel biomarkers of OC and assess their performance in ovarian tumor differential diagnosis. Serum samples were divided based on the histopathological result: epithelial OC (n = 38), borderline ovarian tumors (n = 6), and benign ovarian tumors (BOTs) (n = 62). SFAA profiles were evaluated using aTRAQ methodology based on high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Levels of eleven amino acids significantly differed between OC+borderline and BOTs. The highest area under the receiver operating characteristic curve (AUC of ROC) (0.787) was obtained for histidine. Cystine and histidine were identified as best single markers for early stage OC/BOT and type I OC. For advanced stage OC, seven amino acids differed significantly between the groups and citrulline obtained the best AUC of 0.807. Between type II OC and BOTs, eight amino acids differed significantly and the highest AUC of 0.798 was achieved by histidine and citrulline (AUC of 0.778). Histidine was identified as a potential new biomarker in differential diagnosis of ovarian tumors. Adding histidine to a multimarker panel together with CA125 and HE4 improved the differential diagnosis between OC and BOTs.

Identification and quantification of honeybee venom constituents by multiplatform metabolomics.

Honeybee (Apis mellifera) venom (HBV) has been a subject of extensive proteomics research; however, scarce information on its metabolite composition can be found in the literature. The aim of the study was to identify and quantify the metabolites present in HBV. To gain the highest metabolite coverage, three different mass spectrometry (MS)-based methodologies were applied. In the first step, untargeted metabolomics was used, which employed high-resolution, accurate-mass Orbitrap MS. It allowed obtaining a broad overview of HBV metabolic components. Then, two targeted metabolomics approaches, which employed triple quadrupole MS, were applied to quantify metabolites in HBV samples. The untargeted metabolomics not only confirmed the presence of amines, amino acids, carbohydrates, and organic acids in HBV, but also provided information on venom components from other metabolite classes (e.g., nucleosides, alcohols, purine and pyrimidine derivatives). The combination of three MS-based metabolomics platforms facilitated the identification of 214 metabolites in HBV samples, among which 138 were quantified. The obtaining of the wide free amino acid profiles of HBV is one of the project's achievements. Our study contributed significantly to broadening the knowledge about HBV composition and should be continued to obtain the most comprehensive metabolite profile of HBV.

Urban wastewater analysis as an effective tool for monitoring illegal drugs, including new psychoactive substances, in the Eastern European region.

The use of illicit drugs causes unquestionable societal and economic damage. To implement actions aimed at combating drug abuse, it is necessary to assess illicit drug consumption patterns. The purpose of this paper was to develop, optimize, validate and apply a procedure for determining new psychoactive substances (NPSs) and classic drugs of abuse and their main metabolites in wastewater samples by using solid phase extraction (SPE) and high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Moreover, detailed validation of the procedure was conducted. The developed SPE-HPLC-MS/MS procedure (within the sewage-based epidemiology strategy) allowed for the simultaneous, selective, very sensitive, accurate (recoveries ≥ 80.1%) and precise (CV ≤ 8.1%) determination of new and classic psychoactive substances in wastewater samples. This study is characterized by new scientific elements, especially in terms of the freeze-thaw and post-preparative stability of the selected psychoactive substances. This is the first time that NPSs (mephedrone and ketamine), the main metabolites of heroin (6-acetylmorphine, 6-AM) and marijuana (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, THC-COOH) have been detected and monitored in Poland. This study is also the first to corroborate the data available from the EMCDDA and EUROPOL report and indicates that the retail market for cocaine is expanding in Eastern Europe.

Integrated RNA and metabolite profiling of urine liquid biopsies for prostate cancer biomarker discovery.

Sensitive and specific diagnostic and prognostic biomarkers for prostate cancer (PCa) are urgently needed. Urine samples are a non-invasive means to obtain abundant and readily accessible "liquid biopsies". Herein we used urine liquid biopsies to identify and characterize a novel group of urine-enriched RNAs and metabolites in patients with PCa and normal individuals with or without benign prostatic disease. Differentially expressed RNAs were identified in urine samples by deep sequencing and metabolites in urine were measured by mass spectrometry. mRNA and metabolite profiles were distinct in patients with benign and malignant disease. Integrated analysis of urinary gene expression and metabolite signatures unveiled an aberrant glutamate metabolism and tricarboxylic acid (TCA) cycle node in prostate cancer-derived cells. Functional validation supported a role for glutamate metabolism and glutamate oxaloacetate transaminase 1 (GOT1)-dependent redox balance in PCa, which could be exploited for novel biomarkers and therapies. In this study, we discovered cancer-specific changes in urinary RNAs and metabolites, paving the way for the development of sensitive and specific urinary PCa diagnostic biomarkers either alone or in combination. Our methodology was based on single void urine samples (i.e., without prostatic massage). The integrated analysis of metabolomic and transcriptomic data from these liquid biopsies revealed a glutamate metabolism and tricarboxylic acid cycle node that was specific to prostate-derived cancer cells and cancer-specific metabolic changes in urine.

Choline Salicylate Analysis: Chemical Stability and Degradation Product Identification.

Choline salicylate (CS) as a derivative of acetylsalicylic acid is commonly used in different drug forms. In medicine, it is applied topically to inflammation of the oral cavity mucosa and in laryngology. However, this substance in the form of an ionic liquid has not been investigated enough. There are no literature studies on stability tests constituting a stage of pre-formulation research. HPLC (Nucleosil C18, 4.6 × 150 mm, 5 µm; methanol-water-acetic acid 60:40:1, 230 nm or 270 nm) and UV (276 nm) methods for the determination of CS in 2% (g/mL) aqueous solutions were developed. Under stress conditions, CS susceptibility to hydrolytic degradation in aqueous medium, hydrochloric acid, sodium hydroxide, and hydrogen peroxide, and the effect of light on the stability of CS solutions were studied with HPLC analysis. The degradation degree of CS and the purity of the solutions were also tested. Choline salicylate has been qualified as practically stable in neutral and acid media, stable in an alkaline medium, very stable in an oxidizing environment, and photolabile in solution. The HPLC-MS/MS method was used to identify 2,3- and 2,5-dihydroxybenzoic acids as degradation products of CS under the tested conditions.

Wide spectrum targeted metabolomics identifies potential ovarian cancer biomarkers.

AIMS: Despite of almost a hundred years of research on cancer metabolism, the biological background of cancerogenesis and cancer-related reprogramming of metabolism remains not fully understood. In order to comprehensively and effectively diagnose and treat the deadliest diseases, the mechanisms underlying these diseases have to be discovered urgently. Among the gynecological malignancies, ovarian cancer is the most common cause of death. The aim of the study was to search for potential cancer-related differences in concentrations of metabolites and interactions between them in serum of women with ovarian cancer and benign ovarian tumor in comparison with healthy controls using targeted metabolomics. These metabolites might serve as biomarkers in the future. MAIN METHODS: We used wide spectrum targeted metabolomics to evaluate serum concentrations of metabolites related to ovarian cancer and compared them against benign ovarian tumors and healthy controls. The measurements were performed using high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry technique in highly-selective multiple reaction monitoring mode. KEY FINDINGS: In this study we confirmed our previous findings about the role of histidine and citrulline in ovarian cancer as well as we indicated new lipid compounds (lysoPC a C16:1, PC aa C32:2, PC aa C34:4 and PC aa C 36:6) potentially involved in cancer metabolism. SIGNIFICANCES: We indicated interesting interactions between metabolites for further in-depth research which could potentially serve as clinically useful biomarkers in future. Moreover, the presented work attempts to visualize a possible 3D-network of relationships between the molecules found to be related to ovarian malignancy.

MALDI-TOF-MS Analysis in the Identification of Urine Proteomic Patterns of Gestational Trophoblastic Disease.

Gestational trophoblastic disease (GTD) is a group of highly aggressive, rare tumors. Human chorionic gonadotropin is a common biomarker used in the diagnosis and monitoring of GTD. To improve our knowledge of the pathology of GTD, we performed protein-peptide profiling on the urine of patients affected with gestational trophoblastic neoplasm (GTN). We analyzed urine samples from patients diagnosed with GTN (n = 26) and from healthy pregnant and non-pregnant controls (n = 17) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Ions were examined in a linear mode over a m/z range of 1000⁻10,000. All GTN urine samples were analyzed before and after treatment and compared with those of the controls. The statistical analyses included multivariate classification algorithms as well as ROC curves. Urine sample analyses revealed there were significant differences in the composition of the ions between the evaluated groups. Comparing the pre-treatment and group with the pregnant controls, we identified two discriminatory proteins: hemoglobin subunit α (m/z = 1951.81) and complement C4A (m/z = 1895.43). Then, comparing urine samples from the post-treatment cases with those from the non-pregnant controls, we identified the peptides uromodulin fragments (m/z = 1682.34 and 1913.54) and complement C4A (m/z = 1895.43).

Serum angiogenesis profile in gestational trophoblastic neoplasm using multiplex immunoassay.

AIMS: Gestational trophoblastic neoplasms (GTN) exemplify a rare, mostly curable but highly aggressive disease. It is often associated with a rapid formation of distant metastases and most likely with an intense neoangiogenesis processes. The aim of the study was to analyze markers in serum of patients with GTN before chemotherapy compared to healthy pregnant women. MAIN METHODS: In this study sixteen protein angiogenesis markers were evaluated in serum of 21 patients with GTN before chemotherapy and compared with healthy pregnant women. Markers were measured using BioPlex Pro Human Cancer Biomarker Panel 1 immunoassay. t-Tests and receiver operating characteristic curves were used for statistical analysis. KEY FINDINGS: Receiver operator curve analysis identified six proteins (sTIE-2, osteopontin, sIL-6α, sVEGFR-2, sEGFR, PECAM-1) which had sufficient sensitivity and specificity (AUC > 0,70) to distinguish GTN patients before the treatment from pregnant controls. The levels of three proteins (sTIE-2, osteopontin and sIL-6α) were altered in GTN patients before the treatment as compared to healthy controls (p = 0,0112; p = 0,0442; p = 0,0488, respectively) and thereby may serve as potential disease markers. SIGNIFICANCE: Serum concentration of proteins related to angiogenesis changes in the course of GTN and may appear useful in the diagnostic process of this disease.

A study of low-molecular-weight organic acid urinary profiles in prostate cancer by a new liquid chromatography-tandem mass spectrometry method.

Metabolomic studies constantly require high throughput screenings, and this drives development and optimization of methods that include more analytes in a single run, shorten the analysis time and simplify sample preparation. The aim of the study was to develop a new simple and fast liquid chromatography-tandem mass spectrometry-based methodology for quantitative analysis of a panel of ten organic acids in urine. The metabolites selected for the study include ten molecules potentially associated with cancer development. Chromatographic separation involved a Phenomenex Synergi Hydro-RP column under gradient conditions. Quantitation of the analytes was performed in multiple reaction monitoring mode under negative ionization. Validation parameters were satisfactory and in line with the international guidelines. The methodology enabled us to analyze urine samples collected from prostate cancer (PC) (n = 49) and benign prostate hyperplasia (BPH) (n = 49) patients. The obtained concentrations were normalized with urinary specific gravity (USG) prior to statistical analysis. Five analytes were quantified in all urine samples and we observed the following USG-normalized concentration ranges: citric acid (146.5-6339.8), 3-hydroxyisobutyric acid (22.5-431.7), 2-ketoglutaric acid (4.4-334.4), lactic acid (10.1-786.3), succinic acid (4.1-500.5). 3-hydroxyisobutyric acid significantly decreased between two groups of prostate cancer patients: ≥7 Gleason patients and <7 Gleason patients. Quick sample preparation limited to "dilute and shoot" makes the developed methodology a great tool for future metabolomic studies, especially for detecting disturbances in energy metabolism (Krebs cycle) and amino acids metabolism. The research also broadens our knowledge on the alteration of selected organic acids in PC and BPH patients.

Pilot Study of Metabolomics and Psychoneurological Symptoms in Women With Early Stage Breast Cancer.

Many women with breast cancer experience symptoms of pain, fatigue, and depression, collectively known as psychoneurologic (PN) symptoms, during and after chemotherapy treatment. Evidence that inflammatory dysfunction related to cancer and its treatments contributes to the development and persistence of PN symptoms through several interrelated pathways is accumulating. However, a major limiting factor in more precisely identifying the biological mechanisms underlying these symptoms is the lack of biological measures that represent a holistic spectrum of biological responses. Metabolomics allows for examination of multiple, co-occurring metabolic pathways and provides a systems-level perspective on biological mechanisms that may contribute to PN symptoms. METHODS: In this pilot study, we performed serum metabolome analysis using liquid chromatography high-resolution mass spectrometry of global and targeted metabolomics from the tryptophan pathway from archived samples from 19 women with early-stage breast cancer. We used paired t tests to compare metabolite concentrations and Pearson's correlation coefficients to examine concomitant changes in metabolite concentrations and PN symptoms before and after chemotherapy. RESULTS: Levels of pain, fatigue, and depression increased after chemotherapy. Compared with pre-chemotherapy, global metabolites post-chemotherapy were characterized by higher concentrations of acetyl-l-alanine and indoxyl sulfate and lower levels of 5-oxo-l-proline. Targeted analysis indicated significantly higher kynurenine levels and kynurenine/tryptophan ratios post-chemotherapy. Symptoms of pain and fatigue had strong associations with multiple global and several targeted metabolites. CONCLUSION: Results demonstrated that metabolomics may be useful for elucidating biological mechanisms associated with the development and severity of PN symptoms, specifically pain and fatigue, in women with early-stage breast cancer.

Usefulness of Amino Acid Profiling in Ovarian Cancer Screening with Special Emphasis on Their Role in Cancerogenesis.

The aim of this study was to quantitate 42 serum-free amino acids, propose the biochemical explanation of their role in tumor development, and identify new ovarian cancer (OC) biomarkers for potential use in OC screening. The additional value of this work is the schematic presentation of the interrelationship between metabolites which were identified as significant for OC development and progression. The liquid chromatography-tandem mass spectrometry technique using highly-selective multiple reaction monitoring mode and labeled internal standards for each analyzed compound was applied. Performed statistical analyses showed that amino acids are potentially useful as OC biomarkers, especially as variables in multi-marker models. For the distinguishing metabolites the following metabolic pathways involved in cancer growth and development were proposed: histidine metabolism; tryptophan metabolism; arginine biosynthesis; arginine and proline metabolism; and alanine, aspartate and glutamine metabolism. The presented research identifies histidine and citrulline as potential new OC biomarkers. Furthermore, it provides evidence that amino acids are involved in metabolic pathways related to tumor growth and play an important role in cancerogenesis.

Mass spectrometry as a tool for biomarkers searching in gynecological oncology.

Tumors of the female reproductive tract are an important target for the development of diagnostic, prognostic and therapeutic strategies. Recent research has turned to proteomics based on mass spectrometry techniques, to achieve more effective diagnostic results. Mass spectrometry (MS) enables identification and quantification of multiple molecules simultaneously in a single experiment according to mass to charge ratio (m/z). Several proteomic strategies may be applied to establish the function of a particular protein/peptide or to identify a novel disease and specific biomarkers related to it. Therefore, MS could facilitate treatment in patients with tumors by helping researchers discover new biomarkers and narrowly targeted drugs. This review presents a comprehensive discussion of mass spectrometry as a tool for biomarkers searching that may lead to the discovery of easily available diagnostic tests in gynecological oncology with emphasis on clinical proteomics over the past decade. The article provides an insight into different MS based proteomic approaches.

Amino Acid Profiles of Serum and Urine in Search for Prostate Cancer Biomarkers: a Pilot Study.

There is a great interest in searching for diagnostic biomarkers in prostate cancer patients. The aim of the pilot study was to evaluate free amino acid profiles in their serum and urine. The presented paper shows the first comprehensive analysis of a wide panel of amino acids in two different physiological fluids obtained from the same groups of prostate cancer patients (n = 49) and healthy men (n = 40). The potential of free amino acids, both proteinogenic and non-proteinogenic, as prostate cancer biomarkers and their utility in classification of study participants have been assessed. Several metabolites, which deserve special attention in the further metabolomic investigations on searching for prostate cancer markers, were indicated. Moreover, free amino acid profiles enabled to classify samples to one of the studied groups with high sensitivity and specificity. The presented research provides a strong evidence that ethanolamine, arginine and branched-chain amino acids metabolic pathways can be a valuable source of markers for prostate cancer. The altered concentrations of the above-mentioned metabolites suggest their role in pathogenesis of prostate cancer and they should be further evaluated as clinically useful markers of prostate cancer.

Determination of 16 serum angiogenic factors in stage I non-small cell lung cancer using a bead-based multiplex immunoassay.

The aim of the study was to identify significant abnormalities in angiogenic factor profiles occurring at early-stage non-small cell lung cancer (NSCLC). Contrary to the previous studies, our research included patients with only stage I NSCLC, which allowed for estimating the utility of circulating angiogenic factors in early NSCLC detection. The investigation was performed in serum samples collected from individuals with untreated NSCLC (n=41) and a matched control group of healthy individuals (n=61). All patients had histopathologically-confirmed stage IA or IB NSCLC. Serum concentrations of 16 angiogenesis markers comprising growth factors, receptors, cytokines, chemokines and hormones, were measured using a bead-based multiplex immunoassay. Among the determined proteins, osteopontin, platelet-derived growth factor-AB and -BB (PDGF-AB/BB) and hepatocyte growth factor (HGF) demonstrated the largest increase in concentration in NSCLC patients as compared with the non-cancer group. ROC curve analysis confirmed their high discriminatory abilities in detection of early NSCLC (AUC=0.809, 95%CI 0.725-0.881). Serum levels of the studied angiogenic factors differed slightly between patients with lung adenocarcinoma and squamous cell carcinoma. The study demonstrated that osteopontin, PDGF-AB/BB, and HGF might add up to the methods used in NSCLC diagnosis. Future research should address the question about their specificity and performance in early NSCLC detection in a combination with various putative lung cancer markers.

Study of early stage non-small-cell lung cancer using Orbitrap-based global serum metabolomics.

PURPOSE: The aim of the project was to apply ultra-high-performance liquid chromatography-quadrupole-Orbitrap-high-resolution mass spectrometry for serum metabolite profiling of non-small-cell lung cancer (NSCLC). This Orbitrap-based methodology has been applied for a study of NSCLC potential markers for the first time. METHODS: After extraction using protein precipitation, sera were separated on the ACE Excel 2 C18-PFP (100 × 2.1 mm, 2.0 µm) column using gradient elution and analyzed within the range of 70-1000 m/z. Only patients with early stage disease (stages IA-IIB) were included in the study, providing opportunity to find biomarkers for early lung cancer detection. The resulting metabolite profiles were subjected to univariate and multivariate statistical tests. RESULTS: 36 features were found significantly changed between NSCLC group and controls after FDR adjustment and 19 were identified using various metabolite databases (in-house library, HMDB, mzCloud). The study revealed a number of NSCLC biomarker candidates which belong to such compound classes as acylcarnitines, organic acids, and amino acids. Multivariate ROC curve built using 12 identified metabolites was characterized by AUC = 0.836 (0.722-0.946). There were no significant differences in the serum metabolite profiles between two most common histological types of lung cancer-adenocarcinoma and squamous cell carcinoma. CONCLUSIONS: Through identification of novel potential tumor markers, Orbitrap-based global metabolic profiling is a useful strategy in cancer research. Our study can accelerate development of new diagnostic and therapeutic strategies in NSCLC. The metabolites involved in discrimination between NSCLC patients and the control subjects should be further explored using a targeted approach.

Evaluation of serum amino acid profiles' utility in non-small cell lung cancer detection in Polish population.

OBJECTIVES: Data from studies performed in Japanese and Korean populations suggest that free amino acid profiles have the potential to aid in non-small cell lung cancer (NSCLC) detection. However, there is still no data regarding abnormalities of free amino acids and their usefulness in NSCLC detection in European populations. The aim of the study was an evaluation of utility of amino acid profiles in NSCLC detection in Polish patients. MATERIALS AND METHODS: Levels of 31 free amino acids were determined in 153 serum samples applying a liquid chromatography-tandem mass spectrometry-based methodology. Patients with I stage lung cancer represented a significant part of the studied group (46.7%). The obtained metabolite profiles along with clinical data were subjected to multivariate statistical tests. RESULTS: The presented study indicated that the increased serum level of phenylalanine and decreased level of citrulline are among the most robust cancer signatures in blood of NSCLC group. In addition, increased levels of aspartic acid and ß-alanine were also recognized as important features of NSCLC. Amino acid selected based on studies of Asian patients were found to have insufficient specificity in NSCLC detection in the studied population. Therefore, we proposed a new set of 6 amino acids (aspartic acid, ß-alanine, histidine, asparagine, phenylalanine and serine), which ensured higher accuracy in sample classification (from 90.3% to 77.1% depending of histological type). CONCLUSION: We indicated that some of the free amino acid alterations occur in serum of NSCLC patients in early stage of disease and thus they can be valuable components of a blood multi-marker panel for NSCLC detection.

The correlation between anti phospholipase A2 specific IgE and clinical symptoms after a bee sting in beekeepers.

INTRODUCTION: Beekeepers are a group of people with high exposure to honeybee stings and with a very high risk of allergy to bee venom. Therefore, they are a proper population to study the correlations between clinical symptoms and results of diagnostic tests. AIM: The primary aim of our study was to assess the correlations between total IgE, venom- and phospholipase A2-specific IgE and clinical symptoms after a bee sting in beekeepers. The secondary aim was to compare the results of diagnostic tests in beekeepers and in individuals with standard exposure to bees. MATERIAL AND METHODS: Fifty-four individuals were divided into two groups: beekeepers and control group. The levels of total IgE (tIgE), venom-specific IgE (venom sIgE), and phospholipase A2-specific IgE (phospholipase A2 sIgE) were analyzed. RESULTS: Our study showed no statistically significant correlation between the clinical symptoms after a sting and tIgE in the entire analyzed group. There was also no correlation between venom sIgE level and clinical symptoms either in beekeepers or in the group with standard exposure to bees. We observed a statistically significant correlation between phospholipase A2 sIgE level and clinical signs after a sting in the group of beekeepers, whereas no such correlation was detected in the control group. Significantly higher venom-specific IgE levels in the beekeepers, as compared to control individuals were shown. CONCLUSIONS: In beekeepers, the severity of clinical symptoms after a bee sting correlated better with phospholipase A2 sIgE than with venom sIgE levels.

CREATININE DETERMINATION IN URINE BY LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONIZATION-TANDEM MASS SPECTROMETRY METHOD.

Creatinine determination in urine is used to estimate the completeness of the 24-h urine collection, compensation for variable diuresis and as a preliminary step in protein profiling in urine. Despite the fact that a wide range of methods of measuring creatinine level in biofluids has been developed, many of them are adversely affected by interfering substances. A new liquid chromatography-tandem mass spectrometry method for creatinine determination in urine has been developed. Chromatographic separation was performed by applying C18 column and a gradient elution. Analyses were carried out on a triple quadrupole mass spectrometer equipped with an electrospray ion source. The developed method was fully validated according to the international guidelines. The quantification range of the method was 5-1500 ng/mL, which corresponds to 1-300 mg/dL in urine. Limit of detection and quantitation were 2 and 5 ng/mL, respectively. Additionally, the comparison of creatinine determination by newly developed method to the colorimetric method was performed. The method enables the determination of creatinine in urine samples with a minimal sample preparation, excellent sensitivity and prominent selectivity. Since mass spectrometry allows to measure a number of compounds simultaneously, a future perspective would be to incorporate the determination of other clinically important compounds excreted in urine.

Immune and clinical response to honeybee venom in beekeepers.

OBJECTIVE: The aim of the study was to assess immune response to honeybee venom in relation to the degree of exposure, time after a sting and clinical symptoms. MATERIALS AND METHOD: Fifty-four volunteers were divided into 2 groups: beekeepers and a control group. The serum levels of total IgE (tIgE), bee venom-specific IgE (venom sIgE), phospholipase A2-specific IgE (phospholipase A2 sIgE), tryptase and venom-specific IgG4 (venom sIgG4) were determined. In beekeepers, diagnostic tests were performed within 3 hours following a sting and were repeated after a minimum of 6 weeks from the last sting. In individuals from the control group, the tests were performed only once, without a sting. RESULTS: The tests showed significant differences in venom sIgE (beekeepers' median = 0.34 kUA/l, control group median = 0.29 kUA/l), baseline serum tryptase (beekeepers' median = 4.25 µg/l, control group median = 2.74 µg/l) and sIgG4 (beekeepers' median = 21.2 mgA/l, control group median = 0.14 mgA/l), confirming higher levels of the tested substances in the beekeepers than in the control group. A significant positive correlation was observed between phospholipase A2 sIgE concentration and severity of clinical symptoms after a sting in the group of beekeepers. It was also demonstrated that the clinical symptoms after a sting became less severe with increasing age of the beekeepers. CONCLUSIONS: The differences in the immune response to a bee sting between the beekeepers and individuals not exposed to bees were probably due to the high exposure of the beekeepers to honeybee venom allergens. This may suggest a different approach to the bee venom allergy diagnostic tests in this occupational group.

Estimation of drug abuse in 9 Polish cities by wastewater analysis.

The aim of this work was to measure illicit drug residues in raw sewage samples collected from nine Polish cities in order to determine trends in illicit drug use in these urban populations. This is the first study involving an analysis of samples from several sewage treatment plants in Poland and covering such a large population. Concentration of illicit drugs was determined using a high performance liquid chromatography-tandem mass spectrometry method. The samples were subjected to a multistep preparation procedure with a solid phase extraction as a main pre-treatment step. Among the selected drugs investigated in the study, amphetamine was found in the greatest amounts in all sewage samples and consequently was the most prevalent drug of abuse. Higher loads of illicit drug residues were found during weekends compared to the weekdays, especially for 3,4-methylenedioxymethamphetamine (ecstasy) and benzoylecgonine, main metabolite of cocaine.