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.

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.

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.

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.

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.

HPLC and HPLC/MS/MS Studies on Stress, Accelerated and Intermediate Degradation Tests of Antivirally Active Tricyclic Analog of Acyclovir.

The degradation behavior of a tricyclic analog of acyclovir [6-(4-MeOPh)-TACV] was determined in accordance with International Conference on Harmonization guidelines for good clinical practice under different stress conditions (neutral hydrolysis, strong acid/base degradation, oxidative decomposition, photodegradation, and thermal degradation). Accelerated [40±2°C/75%±5% relative humidity (RH)] and intermediate (30±2°C/65%±5% RH) stability tests were also performed. For observation of the degradation of the tested compound the RP-HPLC was used, whereas for the analysis of its degradation products HPLC/MS/MS was used. Degradation of the tested substance allowed its classification as unstable in neutral environment, acidic/alkaline medium, and in the presence of oxidizing agent. The tested compound was also light sensitive and was classified as photolabile both in solution and in the solid phase. However, the observed photodegradation in the solid phase was at a much lower level than in the case of photodegradation in solution. The study showed that both air temperature and RH had no significant effect on the stability of the tested substance during storage for 1 month at 100°C (dry heat) as well as during accelerated and intermediate tests. Based on the HPLC/MS/MS analysis, it can be concluded that acyclovir was formed as a degradation product of 6-(4-MeOPh)-TACV.

A Combined Metabolomic and Proteomic Analysis of Gestational Diabetes Mellitus.

The aim of this pilot study was to apply a novel combined metabolomic and proteomic approach in analysis of gestational diabetes mellitus. The investigation was performed with plasma samples derived from pregnant women with diagnosed gestational diabetes mellitus (n = 18) and a matched control group (n = 13). The mass spectrometry-based analyses allowed to determine 42 free amino acids and low molecular-weight peptide profiles. Different expressions of several peptides and altered amino acid profiles were observed in the analyzed groups. The combination of proteomic and metabolomic data allowed obtaining the model with a high discriminatory power, where amino acids ethanolamine, L-citrulline, L-asparagine, and peptide ions with m/z 1488.59; 4111.89 and 2913.15 had the highest contribution to the model. The sensitivity (94.44%) and specificity (84.62%), as well as the total group membership classification value (90.32%) calculated from the post hoc classification matrix of a joint model were the highest when compared with a single analysis of either amino acid levels or peptide ion intensities. The obtained results indicated a high potential of integration of proteomic and metabolomics analysis regardless the sample size. This promising approach together with clinical evaluation of the subjects can also be used in the study of other diseases.

METABOLOMICS IN MEDICAL SCIENCES--TRENDS, CHALLENGES AND PERSPECTIVES.

Metabolomics is the latest of the "omic" technologies that involves comprehensive analysis of small molecule metabolites of an organism or a specific biological sample. Metabolomics provides an insight into the cell status and describes an actual health condition of organisms. Analysis of metabolome offers a unique opportunity to study the influence of genetic variation, disease, applied treatment or diet on endogenous metabolic state of organisms. There are many areas that might benefit from metabolomic research. In the article some applications of this novel "omic" technology in the field of medical sciences are presented. One of the most popular aims of metabolomic studies is biomarker discovery. Despite using the state-of-art analytical techniques along with advanced bioinformatic tools, metabolomic experiments encounter numerous difficulties and pitfalls. Challenges that researchers in the field of analysis of metabolome have to face include i.a., technical limitations, bioinformatic challenges and integration with other "omic" sciences. One of the grand challenges for studies in the field of metabolomics is to tackle the problem of data analysis, which is probably the most time consuming stage of metabolomic workflow and requires close collaboration between analysts, clinicians and experts in chemometric analysis. Implementation of metabolomics into clinical practice will be dependent on establishment of standardized protocols in analytical performance and data analysis and development of fit-for-purpose biomarker method validation. Metabolomics allows to achieve a sophisticated level of information about biological systems and opens up new perspectives in many fields of medicine, especially in oncology. Apart from its extensive cognitive significance, metabolomics manifests also a practical importance as it may lead to design of new non-invasive, sensitive and specific diagnostic techniques and development of new therapies.

Changes in Plasma Free Amino Acid Profile in Endurance Athletes over a 9-Month Training Cycle.

We aimed to evaluate long-term changes in proteinogenic and non-proteinogenic plasma free amino acids (PFAA). Eleven male endurance triathletes participated in a 9-month study. Blood was collected at rest, immediately after exhaustive exercise, and during 30-min recovery, in four consecutive training phases: transition, general, specific, and competition. Twenty proteinogenic and 22 non-proteinogenic PFAAs were assayed using the LC-ESI-MS/MS technique. The structured training modified the patterns of exercise-induced PFAA response, with the competition phase being the most distinct from the others. Branched-chain amino acids (p = 0.002; η2 = 0.216), phenylalanine (p = 0.015; η2 = 0.153), methionine (p = 0.002; η2 = 0.206), and lysine (p = 0.006; η2 = 0.196) declined more rapidly between rest and exhaustion in the competition phase. Glutamine (p = 0.008; η2 = 0.255), glutamate (p = 0.006; η2 = 0.265), tyrosine (p = 0.001; η2 = 0.195), cystine (p = 0.042; η2 = 0.183), and serine (p < 0.001; η2 = 0.346) levels were reduced in the competition phase. Arginine (p = 0.046; η2 = 0.138) and aspartate (p = 0.011; η2 = 0.171) levels were highest during exercise in the transition phase. During the competition phase, α-aminoadipic acid (p = 0.023; η2 = 0.145), ß-aminoisobutyric acid (p = 0.007; η2 = 0.167), ß-alanine (p < 0.001; η2 = 0.473), and sarcosine (p = 0.017; η2 = 0.150) levels increased, whereas phosphoethanolamine (p = 0.037; η2 = 0.189) and taurine (p = 0.008; η2 = 0.251) concentrations decreased. Overtraining indicators were not elevated. The altered PFAA profile suggests adaptations within energy metabolic pathways such as the tricarboxylic acid cycle, oxidative phosphorylation, ammonia neutralization, the purine nucleotide cycle, and buffering of intracellular H+ ions. The changes seem to reflect normal adaptations.

A new method for determination of hyaluronidase activity in biological samples using capillary zone electrophoresis.

The aim of the study was to develop a new capillary zone electrophoresis (CZE) method for determination of enzymatic activity of hyaluronidase. The method permits monitoring of the process of hyaluronic acid digestion by hyaluronidase. Studies were performed using CZE instrument equipped with capillary of 64.5 cm total length, 56 cm effective length and internal diameter 75 µm. Separation was performed in the phosphate buffer (pH 8.10) in the electric field of 20 kV, λ = 220 nm. The procedure was based on mixing a known quantity of hyaluronic acid and an aliquot of hyaluronidase solution, followed by obtaining CZE profiles after a known period of incubation (0.5 h). The activity of hyaluronidase was calculated using multiple regression analysis in which sizes of the peaks of the main degradation products were used. The newly developed method was fully validated and it is appropriate to evaluate the activity of hyaluronidase originating from different sources with high precision and accuracy. t-Tests showed that there were no significant differences between results obtained using turbidimetric, viscosimetric and the new CZE method. The developed method is characterized by a short duration of analysis, low volume of analyzed sample, small amount of buffers used and low cost of analysis.

New CZE-DAD method for honeybee venom analysis and standardization of the product.

The aim of this study was to develop a new precise and accurate CZE-DAD method for honeybee venom analysis using cytochrome c as an internal standard. The 64.5 cm total length, 56 cm effective length, 75 µm ID, and 360 µm OD uncoated fused-silica capillary was used. The samples were injected into the capillary under a 50-mbar pressure for 7 s. There were 15 kV of electric field across the capillary applied. The current intensity was 26 µA. The separation was carried out at 25 °C. The analysis was run with the normal electrode polarity. The following steps and parameters were taken into account for the validation of the developed method: selectivity, precision, accuracy, linearity, limit of detection and limit of quantitation. All steps of the validation procedure proved that the developed analytical procedure was suitable for its intended purpose. Possibly this was the first study in which several honeybee venom components were separated and five of them were identified by capillary zone electrophoresis. In addition, the developed method was applied for quantitative analysis of 38 honeybee venom samples. The content (relative to the dry venom mass) of analyzed peptides in honeybee venom samples collected in 2002-2007 was as follows: apamine from 0.93% to 4.34% (mean, 2.85 ± 0.79%); mast cell degranulating peptide (MCDP) from 1.46% to 4.37% (mean, 2.82 ± 0.64%); phospholipase A(2) from 7.41% to 20.25% (mean, 12.95 ± 3.09%); melittin from 25.40% to 60.27%, (mean, 45.91 ± 9.78%). The results were compared with the experimental data obtained for the same venom samples analyzed earlier by the HPLC method. It was stated that HPCE and HPLC data did not differ significantly and that the HPCE method was the alternative for the HPLC method. Moreover, using the results obtained principal component analysis (PCA) was applied to clarify the general distribution patterns or similarities of four major honeybee venom constituents collected from two different bee strains in various months and years. PCA has shown that the strain of bee appears to be the only criteria for bee venom sample classification. Strong correlations between apamine, MCDP, phospholipase A(2), and melittin were confirmed. These correlations have to be taken into account in the honeybee venom standardization. The developed method due to its simplicity can be easily automated and incorporated into routine operations both in the bee venom identification, quality control, and standardization of the product.

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.

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.