Short overview on metabolomics approach to study pathophysiology of oxidative stress in cancer

Association of oxidative stress with carcinogenesis is well known, but not understood well, as is pathophysiology of oxidative stress generated during different types of anti-cancer treatments. Moreover, recent findings indicate that cancer associated lipid peroxidation might eventually help defending adjacent nonmalignant cells from cancer invasion. Therefore, untargeted metabolomics studies designed for advanced translational and clinical studies are needed to understand the existing paradoxes in oncology, including those related to controversial usage of antioxidants aiming to prevent or treat cancer. In this short review we have tried to put emphasis on the importance of pathophysiology of oxidative stress and lipid peroxidation in cancer development in relation to metabolic adaptation of particular types of cancer allowing us to conclude that adaptation to oxidative stress is one of the main driving forces of cancer pathophysiology. With the help of metabolomics many novel findings are being achieved thus encouraging further scientific breakthroughs. Combined with targeted qualitative and quantitative methods, especially immunochemistry, further research might reveal bio-signatures of individual patients and respective malignant diseases, leading to individualized treatment approach, according to the concepts of modern integrative medicine

Metabolomic and transcriptomic response to imatinib treatment of gastrointestinal stromal tumour in xenograft-bearing mice

Background Although imatinib is a well-established first-line drug for treating a vast majority of gastrointestinal stromal tumours (GIST), GISTs acquire secondary resistance during therapy. Multi-omics approaches provide an integrated perspective to empower the development of personalised therapies through a better understanding of functional biology underlying the disease and molecular-driven selection of the best-targeted individualised therapy. In this study, we applied integrative metabolomic and transcriptomic analyses to elucidate tumour biochemical processes affected by imatinib treatment. Materials and methods A GIST xenograft mouse model was used in the study, including 10 mice treated with imatinib and 10 non-treated controls. Metabolites in tumour extracts were analysed using gas chromatography coupled with mass spectrometry (GC-MS). RNA sequencing was also performed on the samples subset (n=6). Results Metabolomic analysis revealed 21 differentiating metabolites, whereas next-generation RNA sequencing data analysis resulted in 531 differentially expressed genes. Imatinib significantly changed the profile of metabolites associated mainly with purine and pyrimidine metabolism, butanoate metabolism, as well as alanine, aspartate, and glutamate metabolism. The related changes in transcriptomic profiles included genes involved in kinase activity and immune responses, as well as supported its impact on the purine biosynthesis pathway. Conclusions Our multi-omics study confirmed previously known pathways involved in imatinib anticancer activity as well as correlated imatinib-relevant downregulation of expression of purine biosynthesis pathway genes with the reduction of respectful metabolites. Furthermore, considering the importance of the purine biosynthesis pathway for cancer proliferation, we identified a potentially novel mechanism for the anti-tumour activity of imatinib. Based on the results, we hypothesise metabolic modulations aiming at the reduction in purine and pyrimidine pool may ensure higher imatinib efficacy or re-sensitise imatinib-resistant tumours.publishedVersio

Evaluation of cysteine metabolism in the rat liver and kidney following intravenous cocaine administration and abstinence

Many toxic effects of cocaine are attributed to reactive oxygen species (ROS) generated during its metabolism. Recently, it has been suggested that the biological action of ROS is often confused with endogenously generated reactive sulfur species (RSS). The aim of this study was to evaluate the impact of cocaine on thiols and RSS in the rat liver and kidney in the drug self-administration (SA) paradigm and the cocaine yoked delivery model (YC) followed by drug abstinence with extinction training. The level of thiols as well as RSS formed during anaerobic metabolism of cysteine and sulfate were assayed. In addition, the activity of enzymes involved in RSS formation and glutathione metabolism were determined. In the liver, following direct cocaine administration (SA and YC), the RSS levels decreased, while in the kidneys, cocaine increased the RSS contents in both groups. These changes were maintained in these tissues during drug abstinence. The level of sulfates was changed by cocaine only in the liver. In the kidney, cocaine shifted cysteine metabolism towards an anaerobic pathway. Our study demonstrates for the first time the changes in cysteine metabolism and thiol levels in the liver and kidney of rats after cocaine self-administration and abstinence

New Biochemical Insights into the Mechanisms of Pulmonary Arterial Hypertension in Humans

Diagnosis of pulmonary arterial hypertension (PAH) is difficult due to the lack of specific clinical symptoms and biomarkers, especially at early stages. We compared plasma metabolic fingerprints of PAH patients (n = 20) with matched healthy volunteers (n = 20) using, for the first time, untargeted multiplatform metabolomics approach consisting of high-performance liquid and gas chromatography coupled with mass spectrometry. Multivariate statistical analyses were performed to select metabolites that contribute most to groups' classification (21 from liquid in both ionization modes and 9 from gas chromatography-mass spectrometry). We found metabolites related to energy imbalance, such as glycolysis-derived metabolites, as well as metabolites involved in fatty acid, lipid and amino acid metabolism. We observed statistically significant changes in threitol and aminomalonic acid in PAH patients, which could provide new biochemical insights into the pathogenesis of the disease. The results were externally validated on independent case and control cohorts, confirming up to 16 metabolites as statistically significant in the validation study. Multiplatform metabolomics, followed by multivariate chemometric data analysis has a huge potential for explaining pathogenesis of PAH and for searching potential and new more specific and less invasive markers of the disease.This research was supported by the Polish National Science Center (2014/13/N/NZ7/04231), the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2014-58920R), by the Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) (PI14-01427), and by the quality-promoting subsidy from the Ministry of Science and Higher Education of Poland, Leading National Research Centre (KNOW programme 2012-2017). The CNIC is supported by the Spanish Ministry of Economy and Competitiveness and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

New biochemical insights into the mechanisms of pulmonary arterial hypertension in humans

Diagnosis of pulmonary arterial hypertension (PAH) is difficult due to the lack of specific clinical symptoms and biomarkers, especially at early stages. We compared plasma metabolic fingerprints of PAH patients (n = 20) with matched healthy volunteers (n = 20) using, for the first time, untargeted multiplatform metabolomics approach consisting of high-performance liquid and gas chromatography coupled with mass spectrometry. Multivariate statistical analyses were performed to select metabolites that contribute most to groups' classification (21 from liquid in both ionization modes and 9 from gas chromatography-mass spectrometry). We found metabolites related to energy imbalance, such as glycolysis-derived metabolites, as well as metabolites involved in fatty acid, lipid and amino acid metabolism. We observed statistically significant changes in threitol and aminomalonic acid in PAH patients, which could provide new biochemical insights into the pathogenesis of the disease. The results were externally validated on independent case and control cohorts, confirming up to 16 metabolites as statistically significant in the validation study. Multiplatform metabolomics, followed by multivariate chemometric data analysis has a huge potential for explaining pathogenesis of PAH and for searching potential and new more specific and less invasive markers of the disease

Metabolomic fingerprinting in the comprehensive study of liver changes associated with onion supplementation in hypercholesterolemic wistar rats

The consumption of functional ingredients has been suggested to be a complementary tool for the prevention and management of liver disease. In this light, processed onion can be considered as a source of multiple bioactive compounds with hepatoprotective properties. The liver fingerprint of male Wistar rats (n = 24) fed with three experimental diets (control (C), high-cholesterol (HC), and high-cholesterol enriched with onion (HCO) diets) was obtained through a non-targeted, multiplatform metabolomics approach to produce broad metabolite coverage. LC-MS, CE-MS and GC-MS results were subjected to univariate and multivariate analyses, providing a list of significant metabolites. All data were merged in order to figure out the most relevant metabolites that were modified by the onion ingredient. Several relevant metabolic changes and related metabolic pathways were found to be impacted by both HC and HCO diet. The model highlighted several metabolites (such as hydroxybutyryl carnitine and palmitoyl carnitine) modified by the HCO diet. These findings could suggest potential impairments in the energy−lipid metabolism, perturbations in the tricarboxylic acid cycle (TCA) cycle and β-oxidation modulated by the onion supplementation in the core of hepatic dysfunction. Metabolomics shows to be a valuable tool to evaluate the effects of complementary dietetic approaches directed to hepatic damage amelioration or non-alcoholic fatty liver disease (NAFLD) prevention.This work was supported by the Spanish Ministry of Science and Innovation (AGL2010-15910 (subprogram ALI)) and the Spanish Ministry of Economy and Competitiveness (MINECO-CTQ2014-55279-R).We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

A Robust Method for Sample Preparation of Gastrointestinal Stromal Tumour for LC/MS Untargeted Metabolomics

Gastrointestinal stromal tumour has already been well explored at the genome level; however, little is known about metabolic processes occurring in the sarcoma. Sample preparation is a crucial step in untargeted metabolomics workflow, highly affecting the metabolome coverage and the quality of the results. In this study, four liquid-liquid extraction methods for the isolation of endogenous compounds from gastrointestinal stromal tumours were compared and evaluated. The protocols covered two-step or stepwise extraction with methyl-tert-butyl ether (MTBE) or dichloromethane. The extracts were subjected to LC-MS analysis by the application of reversed-phase and hydrophilic interaction liquid chromatography to enable the separation and detection of both polar and nonpolar analytes. The extraction methods were compared in terms of efficiency (total number of detected metabolites) and reproducibility. The method was based on the stepwise extraction with MTBE, methanol, and water proved to be the most reproducible, and thus, its robustness to fluctuations in experimental conditions was assessed employing Plackett–Burman design and hierarchical modelling. While most studied factors had no effect on the metabolite abundance, the highest coefficient value was observed for the volume of MTBE added during extraction. Herein, we demonstrate the application and the feasibility of the selected protocol for the analysis of gastrointestinal stromal tumour samples. The method selected could be considered as a reference for the best characterization of underlying molecular changes associated with complex tissue extracts of GIST

Common Strategies and Factors Affecting Off-Line Breath Sampling and Volatile Organic Compounds Analysis Using Thermal Desorption-Gas Chromatography-Mass Spectrometry (TD-GC-MS)

An analysis of exhaled breath enables specialists to noninvasively monitor biochemical processes and to determine any pathological state in the human body. Breath analysis holds the greatest potential to remold and personalize diagnostics; however, it requires a multidisciplinary approach and collaboration of many specialists. Despite the fact that breath is considered to be a less complex matrix than blood, it is not commonly used as a diagnostic and prognostic tool for early detection of disordered conditions due to its problematic sampling, analysis, and storage. This review is intended to determine, standardize, and marshal experimental strategies for successful, reliable, and especially, reproducible breath analysi

LC–MS-Based Metabolomics Identification of Novel Biomarkers of Chorioamnionitis and Its Associated Perinatal Neurological Damage

Chorioamnionitis is a complication of pregnancy associated with significant maternal and perinatal long-term adverse outcomes. We apply high-throughput amniotic fluid (AF) metabolomics analysis for better understanding the pathophysiological mechanism of chorioamnionitis and its associated perinatal neurological injury and to provide meaningful information about new potential biomarkers. AF samples (<i>n</i> = 40) were collected from women at risk of chorioamnionits. Detailed clinical information on each pregnancy was obtained from obstetrical and neonatal medical examination. Liquid chromatography (LC)/mass spectrometry (MS) followed by data alignment and filtration as well as univariate and multivariate statistical analysis was performed. Statistically significant differences were found in 60 masses in positive and 115 in negative ionization mode obtained with LC/quadrupole time-of-flight MS (LC–QTOF-MS) between women with and without chorioamnionitis. Identified compounds were mainly related to glycerophospholipids and sphingolipids metabolism. From them, LPE(16:0)/LPE­(P-16:0) and especially lactosylceramides emerged as the best biomarker candidates. Sulfocholic acid, trioxocholenoic acids, and LPC(18:2) were particularly increased in women with chorioamnionitis whose newborns developed perinatal brain damage. Therefore, we propose LPE(16:0)/LPE­(P-16:0) and lactosylceramides as biomarkers for chorioamnionitis as well as LPC(18:2), trioxocholenoic acid, and sulfocholic acid for its associated perinatal brain damage. Metabolomics fingerprinting of AF enables the prediction of pregnancy-related disorders and the development of new diagnostics strategies

Metabolomics fingerprinting of liver in the comprehensive evaluation of onion as a functional ingredient in the prevention of metabolic impairments associated to diet-induced hypercholesterolaemia

Resumen del trabajo presentado al 24th International Symposium on Electro- and Liquid Phase- Separation Techniques (ITP) - XI Polska Konferencja Chromatograficzna (PKChrom), celebrados en Gdansk (Polonia) del 10 al 13 de septiembre de 2017.Functional ingredients has been suggested to be a complementary tool for the prevention and management of diseases. Metabolomics is capable of gathering comprehensive data, and appear to best enhance our understanding of the role of food in health and disease. Employment of metabolomics to nutrition research is increasing and applications range from assessing novel biomarkers of dietary intake to intervention studies. The aim was to combine untargeted metabolomics platforms to obtain the liver fingerprint of induced hypercholesterolemia in Wistar rats. The extent to which processed onion may affect the liver regulation and prevent the induced metabolic imbalance was assessed. The liver fingerprint of male Wistar rats (n = 24) fed with three experimental diets: control (C), highcholesterol: 2 % cholesterol and 0.5 % cholic acid (HC), and high-cholesterol enriched with onion (HCO) was obtained with LC-MS, GC-MS and CE-MS analysis, in order to achieve a broad metabolite coverage. Raw data acquired were processed to provide structured data in an appropriate format for data analysis. Reprocessed data were subjected to univariate and multivariate statistical analyses, providing a list of significant metabolites. All results were merged in order to determine the most relevant metabolites that were modified by the onion ingredient. 1169 and 490 features (ESI(+/-) ionization mode, respectively) were obtained after alignment, manual screening and integration correction by LC-MS and 129 features by CE-MS analysis. 75 metabolites were identified with the target metabolite Fiehn RTL library (G1676AA, Agilent), the CEMBIOlibrary and the NIST mass spectra library. In total 618 features in ESI(+) and 369 in ESI(-) LC-MS mode, 67 features in CE-MS and 33 metabolites in GC-MS were found to be statistically significant. Several relevant metabolic changes and related metabolic pathways were found to be impacted by both HC and HCO diet. The model highlighted metabolites, such as e.g., hydroxybutyryl carnitine and palmitoyl carnitine, modified by the HCO diet. These findings could suggest potential impairments in the energy-lipid metabolism, perturbations in the tricarboxylic acid cycle (TCA) cycle and β-oxidation modulated by the onion supplementation in the core of hepatic dysfunction. Processed onion can be considered as a source of multiple bioactive compounds with hepatoprotective properties. Metabolomics shows to be a valuable tool to evaluate the effects of complementary dietetic approaches directed to hepatic damage amelioration or non-alcoholic fatty liver disease (NAFLD) prevention.This work was supported by the Spanish Ministry of Science and Innovation (AGL2010-15910 (subprogram ALI)) and the Spanish Ministry of Economy and Competitiveness (MINECO-CTQ2014-55279-R).Peer Reviewe