A Review of Wastewater-Based Epidemiology Studies for the Assessment of Over-the-Counter Medicines Used as Recreational Drugs: The Example of Dextromethorphan.

The 'recreational use' of selected over-the-counter (OTC) medicines is an unofficial activity. The traditional surveys assessing the use of drugs are affected by the bias of underreporting and are thus unreliable. The development of analytical techniques helps to monitor the substances at trace levels, such as in wastewater, and might be applied to estimate the consumption of an analyte of interest and ensure additional, evidence-based information complementary to population surveys. We reviewed studies focused on evaluating the estimated consumption of drugs as a reliable and unbiased source of evidence-based information (called wastewater-based epidemiology, WBE) to monitor the scale of this phenomenon. We found there is a need to test not only narcotics in the environment but also medicines that may be abused or recreationally used. The reviewed studies show methods that might provide reliable information about consumption of drugs, narcotics, and OTC medications for proposing targeted, preventive actions. Moreover, as all the selected studies were based on mass spectrometry, there is a potential to include the dextromethorphan and/or related compounds as part of the screening for narcotics and OTC drugs that can be socially harmful, overused, or misused. This article reviews the analytical methods for detecting dextromethorphan and/or its transformation products in environmental water samples.

Changes in Serum Protein-Peptide Patterns in Atopic Children Allergic to Plant Storage Proteins.

Next to cow's milk and eggs, plant foods, i.e., legumes, tree nuts and cereal grains, most often sensitise atopic children. Storage proteins constitutes the most relevant protein fraction of plant foods, causing primary sensitisation. They exhibit strong allergenic properties and immunogenicity. Our goal was to analyse sensitisation to 26 plant storage proteins in a group of 76 children aged 0-5 years with chronic symptoms of atopic dermatitis using Allergy Explorer ALEX2 and to discover changes in serum protein-peptide patterns in allergic patients with the use of MALDI-TOF-MS. We reported that 25% of children were allergic to 2S albumins, 19.7% to 7S globulins, 13.2% to 11S globulins and 1.3% to cereal prolamins. The most common allergenic molecules were Ara h 1 (18.4%), Ara h 2 (17.1%), Ara h 6 (15.8%) and Ara h 3 (11.8%) from peanuts, and the mean serum sIgE concentrations in allergic patients were 10.93 kUA/L, 15.353 kUA/L, 15.359 kUA/L and 9.038 kUA/L, respectively. In children allergic to storage proteins compared to the other patients (both allergic and non-allergic), the cell cycle control protein 50A, testis-expressed sequence 13B, DENN domain-containing protein 5A and SKI family transcriptional corepressor 2 were altered. Our results indicate that the IgE-mediated allergy to storage proteins is a huge problem in a group of young, atopic children, and show the potential of proteomic analysis in the prediction of primary sensitisation to plant foods. It is the next crucial step for understanding the molecular consequences of allergy to storage proteins.

Serum Proteomic Profiles of Patients with High and Low Risk of Endometrial Cancer Recurrence.

Endometrial cancer is the most common gynecological cancer worldwide. Classifying endometrial cancer into low- or high-risk groups based on the following features is recommended: tumor grade, lymphovascular space invasion, myometrial involvement, and non-endometrioid histology. Despite the recent progress in molecular profiling of endometrial cancer, a substantial group of patients are misclassified based on the current criteria. This study aimed to identify proteins that could be used as biomarkers for the stratification of endometrial cancer patients into low- or high-risk groups. The proteomic analysis of serum samples from endometrial cancer patients was performed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The data were then analyzed using chemometric algorithms to identify potential biomarkers. Nineteen precursor ions were identified as fragments of eighteen proteins which included (1) connective tissue matrix proteins, (2) cytoskeletal proteins, and (3) innate immune system molecules and stress proteins. These biomarkers could be used to stratify the high- and low-risk patients, thus enabling more precise treatment decisions.

Mass spectrometry imaging in gynecological cancers: the best is yet to come.

Mass spectrometry imaging (MSI) enables obtaining multidimensional results simultaneously in a single run, including regiospecificity and m/z values corresponding with specific proteins, peptides, lipids, etc. The knowledge obtained in this way allows for a multifaceted analysis of the studied issue, e.g., the specificity of the neoplastic process and the search for new therapeutic targets. Despite the enormous possibilities, this relatively new technique in many aspects still requires the development or standardization of analytical protocols (from collecting biological material, through sample preparation, analysis, and data collection, to data processing). The introduction of standardized protocols for MSI studies, with its current potential to extend diagnostic and prognostic capabilities, can revolutionize clinical pathology. As far as identifying ovarian cancer subtypes can be challenging, especially in poorly differentiated tumors, developing MSI-based algorithms may enhance determining prognosis and tumor staging without the need for extensive surgery and optimize the choice of subsequent therapy. MSI might bring new solutions in predicting response to treatment in patients with endometrial cancer. Therefore, MSI may help to revolutionize the future of gynecological oncology in terms of diagnostics, treatment, and predicting the response to therapy. This review will encompass several aspects, e.g., contemporary discoveries in gynecological cancer research utilizing MSI, indicates current challenges, and future perspectives on MSI.

Mass Spectrometry-Based Identification of Bioactive Bee Pollen Proteins: Evaluation of Allergy Risk after Bee Pollen Supplementation.

Bee pollen, because of its high content of nutrients, is a very valuable medicinal and nutritional product. However, since its composition is not completely studied, the consumption of this product may cause adverse effects, including allergic reactions. Therefore, this study aimed to discover and characterize the bioactive proteins of bee pollen collected in Poland, focusing mainly on the allergens. For this purpose, the purified and concentrated pollen aqueous solutions were analyzed using the nanoLC-MALDI-TOF/TOF MS analytical platform. As a result of the experiments, 197 unique proteins derived from green plants (Viridiplantae) and 10 unique proteins derived from bees (Apis spp.) were identified. Among them, potential plant allergens were discovered. Moreover, proteins belonging to the group of hypothetical proteins, whose expression had not been confirmed experimentally before, were detected. Because of the content of bioactive compounds-both beneficial and harmful-there is a critical need to develop guidelines for standardizing bee pollen, especially intended for consumption or therapeutic purposes. This is of particular importance because awareness of the allergen content of bee pollen and other bee products can prevent health- or life-threatening incidents following the ingestion of these increasingly popular "superfoods".

New Gene Markers Expressed in Porcine Oviductal Epithelial Cells Cultured Primary In Vitro Are Involved in Ontological Groups Representing Physiological Processes of Porcine Oocytes.

Changes that occur within oviducts after fertilization are dependent on post-ovulation events, including oocyte-oviduct interactions. Although general processes are well-defined, the molecular basis are poorly understood. Recently, new marker genes involved in 'cell development', 'cell growth', 'cell differentiation' and 'cell maturation' processes have been identified in porcine oocytes. The aim of the study was to assess the expression profile of genes in primary in vitro cultured oviductal epithelial cells (OECs), clustered in Gene Ontology groups which enveloped markers also identified in porcine oocytes. OECs (from 45 gilts) were surgically removed and cultured in vitro for ≤ 30 days, and then subjected to molecular analyses. The transcriptomic and proteomic profiles of cells cultured during 7, 15 and 30 days were investigated. Additionally, morphological/histochemical analyzes were performed. The results of genes expression profiles were validated after using RT-qPCR. The results showed a significant upregulation of UNC45B, NOX4, VLDLR, ITGB3, FMOD, SGCE, COL1A2, LOX, LIPG, THY1 and downregulation of SERPINB2, CD274, TXNIP, CELA1, DDX60, CRABP2, SLC5A1, IDO1, ANPEP, FST. Detailed knowledge of the molecular pathways occurring in the OECs and the gametes that contact them may contribute both to developments of basic science of physiology, and new possibilities in advanced biotechnology of assisted reproduction.

Multielemental Analysis of Bee Pollen, Propolis, and Royal Jelly Collected in West-Central Poland.

Beehive products possess nutritional value and health-promoting properties and are recommended as so-called "superfoods". However, because of their natural origin, they may contain relevant elemental contaminants. Therefore, to assess the quality of bee products, we examined concentrations of a broad range of 24 selected elements in propolis, bee pollen, and royal jelly. The quantitative analyses were performed with inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES) techniques. The results of our research indicate that bee products contain essential macronutrients (i.e., K, P, and S) and micronutrients (i.e., Zn and Fe) in concentrations depending on the products' type. However, the presence of toxic heavy metals makes it necessary to test the quality of bee products before using them as dietary supplements. Bearing in mind that bee products are highly heterogenous and, depending on the environmental factors, differ in their elemental content, it is necessary to develop standards regulating the acceptable levels of inorganic pollutants. Furthermore, since bees and their products are considered to be an effective biomonitoring tool, our results may reflect the environment's condition in west-central Poland, affecting the health and well-being of both humans and bees.

Rapid and Accurate Approach for Honeybee Pollen Analysis Using ED-XRF and FTIR Spectroscopy.

Since honeybee pollen is considered a "perfectly complete food" and is characterized by many beneficial properties (anti-inflammatory, antioxidant, anti-bacterial, etc.), it has begun to be used for therapeutic purposes. Consequently, there is a high need to develop methods for controlling its composition. A thorough bee pollen analysis can be very informative regarding its safety for consumption, the variability of its composition, its biogeographical origin, or harvest date. Therefore, in this study, two reliable and non-destructive spectroscopy methods, i.e., ED-XRF and ATR-FTIR, are proposed as a fast approach to characterize bee pollen. The collected samples were derived from apiaries located in west-central Poland. Additionally, some commercially available samples were analyzed. The applied methodology was optimized and combined with sophisticated chemometric tools. Data derived from IR analyses were also subjected to two-dimensional correlation spectroscopy. The developed ED-XRF method allowed the reliable quantification of eight macro- and micro-nutrients, while organic components were characterized by IR spectroscopy. Principal component analysis, cluster analysis, and obtained synchronous and asynchronous maps allowed the study of component changes occurring dependently on the date and location of harvest. The proposed approach proved to be an excellent tool to monitor the variability of the inorganic and organic content of bee pollen.

Promising Antimicrobial Properties of Bioactive Compounds from Different Honeybee Products.

Bee products have been known for centuries for their versatile healing properties. In recent decades they have become the subject of documented scientific research. This review aims to present and compare the impact of bee products and their components as antimicrobial agents. Honey, propolis, royal jelly and bee venom are bee products that have antibacterial properties. Sensitivity of bacteria to these products varies considerably between products and varieties of the same product depending on their origin. According to the type of bee product, different degrees of activity were observed against Gram-positive and Gram-negative bacteria, yeasts, molds and dermatophytes, as well as biofilm-forming microorganisms. Pseudomonas aeruginosa turned out to be the most resistant to bee products. An analysis of average minimum inhibitory concentration values for bee products showed that bee venom has the strongest bacterial effectiveness, while royal jelly showed the weakest antibacterial activity. The most challenging problems associated with using bee products for medical purposes are dosage and safety. The complexity and variability in composition of these products raise the need for their standardization before safe and predictable clinical uses can be achieved.

Mining the Royal Jelly Proteins: Combinatorial Hexapeptide Ligand Library Significantly Improves the MS-Based Proteomic Identification in Complex Biological Samples.

Royal jelly (RJ) is a complex, creamy secretion produced by the glands of worker bees. Due to its health-promoting properties, it is used by humans as a dietary supplement. However, RJ compounds are not fully characterized yet. Hence, in this research, we aimed to broaden the knowledge of the proteomic composition of fresh RJ. Water extracts of the samples were pre-treated using combinatorial hexapeptide ligand libraries (ProteoMinerTM kit), trypsin-digested, and analyzed by a nanoLC-MALDI-TOF/TOF MS system. To check the ProteoMinerTM performance in the MS-based protein identification, we also examined RJ extracts that were not prepared with the ProteoMinerTM kit. We identified a total of 86 proteins taxonomically classified to Apis spp. (bees). Among them, 74 proteins were detected in RJ extracts pre-treated with ProteoMinerTM kit, and only 50 proteins were found in extracts non-enriched with this technique. Ten of the identified features were hypothetical proteins whose existence has been predicted, but any experimental evidence proves their in vivo expression. Additionally, we detected four uncharacterized proteins of unknown functions. The results of this research indicate that the ProteoMinerTM strategy improves proteomic identification in complex biological samples. Broadening the knowledge of RJ composition may contribute to the development of standards and regulations, enhancing the quality of RJ, and consequently, the safety of its supplementation.

Overexpression of Prolidase Induces Autophagic Death in MCF-7 Breast Cancer Cells.

BACKGROUND/AIMS: Proline availability for proline dehydrogenase/proline oxidase (PRODH/POX) may represent switching mechanism between PRODH/POX-dependent apoptosis and autophagy. The aim of the study was to evaluate the impact of overexpression of prolidase (proline releasing enzyme) on apoptosis/autophagy in breast cancer MCF-7 cells. METHODS: The model of MCF-7 cells with prolidase overexpression (MCF-7PL) was obtained. In order to targeting proline for PRODH/POX-dependent pathways substrate for prolidase, glycyl-proline (GP) was provided and proline utilization for collagen biosynthesis was blocked using 2-methoxyestradiol (MOE). Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. DNA, collagen and total protein biosynthesis were determined by radiometric method. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. RESULTS: Prolidase overexpression in MCF-7PL cells contributed to 10-fold increase in the enzyme activity, 3-fold increase in cytoplasmic proline level and decrease in cell viability and DNA biosynthesis compared to wild type MCF-7 cells. In MCF-7PL cells MOE and GP significantly decreased the number of living cells. MOE inhibited DNA biosynthesis in both cell lines while GP evoked inhibitory effect on the process only in MCF-7PL cells. In both cell lines, MOE or MOE+GP inhibited DNA and collagen biosynthesis. Although GP in MCF-7 cells stimulated collagen biosynthesis, it inhibited the process in MCF-7PL cells. The effects of studied compounds in MCF-7PL cells were accompanied by increase in the expression of Atg7, LC3A/B, Beclin-1, HIF-1α and decrease in the expression of PRODH/POX, active caspases-3 and -9. CONCLUSION: The data suggest that overexpression of prolidase in MCF-7 cells contributes to increase in intracellular proline concentration and PRODH/POX-dependent autophagic cell death.

Population analysis to assess the influence of age and body weight on pharmacokinetics and pharmacodynamics of dexmedetomidine in New Zealand White rabbits.

The purpose of this work was i) to develop a population pharmacokinetic (PK) and pharmacodynamic (PD) model of dexmedetomidine (DEX) in New Zealand White rabbits, ii) to investigate the influence of the age and weight of the animals on the model parameters, and iii) to assess the linearity of DEX PKs in the examined dose range. This was a prospective, crossover study, using a total of 18 New Zealand White rabbits. DEX was administered as a single intravenous bolus injection in the doses from 25 to 300 µg kg-1 . Each New Zealand White rabbit was given the same dose of drug in its three developmental stages. To determine the DEX PK, seven blood samples were taken from each animal. The pedal withdrawal reflex was the PD response used to assess the degree of sedation. Nonlinear mixed effects modelling was used for the population PK/PD analysis. The typical value of elimination clearance was 0.061 L min-1 and was 35% higher in younger New Zealand White rabbits compared with older animals. The PK of DEX was linear in the examined concentration range. Age-related changes in sensitivity to DEX were not detected. The results suggest that due to the pharmacokinetics, younger animals will have lower DEX concentrations and a shorter duration of sedation than older animals given the same doses of DEX per kg of body weight.

Development of an LC-MS Targeted Metabolomics Methodology to Study Proline Metabolism in Mammalian Cell Cultures.

A growing interest in metabolomics studies of cultured cells requires development not only untargeted methods capable of fingerprinting the complete metabolite profile but also targeted methods enabling the precise and accurate determination of a selected group of metabolites. Proline metabolism affects many crucial processes at the cellular level, including collagen biosynthesis, redox balance, energetic processes as well as intracellular signaling. The study aimed to develop a robust and easy-to-use targeted metabolomics method for the determination of the intracellular level of proline and the other two amino acids closely related to proline metabolism: glutamic acid and arginine. The method employs hydrophilic interaction liquid chromatography followed by high-resolution, accurate-mass mass spectrometry for reliable detection and quantification of the target metabolites in cell lysates. The sample preparation consisted of quenching by the addition of ice-cold methanol and subsequent cell scraping into a quenching solution. The method validation showed acceptable linearity (r > 0.995), precision (%RSD < 15%), and accuracy (88.5-108.5%). Pilot research using HaCaT spontaneously immortalized human keratinocytes in a model for wound healing was performed, indicating the usefulness of the method in studies of disturbances in proline metabolism. The developed method addresses the need to determine the intracellular concentration of three key amino acids and can be used routinely in targeted mammalian cell culture metabolomics research.

"Biological Adhesion" is a Significantly Regulated Molecular Process during Long-Term Primary In Vitro Culture of Oviductal Epithelial Cells (Oecs): A Transcriptomic and Proteomic Study.

Oviductal epithelial cells (OECs) actively produce stimulating and protecting factors, favoring survival and viability of gametes and early embryos. The oviduct participates in the initial reproductive events, which strongly depends on adhesion. The analysis of differential gene expression in OECs, during long-term in vitro culture, enables recognition of new molecular markers regulating several processes, including "biological adhesion". Porcine oviducts were stained with hematoxylin and eosin, as well as with antibodies against epithelial markers. Then, OECs were long-term in vitro cultured and after 24 h, 7, 15, and 30 days of culture were subjected to transcriptomic and proteomic assays. Microarrays were employed to evaluate gene expression, with Matrix-assisted laser desorption/ionization-time of light (MALDI-TOF) mass spectrometry applied to determine the proteome. The results revealed proper morphology of the oviducts and typical epithelial structure of OECs during the culture. From the set of differentially expressed genes (DEGs), we have selected the 130 that encoded proteins detected by MALDI-TOF MS analysis. From this gene pool, 18 significantly enriched gene ontology biological processes (GO BP) terms were extracted. Among them we focused on genes belonging to "biological adhesion" GO BP. It is suggested that increased expression of studied genes can be attributed to the process of intensive secretion of substances that exhibit favorable influence on oviductal environment, which prime gametes adhesion and viability, fertilization, and early embryo journey.

Pharmacokinetics of dexmedetomidine during analgosedation in ICU patients.

Dexmedetomidine (DEX) is a fairly new alfa2-agonist which has been increasingly used in recent years for analgosedation, mostly because it offers a unique ability of providing both moderate level of sedation and analgesia without respiratory depression. Despite of many papers published, there are still only a few concerning the PK of the drug given as long-term infusion in ICU patients. The aim of this work was to characterize the population pharmacokinetics of dexmedetomidine and to investigate the potential benefits of individualization of drug dosing based on patient characteristics in the heterogeneous group of medical and surgical patients staying in intensive care unit. This study was performed in the group of 17 males and 10 females patients with a median age of 59.5 years and median body weight of 75 kg. Blood samples for dexmedetomidine assay were collected from arterial catheter, during and after discontinuation of a standard infusion, that ranged from 24 to 102 h. The following covariates were examined to influence dexmedetomidine PK: age, sex, body weight, patients' health status described by Sequential Organ Failure Assessment Score (SOFA), inotropes usage, and infusion duration. The dexmedetomidine PK was best described by a two-compartment model. The typical values of PK parameters were estimated as 27 L for the volume of the central compartment, 87.6 L for the volume of the peripheral compartment, 38.5 L/h (9.2 mL/min/kg for a 70 kg patient) for systemic clearance and 46.4 L/h for the distribution clearance. Those values are consistent with literature findings. We were unable to show any significant relationship between collected covariates and dexmedetomidine PK. This study does not provide sufficient evidence to support the individualization of dexmedetomidine dosing based on age, sex, body weight, SOFA, and infusion duration.

Understanding Ovarian Cancer: iTRAQ-Based Proteomics for Biomarker Discovery.

Despite many years of studies, ovarian cancer remains one of the top ten cancers worldwide. Its high mortality rate is mainly due to lack of sufficient diagnostic methods. For this reason, our research focused on the identification of blood markers whose appearance would precede the clinical manifestation of the disease. ITRAQ-tagging (isobaric Tags for Relative and Absolute Quantification) coupled with mass spectrometry technology was applied. Three groups of samples derived from patients with: ovarian cancer, benign ovarian tumor, and healthy controls, were examined. Mass spectrometry analysis allowed for highlighting the dysregulation of several proteins associated with ovarian cancer. Further validation of the obtained results indicated that five proteins (Serotransferrin, Amyloid A1, Hemopexin, C-reactive protein, Albumin) were differentially expressed in ovarian cancer group. Interestingly, the addition of Albumin, Serotransferrin, and Amyloid A1 to CA125 (cancer antigen 125) and HE4 (human epididymis protein4) improved the diagnostic performance of the model discriminating between benign and malignant tumors. Identified proteins shed light on the molecular signaling pathways that are associated with ovarian cancer development and should be further investigated in future studies. Our findings indicate five proteins with a strong potential to use in a multimarker test for screening and detection of ovarian cancer.

Proteomic pattern of cervico-vaginal fluid (CVF) in an ovarian cancer diagnosis - pilot study.

OBJECTIVES: High grade serous ovarian cancer (HGSC) is the most common type of ovarian cancer and is responsible for about 90% of ovarian cancer deaths. The diagnostic tests currently used do not increase the detection rates for ovarian cancer. There is a great necessity to develop new and non-invasive diagnostic tests for ovarian cancer (OC). Cervico-vaginal fluid (CVF) seems to be a potential and valuable source of biomarkers for genital tract diseases including ovarian cancer. The aim of our pilot study was to undertake a preliminary proteomic analysis of CVF derived from ovarian cancer patients and to compare these with results from a control group. MATERIAL AND METHODS: We analysed and compared samples from a group of ovarian cancer patients and a control group of healthy patients. The study used MALDI-TOF coupled with nanoLC and ClinProTools software for MS, MS/MS spectra collection and proteomic analysis. RESULTS: We identified 404 different proteins in the OC group and 417 proteins in the control group. 239 of the proteins were found to be common to both study groups, 165 proteins were unique to the OC subjects, and 178 proteins were unique to the control subjects. We selected three proteins as the OC markers with the greatest potential: cysteine-rich secretory protein 3, fibronectin and Ly6/PLAUR domain-containing protein 3. CONCLUSIONS: The proteins we selected seem to possess great potential as markers for the screening and early detection of OC, especially in non-invasive and low-cost diagnostic tests. However, our findings require more advanced and validated proteomic analysis to confirm the suitability of the selected proteins in everyday medical diagnoses.

Assessment of diagnostic utility of multivariate diagnostic models in differential diagnosis of ovarian tumors.

INTRODUCTION: Ovarian cancer (OC) diagnosis remains a clinical challenge due to lack of early symptoms and insufficient accuracy of the available diagnostic methods. The purpose of this study was to determine whether osteopontin could be useful in differential diagnosis of ovarian tumors. MATERIAL AND METHODS: Serum samples from 92 patients qualified for surgical treatment due to ovarian mass were divided into 2 groups according to the histopathological result: OC including borderline ovarian tumors (n = 39) and benign ovarian tumors (BOTs) (n = 53). CA125, HE4 and osteopontin concentrations were measured in all patients. Areas under the receiver operating characteristic curves (AUC of ROC) were used to compare the discriminative ability of the univariate and multivariate diagnostic models. RESULTS: The addition of osteopontin to ROMA significantly improved the diagnostic performance of the test in 3 of the 5 analyses: 1) in the OC vs BOT group (from AUC of 0.955 to 0.975), 2) in premenopausal women OC vs BOT (from AUC of 0.828 to 0.892) and 3) in the FIGO I-II stage OC vs BOT (from AUC of 0.865 to 0.895). It did not alter the diagnostic performance of multifactor tests in the group of postmenopausal women nor in OC FIGO III-IV stage group. Osteopontin was also the best single marker to differentiate between early stage OC and BOTs (AUC of 0.863). CONCLUSIONS: Osteopontin improves the diagnostic performance of a multimarker OC diagnostic test and could be useful in differential diagnosis of ovarian tumors, especially in pre-menopausal women and for early stage OC.

MALDI-TOF-MS analysis in discovery and identification of serum proteomic patterns of ovarian cancer.

BACKGROUND: Due to high mortality and lack of efficient screening, new tools for ovarian cancer (OC) diagnosis are urgently needed. To broaden the knowledge on the pathological processes that occur during ovarian cancer tumorigenesis, protein-peptide profiling was proposed. METHODS: Serum proteomic patterns in samples from OC patients were obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Eighty nine serum samples (44 ovarian cancer and 45 healthy controls) were pretreated using solid-phase extraction method. Next, a classification model with the most discriminative factors was identified using chemometric algorithms. Finally, the results were verified by external validation on an independent test set of samples. RESULTS: Main outcome of this study was an identification of potential OC biomarkers by applying liquid chromatography coupled with tandem mass spectrometry. Application of this novel strategy enabled the identification of four potential OC serum biomarkers (complement C3, kininogen-1, inter-alpha-trypsin inhibitor heavy chain H4, and transthyretin). The role of these proteins was discussed in relation to OC pathomechanism. CONCLUSIONS: The study results may contribute to the development of clinically useful multi-component diagnostic tools in OC. In addition, identifying a novel panel of discriminative proteins could provide a new insight into complex signaling and functional networks associated with this multifactorial disease.

Diagnostic Value of Serum Angiogenesis Markers in Ovarian Cancer Using Multiplex Immunoassay.

As cancer development involves pathological vessel formation, 16 angiogenesis markers were evaluated as potential ovarian cancer (OC) biomarkers. Blood samples collected from 172 patients were divided based on histopathological result: OC (n = 38), borderline ovarian tumours (n = 6), non-malignant ovarian tumours (n = 62), healthy controls (n = 50) and 16 patients were excluded. Sixteen angiogenesis markers were measured using BioPlex Pro Human Cancer Biomarker Panel 1 immunoassay. Additionally, concentrations of cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) were measured in patients with adnexal masses using electrochemiluminescence immunoassay. In the comparison between OC vs. non-OC, osteopontin achieved the highest area under the curve (AUC) of 0.79 (sensitivity 69%, specificity 78%). Multimarker models based on four to six markers (basic fibroblast growth factor-FGF-basic, follistatin, hepatocyte growth factor-HGF, osteopontin, platelet-derived growth factor AB/BB-PDGF-AB/BB, leptin) demonstrated higher discriminatory ability (AUC 0.80-0.81) than a single marker (AUC 0.79). When comparing OC with benign ovarian tumours, six markers had statistically different expression (osteopontin, leptin, follistatin, PDGF-AB/BB, HGF, FGF-basic). Osteopontin was the best single angiogenesis marker (AUC 0.825, sensitivity 72%, specificity 82%). A three-marker panel consisting of osteopontin, CA125 and HE4 better discriminated the groups (AUC 0.958) than HE4 or CA125 alone (AUC 0.941 and 0.932, respectively). Osteopontin should be further investigated as a potential biomarker in OC screening and differential diagnosis of ovarian tumours. Adding osteopontin to a panel of already used biomarkers (CA125 and HE4) significantly improves differential diagnosis between malignant and benign ovarian tumours.