Search

Background: Understanding the multifactorial nature of major depressive disorder (MDD) is crucial for tailoring treatments. However, the complex interplay of various factors underlying the development and progression of MDD poses significant challenges. Our previous study demonstrated improvements in cognitive functions in MDD patients undergoing treatment with selective serotonin reuptake inhibitors (SSRIs) supplemented with Lactobacillus plantarum 299v (LP299v)., Methods: To elucidate the biochemical mechanisms underlying cognitive functions improvements, we explored underlying metabolic changes. We employed multi-platform metabolomics, including LC-QTOF-MS and CE-TOF-MS profiling, alongside chiral LC-QqQ-MS analysis for amino acids., Results: Supplementation of SSRI treatment with LP299v intensified the reduction of long-chain acylcarnitines, potentially indicating improved mitochondrial function. LP299v supplementation reduced N-acyl taurines more than four times compared to the placebo, suggesting a substantial impact on restoring biochemical balance. The LP299v-supplemented group showed increased levels of oxidized glycerophosphocholine (oxPC). Additionally, LP299v supplementation led to higher levels of sphingomyelins, L-histidine, D-valine, and p-cresol., Limitations: This exploratory study suggests potential metabolic pathways influenced by LP299v supplementation. However, the need for further research hinders the ability to draw definitive conclusions., Conclusions: Observed metabolic changes were linked to mitochondrial dysfunction, inflammation, oxidative stress, and gut microbiota disruption. Despite the subtle nature of this alterations, our research successfully detected these differences and connected them to the metabolic disruptions associated with MDD. Our findings emphasise the intricate relationship between metabolism, gut microbiota, and mental health prompting further research into the mechanisms of action of probiotics in MDD treatment., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Primary Hyperparathyroidism (PHPT) is characterized by excessive parathormone (PTH) secretion and disrupted calcium homeostasis. Untargeted metabolomics offers a valuable approach to understanding the complex metabolic alterations associated with different diseases, including PHPT. Plasma untargeted metabolomics was applied to investigate the metabolic profiles of PHPT patients compared to a control group. Two complementary liquid-phase separation techniques were employed to comprehensively explore the metabolic landscape in this retrospective, single-center study. The study comprised 28 female patients diagnosed following the current guidelines of PHPT diagnosis and a group of 30 healthy females as a control group. To evaluate their association with PHPT, we identified changes in plasma metabolic profiles in patients with PHPT compared to the control group. The primary outcome measure included detecting plasma metabolites and discriminating PHPT patients from controls. The study unveiled specific metabolic imbalances that may link L-amino acids with peptic ulcer disease, gamma-glutamyls with oxidative stress, and asymmetric dimethylarginine (ADMA) with cardiovascular complications. Several metabolites, such as gamma-glutamyls, caffeine, sex hormones, carnitine, sphingosine-1-phosphate (S-1-P), and steroids, were connected with reduced bone mineral density (BMD). Metabolic profiling identified distinct metabolic patterns between patients with PHPT and healthy controls. These findings provided valuable insights into the pathophysiology of PHPT., (© 2024. The Author(s).)

Nutraceuticals have gained increasing interest, prompting the need to investigate plant extracts for their beneficial properties and potential side effects. This study aimed to assess the nutraceutical effects of environmentally clean extracts from Rosmarinus officinalis and Gongolaria abies-marina (formerly Cystoseira abies-marina (Phaeophyceae)) on the metabolic profile of streptozotocin-induced diabetic rats. We conducted untargeted LC-QTOF-MS metabolic profiling on six groups of rats: three diabetic groups receiving either a placebo, R. officinalis, or G. abies-marina extracts, and three corresponding control groups. The metabolic analysis revealed significant alterations in the levels of various glycerophospholipids, sterol lipids, and fatty acyls. Both extracts influenced the metabolic profile, partially mitigating diabetes-induced changes. Notably, G. abies-marina extract had a more pronounced impact on the animals' metabolic profiles compared to R. officinalis. In conclusion, our findings suggest that environmentally clean extracts from R. officinalis and G. abies-marina possess nutraceutical potential, as they were able to modulate the metabolic profile in streptozotocin-induced diabetic rats. G. abies-marina extract exhibited a more substantial effect on metabolic alterations induced by diabetes compared to R. officinalis. These results warrant further exploration of these plant extracts for their potential in managing diabetes-related metabolic disturbances., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Introduction: Discrimination between adenocarcinoma (ADC) and squamous cell carcinoma (SCC) subtypes in non-small cell lung cancer (NSCLC) patients is a significant challenge in oncology. Lipidomics analysis provides a promising approach for this differentiation. Methods: In an accompanying paper, we explored oxPCs levels in a cohort of 200 NSCLC patients. In this research, we utilized liquid chromatography coupled with mass spectrometry (LC-MS) to analyze the lipidomics profile of matching tissue and plasma samples from 25 NSCLC patients, comprising 11 ADC and 14 SCC cases. This study builds upon our previous findings, which highlighted the elevation of oxidised phosphatidylcholines (oxPCs) in NSCLC patients. Results: We identified eight lipid biomarkers that effectively differentiate between ADC and SCC subtypes using an untargeted approach. Notably, we observed a significant increase in plasma LPA 20:4, LPA 18:1, and LPA 18:2 levels in the ADC group compared to the SCC group. Conversely, tumour PC 16:0/18:2, PC 16:0/4:0; CHO, and plasma PC 16:0/18:2; OH, PC 18:0/20:4; OH, PC 16:0/20:4; OOH levels were significantly higher in the ADC group. Discussion: Our study is the first to report that plasma LPA levels can distinguish between ADC and SCC patients in NSCLC, suggesting a potential role for LPAs in NSCLC subtyping. This finding warrants further investigation into the mechanisms underlying these differences and their clinical implications., Competing Interests: Author LN was employed by Altium International Sp. z o. o. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sieminska, Miniewska, Mroz, Sierko, Naumnik, Kisluk, Michalska-Falkowska, Reszec, Kozlowski, Nowicki, Moniuszko, Kretowski, Niklinski, Ciborowski and Godzien.)

Introduction: Lung cancer is one of the most frequently studied types of cancer and represents the most common and lethal neoplasm. Our previous research on non-small cell lung cancer (NSCLC) has revealed deep lipid profile reprogramming and redox status disruption in cancer patients. Lung cell membranes are rich in phospholipids that are susceptible to oxidation, leading to the formation of bioactive oxidized phosphatidylcholines (oxPCs). Persistent and elevated levels of oxPCs have been shown to induce chronic inflammation, leading to detrimental effects. However, recent reports suggest that certain oxPCs possess anti-inflammatory, pro-survival, and endothelial barrier-protective properties. Thus, we aimed to measure the levels of oxPCs in NSCLC patients and investigate their potential role in lung cancer. Methods: To explore the oxPCs profiles in lung cancer, we performed in-depth, multi-level metabolomic analyses of nearly 350 plasma and lung tissue samples from 200 patients with NSCLC, including adenocarcinoma (ADC) and squamous cell carcinoma (SCC), the two most prevalent NSCLC subtypes and COPD patients as a control group. First, we performed oxPC profiling of plasma samples. Second, we analyzed tumor and non-cancerous lung tissues collected during the surgical removal of NSCLC tumors. Because of tumor tissue heterogeneity, subsequent analyses covered the surrounding healthy tissue and peripheral and central tumors. To assess whether the observed phenotypic changes in the patients were associated with measured oxPC levels, metabolomics data were augmented with data from medical records. Results: We observed a predominance of long-chain oxPCs in plasma samples and of short-chain oxPCs in tissue samples from patients with NSCLC. The highest concentration of oxPCs was observed in the central tumor region. ADC patients showed higher levels of oxPCs compared to the control group, than patients with SCC. Conclusion: The detrimental effects associated with the accumulation of short-chain oxPCs suggest that these molecules may have greater therapeutic utility than diagnostic value, especially given that elevated oxPC levels are a hallmark of multiple types of cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Godzien, Lopez-Lopez, Sieminska, Jablonowski, Pietrowska, Kisluk, Mojsak, Dzieciol-Anikiej, Barbas, Reszec, Kozlowski, Moniuszko, Kretowski, Niklinski and Ciborowski.)

The heterogeneity of acute myeloid leukemia (AML), a complex hematological malignancy, is caused by mutations in myeloid cells affecting their differentiation and proliferation. Thus, various cytogenetic alterations in AML cells may be characterized by a unique metabolome and require different treatment approaches. In this study, we performed untargeted metabolomics to assess metabolomics differences between AML patients and healthy controls, AML patients with different treatment outcomes, AML patients in different risk groups based on the 2017 European LeukemiaNet (ELN) recommendations for the diagnosis and management of AML, AML patients with and without FLT3-ITD mutation, and a comparison between patients with FLT3-ITD, CBF-AML (Core binding factor acute myelogenous leukemia), and MLL AML (mixed-lineage leukemia gene) in comparison to control subjects. Analyses were performed in serum samples using liquid chromatography coupled with mass spectrometry (LC-MS). The obtained metabolomics profiles exhibited many alterations in glycerophospholipid and sphingolipid metabolism and allowed us to propose biomarkers based on each of the above assessments as an aid for diagnosis and eventual classification, allowing physicians to choose the best-suited treatment approach. These results highlight the application of LC-MS-based metabolomics of serum samples as an aid in diagnostics and a potential minimally invasive prognostic tool for identifying various cytogenetic and treatment outcomes of AML., (© 2023. The Author(s).)

The relationship of high-carbohydrate (HC) meal intake to metabolic syndrome is still not fully explained. Metabolomics has the potential to indicate metabolic pathways altered by HC meals, which may improve our knowledge regarding the mechanisms by which HC meals may contribute to metabolic syndrome development. The fasting and postprandial metabolic response to HC or normo-carbohydrate (NC) meals with/without cinnamon + capsicum intake was evaluated using untargeted metabolomics and compared between normal-weight (NW) and overweight/obese (OW/OB) healthy men. Healthy male participants (age-matched) were divided into two groups (12 subjects per group). One was composed of men with normal weight (NW) and the other of men with overweight/obesity (OW/OB). On separate visits (with 2-3 week intervals), the participants received standardized HC or NC meals (89% or 45% carbohydrates, respectively). Fasting (0 min) and postprandial (30, 60, 120, 180 min) blood were collected for untargeted plasma metabolomics. Based on each metabolic feature's intensity change in time, the area under the curve (AUC) was calculated. Obtained AUCs were analyzed using multivariate statistics. Several metabolic pathways were found dysregulated after an HC meal in people from the OW/OB group but not the NW group. The consumption of HC meals by people with overweight/obesity led to a substantial increase in AUC, mainly for metabolites belonging to phospholipids and fatty acid amides. The opposite was observed for selected sphingolipids. The intake of cinnamon and capsicum normalized the concentration of selected altered metabolites induced by the intake of HC meals. A HC meal may induce an unfavourable postprandial metabolic response in individuals with overweight/obesity, and such persons should avoid HC meals.

Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca 2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Changes in serum or plasma metabolome may reflect gut microbiota dysbiosis, which is also known to occur in patients with prediabetes and type 2 diabetes (T2DM). Thus, developing a robust method for the analysis of microbiota-dependent metabolites (MDMs) is an important issue. Gas chromatography with mass spectrometry (GC-MS) is a powerful approach enabling detection of a wide range of MDMs in biofluid samples with good repeatability and reproducibility, but requires selection of a suitable solvents and conditions. For this reason, we conducted for the first time the study in which, we demonstrated an optimisation of samples preparation steps for the measurement of 75 MDMs in two matrices. Different solvents or mixtures of solvents for MDMs extraction, various concentrations and volumes of derivatizing reagents as well as temperature programs at methoxymation and silylation step, were tested. The stability, repeatability and reproducibility of the 75 MDMs measurement were assessed by determining the relative standard deviation (RSD). Finally, we used the developed method to analyse serum samples from 18 prediabetic (PreDiab group) and 24 T2DM patients (T2DM group) from our 1000PLUS cohort. The study groups were homogeneous and did not differ in age and body mass index. To select statistically significant metabolites, T2DM vs. PreDiab comparison was performed using multivariate statistics. Our experiment revealed changes in 18 MDMs belonging to different classes of compounds, and seven of them, based on the SVM classification model, were selected as a panel of potential biomarkers, able to distinguish between patients with T2DM and prediabetes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mojsak, Maliszewska, Klimaszewska, Miniewska, Godzien, Sieminska, Kretowski and Ciborowski.)

Identification of the NSCLC subtype at an early stage is still quite sophisticated. Metabolomics analysis of tissue and plasma of NSCLC patients may indicate new, and yet unknown, metabolic pathways active in the NSCLC. Our research characterized the metabolomics profile of tissue and plasma of patients with early and advanced NSCLC stage. Samples were subjected to thorough metabolomics analyses using liquid chromatography-mass spectrometry (LC-MS) technique. Tissue and/or plasma samples from 137 NSCLC patients were analyzed. Based on the early stage tissue analysis, more than 200 metabolites differentiating adenocarcinoma (ADC) and squamous cell lung carcinoma (SCC) subtypes as well as normal tissue, were identified. Most of the identified metabolites were amino acids, fatty acids, carnitines, lysoglycerophospholipids, sphingomyelins, plasmalogens and glycerophospholipids. Moreover, metabolites related to N-acyl ethanolamine (NAE) biosynthesis, namely glycerophospho (N-acyl) ethanolamines (GP-NAE), which discriminated early-stage SCC from ADC, have also been identified. On the other hand, the analysis of plasma of chronic obstructive pulmonary disease (COPD) and NSCLC patients allowed exclusion of the metabolites related to the inflammatory state in lungs and the identification of compounds (lysoglycerophospholipids, glycerophospholipids and sphingomyelins) truly characteristic to cancer. Our results, among already known, showed novel, thus far not described, metabolites discriminating NSCLC subtypes, especially in the early stage of cancer. Moreover, the presented results also indicated the activity of new metabolic pathways in NSCLC. Further investigations on the role of NAE biosynthesis pathways in the early stage of NSCLC may reveal new prognostic and diagnostic targets.

Capillary electrophoresis coupled to mass spectrometry is a power tool in untargeted metabolomics studies to analyze charged and polar compounds. However, identification is a challenge due to the variability of migration times and the lack of MS/MS spectra in CE-TOF-MS, the type of instruments most frequently employed. We present here a CE-MS search platform incorporated in CEU Mass Mediator to annotate metabolites with a confidence level L2. For its the development we analyzed 226 compounds using two fragmentor voltages: 100 and 200 V. The information obtained, such as relative migration times (RMT) and in-source fragments, were incorporated into the platform. In addition, we validated the CE-MS search functionality using different types of biological samples such as plasma samples (human, rat, and rabbit), mouse macrophages, and human urine. The RMT tolerance percentage for the search of metabolites has been determined, establishing 5% for all compounds, except for the compounds migrating in the electro-osmotic flow, for which the tolerance should be of 10%. It has also been demonstrated the robustness of the in-source fragmentation, which makes possible the annotation of compounds by means of their fragmentation pattern. As an example, 3-methylhistidine and 1-methilhistidine, whose RMT are very close, have been annotated. Studies of the fragmentation mechanisms of acyl-L-carnitines have shown that in-source fragmentation follows the general fragmentation rules and is a suitable alternative to MS/MS., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Adipose tissue has been the subject of research for a very long time. Many studies perform a comprehensive analysis of different types of adipose tissue with an emphasis on brown adipose tissue. Mass spectrometry-based approaches are particularly useful in the exploration not only of the metabolic composition of adipose tissue but also its function. In the presented review, a complex and critical overview of publications devoted to the analysis of adipose tissue by means of mass spectrometry was performed. Detailed investigation of analytical aspects related to either untargeted or targeted analysis of adipose tissue was performed, leading to the formation of a collection of hints at the available analytical methods. Moreover, a profound analysis of the metabolic composition of brown adipose tissue was performed. Brown adipose tissue metabolome was characterized on structural and functional levels, providing information about its exact metabolic composition but also connecting these molecules and placing them into biochemical pathways. All our work resulted in a very broad picture of the analysis of adipose tissue, starting from the analytical aspects and finishing on the current knowledge about its composition., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Untargeted metabolomics can be a great tool for exploring new scientific areas; however, wrong metabolite annotation questions the credibility and puts the success of the entire research at risk. Therefore, an effort should be made to improve the quality and robustness of the annotation despite of the challenges, especially when final identification with standards is not possible. Through non-targeted analysis of human plasma samples, from a large cancer cohort study using RP-LC-ESI-QTOF-MS/MS, we have resolved MS/MS annotation through spectral matching, directed to hydroxyeicosatetraenoic acids (HETEs) and, MS/MS structural elucidation for newly annotated oxidized lyso-phosphatidylcholines (oxLPCs). The annotation of unknowns is supported with structural information from fragmentation spectra as well as the fragmentation mechanisms involved, necessarily including data from both polarity modes and different collision energies. In this work, we present evidences that various oxidation products show significant differences between cancer patients and control individuals and we establish a workflow to help identify such modifications. We report here the upregulation of HETEs and oxLPCs in patients with neuroendocrine tumors (NETs). To our knowledge, this is the first attempt to determine HETEs in NETs and one of very few studies where oxLPCs are annotated. The obtained results provide an important insight regarding lipid oxidation in NETs, although their physiological functions still have to be established and require further research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Balloon catheter endothelial denudation in New Zealand white rabbits fed high cholesterol diet is a validated atherosclerosis model. Well-characterized in terms of atherosclerosis induction and progression, the metabolic changes associated with the atherosclerosis progression remain indeterminate. Non-targeted metabolomics permits to develop such elucidation and allows to evaluate the metabolic consequences of colchicine treatment, an anti-inflammatory drug that could revert these changes. 16 rabbits underwent 18 weeks of atherosclerosis induction by diet and aortic denudation. Thereafter animals were randomly assigned to colchicine treatment or placebo for 18 weeks while on diet. Plasma samples were obtained before randomization and at 36 weeks. Multiplatform (GC/MS, CE/MS, RP-HPLC/MS) metabolomics was applied. Plasma fingerprints were pre-processed, and the resulting matrixes analyzed to unveil differentially expressed features. Different chemical annotation strategies were accomplished for those significant features. We found metabolites associated with either atherosclerosis progression, or colchicine treatment, or both. Atherosclerosis was profoundly associated with an increase in circulating bile acids. Most of the changes associated with sterol metabolism could not be reverted by colchicine treatment. However, the variations in lysine, tryptophan and cysteine metabolism among others, have shown new potential mechanisms of action of the drug, also related to atherosclerosis progression, but not previously described.

In the liver, obesity is often manifested by the clinical disorder of the Non-Alcoholic Fatty Liver Disease (NAFLD). A proportion of NAFLD patients develop hepatic inflammation, known as Non-Alcoholic Steatohepatitis (NASH), which can end up in cirrhosis, or Hepatocellular Carcinoma (HCC). In this scenario, partial hepatectomy (PH) is an alternative to promote liver regeneration. However, as liver regeneration is impaired in NASH patients, more knowledge about its metabolic condition is needed to improve the regenerative response of the liver in this pathological condition. Although extensively employed, the panoply of molecular alterations involved in the regenerative response of the liver after partial hepatectomy PH is far from being fully characterized. Metabolic fingerprinting (metabolomics) is a powerful tool to help in the elucidation of complex metabolic networks, by means of a blind, naïve approach to study which metabolic nodes (metabolites) show the biggest variations between conditions. The objective of the present study was to gain deeper knowledge about the metabolic processes involved in the NASH animal model, and particularly in the effect of PH by using metabolomics. For achieving such information, twelve 8-week-old male C57BL/6 J mice, fed commercial chow (control diet) or methionine and choline-Deficient diet (MCD) for three weeks were subjected to PH and sacrificed 2 weeks later. Livers were removed and submitted to metabolic profiling analysis through RP-LC/MS (qTOF), GC/MS (qTOF) and CE/MS(TOF). More than 3000 different features were detected and repeated measurements one-way ANOVA analysis was performed to unveil significant features. MCD diet induced changes (p < 0.05) in 46% of the detected features, whereas PH provoked significant changes in 85% of them. Most of the changes were detected through LC/MS and were associated to lipid metabolism. However, changes of metabolites virtually related to other metabolic routes (amino acids, carbohydrates, nucleotides) were found altered and detected by CE/MS and GC/MS. The changes associated to PH show a similar trend regardless of the diet, but in the context of the diet deficient in methionine and choline we have found results that point to a different ratio glycolysis/tricarboxylic acid cycle. Moreover, in the NASH model, the regeneration of the liver structures occurs at the expense of an increased phosphatidylethanolamines/phosphatidylcholines ratio., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The Lipid Annotation Service (LAS) is a representational state transfer (REST) application programming interface (API) service designed to aid researchers performing lipid annotation. It assigns certainty levels (very unlikely, unlikely, likely, and very likely) to the putative annotations received as input and explains the rationale of such assignments. Its rules, obtained from the Centre for Metabolomics and Bioanalysis (CEMBIO) and from a literature review, enable LAS to extract evidence to support or refute the annotations automatically by checking the inter-rule relationships. LAS is the first metabolite annotation tool capable of explaining in natural language (English) the evidence that supports or refutes the annotations. This facilitates the understanding of the results by the user and, thus, increases the user's confidence in the results. Concerning its performance, in an evaluation of blood plasma samples whose compounds had previously been identified using well-established standards, LAS yielded an F-measure higher than 80%.

Lipid oxidation is one of the most important processes occurring in living cells and has been investigated through stable end-products. Currently, new insights into many physiological and pathophysiological processes provide a measurement of the first products of oxidation, e.g., oxidized glycerophosphatidylcholines (oxGPCs). Here, we evaluate the capacity of untargeted global metabolomics to measure oxGPCs in serum samples. This evaluation covered analytical reproducibility and data quality as well as the ability to capture metabolic alterations in diverse conditions. The analytical evaluation was performed based on the quality control samples, while the comparative analysis was based on the model of the development of type 2 diabetes mellitus (T2DM). The novelty of this approach arises not only from the measurement of oxGPCs instead of lipid peroxide-derived aldehydes but also from the stratification of the patients according to body mass index (BMI). Such a scenario was dictated by the fact that, despite the well-known relationship between obesity and T2DM development, there are lean individuals suffering from T2DM as well as obese people with normal glucose homeostasis. Our results provided evidence to support the ability of nontargeted metabolomics to measure oxGPCs. Comparative analysis of measured oxGPCs revealed differences in the level of oxGPCs either between different stages of disease development (insulin resistance, prediabetes) or BMI groups (normal weight, overweight, obese). The obtained results provided new insights into the metabolic processes leading to the development of T2DM and opened new paths in the investigation of the impact of body mass in T2DM progress., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: The transcription factor 7-like 2 (TCF7L2) gene confers one of the strongest genetic predispositions to type 2 diabetes, but diabetes development can be modified by diet., Objective: The aim of our study was to evaluate postprandial metabolic alterations in healthy men with a high genetic risk of diabetes, after two meals with varying macronutrient content., Methods: The study was conducted in 21 homozygous nondiabetic men carrying the high-risk (HR, n = 8, age: 31.2 ± 6.3 y, body mass index (BMI, kg/m2) 28.5 ± 8.1) or low-risk (LR, n = 13, age: 35.2 ± 10.3 y, BMI: 28.1 ± 6.4) genotypes at the rs7901695 locus. During two meal challenge test visits subjects received standardized isocaloric (450 kcal) liquid meals: high-carbohydrate (HC, carbohydrates: 89% of energy) and normo-carbohydrate (NC, carbohydrates: 45% of energy). Fasting (0 min) and postprandial (30, 60, 120, 180 min) plasma samples were analyzed for metabolite profiles through untargeted metabolomics. Metabolic fingerprinting was performed on an ultra-high-performance liquid chromatography (UHPLC) system connected to an iFunnel quadrupole-time-of-flight (Q-TOF) mass spectrometer., Results: In HR-genotype men, after the intake of an HC-meal, we noted a significantly lower area under the curves (AUCs) of postprandial plasma concentrations of most of the phospholipids (-37% to -53%, variable importance in the projection (VIP) = 1.2-1.5), lysophospholipids (-29% to -86%, VIP = 1.1-2.6), sphingolipids (-32% to -47%, VIP = 1.1-1.3), as well as arachidonic (-36%, VIP = 1.4) and oleic (-63%, VIP = 1.3) acids, their metabolites: keto- and hydoxy-fatty acids (-38% to -78%, VIP = 1.3-2.5), leukotrienes (-65% to -83%, VIP = 1.4-2.2), uric acid (-59%, VIP = 1.5), and pyroglutamic acid (-65%, VIP = 1.8). The AUCs of postprandial sphingosine concentrations were higher (125-832%, VIP = 1.9-3.2) after the NC-meal, AUCs of acylcarnitines were lower (-21% to -61%, VIP = 1.1-2.4), and AUCs of fatty acid amides were higher (51-508%, VIP = 1.7-3.1) after the intake of both meals., Conclusions: In nondiabetic men carrying the TCF7L2 HR genotype, subtle but detectable modifications in intermediate lipid metabolism are induced by an HC-meal. This trial was registered at www.clinicaltrials.gov as NCT03792685., (Copyright © American Society for Nutrition 2019.)

The prospero homeobox 1 (PROX1) gene may show pleiotropic effects on metabolism. We evaluated postprandial metabolic alterations dependently on the rs340874 genotypes, and 28 non-diabetic men were divided into two groups: high-risk (HR)-genotype (CC-genotype carriers, n = 12, 35.3 ± 9.5 years old) and low-risk (LR)-genotype (allele T carriers, n = 16, 36.3 ± 7.0 years old). Subjects participated in two meal-challenge-tests with high-carbohydrate (HC, carbohydrates 89%) and normo-carbohydrate (NC, carbohydrates 45%) meal intake. Fasting and 30, 60, 120, and 180 min after meal intake plasma samples were fingerprinted by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In HR-genotype men, the area under the curve (AUC) of acetylcarnitine levels was higher after the HC-meal [+92%, variable importance in the projection (VIP) = 2.88] and the NC-meal (+55%, VIP = 2.00) intake. After the NC-meal, the HR-risk genotype carriers presented lower AUCs of oxidized fatty acids (-81-66%, VIP = 1.43-3.16) and higher linoleic acid (+80%, VIP = 2.29), while after the HC-meal, they presented lower AUCs of ornithine (-45%, VIP = 1.83), sphingosine (-48%, VIP = 2.78), linoleamide (-45%, VIP = 1.51), and several lysophospholipids (-40-56%, VIP = 1.72-2.16). Moreover, lower AUC (-59%, VIP = 2.43) of taurocholate after the HC-meal and higher (+70%, VIP = 1.42) glycodeoxycholate levels after the NC-meal were observed. Our results revealed differences in postprandial metabolites from inflammatory and oxidative stress pathways, bile acids signaling, and lipid metabolism in PROX1 HR-genotype men. Further investigations of diet-genes interactions by which PROX1 may promote T2DM development are needed.

CEU Mass Mediator (CMM, http://ceumass.eps.uspceu.es ) is an online tool that has evolved from a simple interface to query different metabolomic databases (CMM 1.0) to a tool that unifies the compounds from these databases and, using an expert system with knowledge about the experimental setup and the compounds properties, filters and scores the query results (CMM 2.0). Since this last major revision, CMM has continued to grow, expanding the knowledge base of its expert system and including new services to support researchers in the metabolite annotation and identification process. The information from external databases has been refreshed, and an in-house library with oxidized lipids not present in other sources has been added. This has increased the number of experimental metabolites up 332,665 and the number of predicted metabolites to 681,198. Furthermore, new taxonomy and ontology metadata have been included. CMM has expanded its functionalities with a service for the annotation of oxidized glycerophosphocholines, a service for spectral comparison from MS 2 data, and a spectral quality-assessment service to determine the reliability of a spectrum for compound identification purposes. To facilitate the collaboration and integration of CMM with external tools and metabolomic platforms, a RESTful API has been created, and it has already been integrated into the HMDB (Human Metabolome Database). This paper will present the novel functionalities incorporated into version 3.0 of CMM.

The characterization of specialized cell subpopulations in a heterogeneous tissue is essential for understanding organ function in health and disease. A popular method of cell isolation is fluorescence-activated cell sorting (FACS) based on probes that bind surface or intracellular markers. In this study, we analyze the impact of FACS on the cell metabolome of mouse peritoneal macrophages. Compared with directly pelleted macrophages, FACS-treated cells had an altered content of metabolites related to the plasma membrane, activating a mechanosensory signaling cascade causing inflammation-like stress. The procedure also triggered alterations related to energy consumption and cell damage. The observed changes mostly derive from the physical impact on cells during their passage through the instrument. These findings provide evidence of FACS-induced biochemical changes, which should be taken into account in the design of robust metabolic assays of cells separated by flow cytometry.

Although capillary electrophoresis (CE) coupled to mass spectrometry (MS) is a separation technique not extensively implemented, it offers differential possibilities in the study of polar and ionic metabolites in complex matrices with minimum sample treatment. However, in order to get successful results, some efforts at early stages and following specific recommendations are necessary.In this chapter, we describe our updated and well-tested methods for untargeted metabolomics using CE-MS-TOF for common biological samples: urine, serum or plasma, feces, tissues, and cells. Sample treatment, as well as separation and detection conditions are described in detail and other steps in the workflow for untargeted metabolomics are also explained. Special attention is paid to instrumental setup and advices for daily practice.Characteristic electropherograms obtained with each type of sample are depicted as well as groups of metabolites easily measured by this technique. Their global or individual comparisons have been given undoubtedly important information to unveil altered metabolic pathways, diagnosis, and prognosis or biomarker discovery in the study of diseases or conditions over decades.

The biological role of oxidized glycerophosphocholines (oxPCs) is a current topic of research importantly contributing to the understanding of health and disease. Global non-targeted metabolomics offers an interesting approach to expand current knowledge and link oxPCs to new biological functions. Although this strategy is successful, it also has some limitations which are clearly noticeable during the identification process. For this reason, clear rules related to the identification of each group of metabolites are needed. This work attempts to provide the reader with a guideline for the recognition and annotation of oxidation among phosphocholines (PCs). Using several chromatographic characteristics and spectral information from tandem mass spectrometry, rapid and reliable annotation of long and short chain oxPCs can be performed. Some of this knowledge has been implemented in the publicly available annotation tool 'CEU Mass Mediator' (CMM) for semi-automated assignment of oxidation. Additionally, this tool was supplemented with accurate monoisotopic masses of oxPCs, expanding current information in other databases. Moreover, the characterization of oxidization products of PC(16:0/20:4) known as PAPC has been performed, providing a list of accurate mass product ions and neutral losses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

CEU Mass Mediator (CMM) is an on-line tool for aiding researchers when performing metabolite annotation. Its database is comprised of 279,318 real compounds integrated from several metabolomic databases including Human Metabolome Database (HMDB), KEGG and LipidMaps and 672,042 simulated compounds from MINE. In addition, CMM scores the annotations which matched the query parameters using 122 rules based on expert knowledge. This knowledge, obtained from the Centre for Metabolomics and Bioanalysis (CEMBIO) and from a literature review, enables CMM expert system to automatically extract evidence to support or refute the annotations by checking relationships among them. CMM is the first metabolite annotation tool that uses a knowledge-driven approach to provide support to the researcher. This allows to focus on the most plausible annotations, thus saving time and minimizing mistakes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A lack of viable hits, increasing resistance, and limited knowledge on mode of action is hindering drug discovery for many diseases. To optimize prioritization and accelerate the discovery process, a strategy to cluster compounds based on more than chemical structure is required. We show the power of metabolomics in comparing effects on metabolism of 28 different candidate treatments for Leishmaniasis (25 from the GSK Leishmania box, two analogues of Leishmania box series, and amphotericin B as a gold standard treatment), tested in the axenic amastigote form of Leishmania donovani. Capillary electrophoresis-mass spectrometry was applied to identify the metabolic profile of Leishmania donovani, and principal components analysis was used to cluster compounds on potential mode of action, offering a medium throughput screening approach in drug selection/prioritization. The comprehensive and sensitive nature of the data has also made detailed effects of each compound obtainable, providing a resource to assist in further mechanistic studies and prioritization of these compounds for the development of new antileishmanial drugs.

With the World Health Organization reporting over 30,000 deaths and 200,000 to 400,000 new cases annually, visceral leishmaniasis is a serious disease affecting some of the world's poorest people. As drug resistance continues to rise, there is a huge unmet need to improve treatment. Miltefosine remains one of the main treatments for leishmaniasis, yet its mode of action (MoA) is still unknown. Understanding the MoA of this drug and parasite response to treatment could help pave the way for new and more successful treatments for leishmaniasis. A novel method has been devised to study the metabolome and lipidome of Leishmania donovani axenic amastigotes treated with miltefosine. Miltefosine caused a dramatic decrease in many membrane phospholipids (PLs), in addition to amino acid pools, while sphingolipids (SLs) and sterols increased. Leishmania major promastigotes devoid of SL biosynthesis through loss of the serine palmitoyl transferase gene (ΔLCB2) were 3-fold less sensitive to miltefosine than wild-type (WT) parasites. Changes in the metabolome and lipidome of miltefosine-treated L. major mirrored those of L. donovani A lack of SLs in the ΔLCB2 mutant was matched by substantial alterations in sterol content. Together, these data indicate that SLs and ergosterol are important for miltefosine sensitivity and, perhaps, MoA., (Copyright © 2018 Armitage et al.)

Mechanisms responsible for metabolic gains after bariatric surgery are not entirely clear. The purpose of this study was evaluation of metabolic changes after laparoscopic Roux-en-Y gastric bypass or laparoscopic sleeve gastrectomy in semi-annual follow up. The study participants were selected from obese patients with T2DM who underwent one of the mentioned bariatric procedures. Serum metabolic fingerprinting by use of liquid and gas chromatography with mass spectrometry detection was performed on samples obtained from studied patients before, one, and six months post-surgery. Performed analyses resulted in 49 significant and identified metabolites. Comparison of the two described procedures has allowed to detect metabolites linked with numerous pathways, processes and diseases. Based on the metabolites detected and pathways affected, we propose a "gear mechanism" showing molecular changes evoked by both bariatric procedures. Critical evaluation of clinical data and obtained metabolomics results enables us to conclude that both procedures are very similar in terms of general clinical outcome, but they strongly differ from each other in molecular mechanisms leading to the final effect. For the first time general metabolic effect of bariatric procedures is described. New hypotheses concerning molecular mechanisms induced by bariatric surgeries and new gut microbiota modulations are presented., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Metabolite identification is one of the most challenging steps in metabolomics studies and reflects one of the greatest bottlenecks in the entire workflow. The success of this step determines the success of the entire research, therefore the quality at which annotations are given requires special attention. A variety of tools and resources are available to aid metabolite identification or annotation, offering different and often complementary functionalities. In preparation for this article, almost 50 databases were reviewed, from which 17 were selected for discussion, chosen for their online ESI-MS functionality. The general characteristics and functions of each database is discussed in turn, considering the advantages and limitations of each along with recommendations for optimal use of each tool, as derived from experiences encountered at the Centre for Metabolomics and Bioanalysis (CEMBIO) in Madrid. These databases were evaluated considering their utility in non-targeted metabolomics, including aspects such as identifier assignment, structural assignment and interpretation of results., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Highly polar and ionic metabolites, such as sugars, most amino acids, organic acids or nucleotides are not retained by conventional reversed-phase LC columns and polar stationary phases and hydrophilic-interaction LC lacks of robustness, which is still limiting their applications for untargeted metabolomics where reproducibility is a must. Biological samples such as blood, urine or even tissues include many hydrophilic compounds secreted from cells, their analysis is essential for biomarker discovery, disease progression or treatment effects. This review focuses on CE coupled to MS as a mature technique for untargeted metabolomics including sample pretreatment, types of matrices, analytical methods, applications and data treatment strategies for polar compound analysis in biological matrices. The main applications and results of CE-MS in untargeted metabolomics are discussed and presented in a tabulated format.

Quinolinic acid (QUIN), one of the end metabolites in the kynurenine pathway, plays an important role in the pathogenesis of several diseases. Serum QUIN concentration rises in patients with renal dysfunction, liver cirrhosis, and many other inflammatory diseases. In the present study, osmotic minipumps containing QUIN (0.3 and 1mg/day) were implanted intraperitoneally into rats for 28days. Then, the physiological and toxicological variables were evaluated and LC-QTOF-MS serum metabolic fingerprinting was performed. QUIN significantly decreased the serum concentrations of several amino acids (phenylalanine, valine, tyrosine, and tryptophan), pantothenic acid, branched chain C4 acylcarnitine, total cholesterol, and glucose; increased the serum concentrations of amides (pentadecanoic amide, palmitic amide, oleamide, and stearamide), polyamines (spermine and spermidine), sphingosine, and deoxy-prostaglandin; caused alterations in phospholipids. This is the first report of comprehensive metabolites analysis after chronic intraperitoneal administration of QUIN. Further studies could develop new therapeutics for patients with disorders accompanied by increased serum level of QUIN., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A single in-vial dual extraction (IVDE) procedure for the subsequent analysis of lipids and proteins in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions derived from the same biological sample is presented. On the basis of methyl-tert-butyl ether (MTBE) extraction, IVDE leads to the formation of three phases: a protein pellet at the bottom, an aqueous phase with polar compounds, and an ether phase with lipophilic compounds. After sample extraction, performed within a high-performance liquid chromatography vial insert, the ether phase was directly injected for lipid fingerprinting, while the protein pellet, after evaporation of the remaining sample, was used for proteomics analysis. Human HDL and LDL isolates were used to test the suitability of the IVDE methodology for lipid and protein analysis from a single sample in terms of data quality and matching composition to that of HDL and LDL. Subsequently, HDL and LDL fractions isolated from ApoE-KO and wild-type mice were used to validate the capacity of IVDE for revealing changes in lipid and protein abundance. Results indicate that IVDE can be successfully used for the subsequent analysis of lipids and proteins with the advantages of time saving, simplicity, and reduced sample amount.

The origin of missing values can be caused by different reasons and depending on these origins missing values should be considered differently and dealt with in different ways. In this research, four methods of imputation have been compared with respect to revealing their effects on the normality and variance of data, on statistical significance and on the approximation of a suitable threshold to accept missing data as truly missing. Additionally, the effects of different strategies for controlling familywise error rate or false discovery and how they work with the different strategies for missing value imputation have been evaluated. Missing values were found to affect normality and variance of data and k-means nearest neighbour imputation was the best method tested for restoring this. Bonferroni correction was the best method for maximizing true positives and minimizing false positives and it was observed that as low as 40% missing data could be truly missing. The range between 40 and 70% missing values was defined as a "gray area" and therefore a strategy has been proposed that provides a balance between the optimal imputation strategy that was k-means nearest neighbor and the best approximation of positioning real zeros., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Development of type 2 diabetes mellitus (T2DM) is preceded by insulin resistance (IR), which may evolve to impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT). IFG and IGT are considered as prediabetic states (PD). Prediabetes indicates the high risk for the future development of diabetes, it is estimated that up to 70% of prediabetics eventually develop T2DM. The risk of T2DM development is increased in overweight (OW) and obese (OB) people; however normal weight (NW) individuals also suffer from T2DM. The present study was designed to evaluate whether changes in polar metabolites induced by T2DM evolution are different between NW, overweight and obese individuals. CE-MS serum fingerprinting was performed on 197 serum samples obtained from OW, OB, and NW humans whom were IR, prediabetics, diabetics or with normal glucose homeostasis. Metabolic changes evoked by the progression of T2DM differ between obese, overweight, and normal weight subjects. Based on obtained results several metabolites can be proposed as a promising target to track T2DM evolution; BCAA in OW and NW humans, lysine in OB, while acetylcarnitine and methionine independently on body mass index. Validation of obtained results on larger population is required., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The role of non-targeted metabolomics with its discovery power is constantly growing in many different fields of science. However, its biggest advantage of uncovering the unexpected is turning into one of its biggest bottlenecks, particularly in metabolite identification. Among different methods for metabolite identification or ID confirmation, tandem MS analysis plays a very important role. However, this method is limited to only certain types of MS analysers, making for example TOF-MS inaccessible for this type of metabolite identification. To overcome this, in-source fragmentation has been used to fragment molecules and obtain product ions. Since the molecule of interest is not isolated prior to its fragmentation, the acquired spectrum contains many different signals arising from the fragmentation of all compounds present in the sample. Therefore, to assign product ions to their precursors, a novel use of correlation analysis was tested with r ≥0.9 as an assignation of a product ion belonging to the precursor. This method and chosen cut-off was tested on three different sample complexity levels: conducting the analysis on a single standard, mix of co-eluting standards and on a plasma sample. Obtained results clearly proved the effectiveness of the proposed methodology for metabolite ID confirmation. Moreover, the proposed strategy can be successfully applied for semi-quantification of co-eluting molecules with the same monoisotopic mass but that differ in fragmentation pattern. The proposed methodology can greatly improve the robustness and throughput of identification in metabolomics studies by use of TOF-MS, which is crucial to obtain meaningful and trustful results., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lipids are important components of biological systems, and their role can be currently investigated by the application of untargeted, holistic approaches such as metabolomics and lipidomics. Acquired data are analyzed to find significant signals responsible for the differentiation between the investigated conditions. Subsequently, identification has to be performed to bring biological meaning to the obtained results. Lipid identification seems to be relatively easy due to the known characteristic fragments; however, the large number of structural isomers and the formation of different adducts makes it challenging and at risk of misidentification. The inspection of data, acquired for plasma samples by a standard metabolic fingerprinting method, revealed multisignal formations for phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins by the formation of ions such as [M + H](+), [M + Na](+), and [M + K](+) in positive ionization mode and [M - H](-), [M + HCOO](-), and [M + Cl](-) in negative mode. Moreover, sodium formate cluster formation was found for [M + H·HCOONa](+) and [H-H·HCOONa](-). The MS/MS spectrum obtained for each of the multi-ions revealed significant differences in the fragmentation, which were confirmed by the analysis of the samples in two independent research centers. After the inspection of an acquired spectra, a list of characteristic and diagnostic fragments was proposed that allowed for easy, quick, and robust lipid identification that provides information about the headgroup, formed adduct, and fatty acyl composition. This ensures successful identification, which is of great importance for the contextualization of data and results validation.

Application of high-throughput technologies in metabolomics studies increases the quantity of data obtained, which in turn imposes several problems during data analysis. Correctly and clearly addressed biological question and comprehensive knowledge about data structure and properties are definitely necessary to select proper chemometric tools. However, there is a broad range of chemometric tools available for use with metabolomics data, which makes this choice challenging. Precisely performed data treatment enables valuable extraction of information and its proper interpretation. The effect of an error made at an early stage will be enhanced throughout the later stages, which in combination with other errors made at each step can accumulate and significantly affect the data interpretation. Moreover, adequate application of these tools may help not only to detect, but sometimes also to correct, biological, analytical, or methodological errors, which may affect truthfulness of obtained results. This report presents steps and tools used for LC-MS based metabolomics data extraction, reduction, and visualization. Following such steps as data reprocessing, data pretreatment, data treatment, and data revision, authors want to show how to extract valuable information and how to avoid misinterpretation of results obtained. The purpose of this work was to emphasize problematic characteristics of metabolomics data and the necessity for their attentive and precise treatment. The dataset used to illustrate metabolomics data properties and to illustrate major data treatment challenges was obtained utilizing an animal model of control and diabetic rats, both with and without rosemary treatment. Urine samples were fingerprinted employing LC-QTOF-MS., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of metabolomics studies is the comprehensive and quantitative analysis of all metabolites in a cell, tissue or organism. This approach requires sample preparation methods to be fast, reproducible and able to extract a wide range of analytes with different polarities, as well as analytical platforms able to detect the extracted metabolites. Recently, we have developed a one-step extraction method consisting of a lipophilic and hydrophilic layer within a single vial insert, in-vial dual extraction (IVDE). In order to check possible application of this method to real biological case, analysis of plasma samples obtained from three streptozotocin-induced diabetic and three control rats was performed. Analytical validity of the method was proved by the calculation (in quality control samples) of relative standard deviation (RSD) for detected metabolites. The percentage of metabolites with RSD<30% was 93% for Fatty acyls, 80% for Glycerolipids, 93% for Glycerophospholipids, 68% for Sterol lipids, and 91% for Sphingolipids. IVDE allowed for selection of more than 600 different features discriminating two studied groups. For around 40% of these masses putative identification was possible. Adequate, with several considerations described within this paper, application of IVDE method enables wide metabolite coverage from a single 20μL plasma aliquot. Within the features putatively identified, glycerolipids and glycerophospholipids arose as the most important groups of compounds discriminating diabetic rats from controls. All discriminating metabolites give an idea of the large metabolic differences that can be present in non-controlled type 1 diabetes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ozonated autohemotherapy (O3-AHT) is a medical approach during which blood obtained from the patient is ozonated and injected back into the body. Despite an increasing number of evidence that O3-AHT is safe, this type of therapy remains controversial. To extend knowledge about the changes in blood evoked by O3-AHT, LC-MS- and GC-MS-based metabolic fingerprinting was used to compare plasma samples obtained from blood before and after the treatment with potentially therapeutic concentrations of ozone. The procedure was performed in PVC bags utilized for blood storage to study also possible interactions between ozone and plastic. By use of GC-MS, an increase in lactic acid and pyruvic acid was observed, which indicated an increased rate of glycolysis. With LC-MS, changes in plasma antioxidants were observed. Moreover, concentrations of lipid oxidation products (LOP) and lysophospholipids were increased after ozone treatment. This is the first report of increased LOPs metabolites after ozonation of blood. Seven metabolites detected by LC-QTOF-MS only in ozonated samples could be considered as novel biomarkers of oxidative stress. Several plasticizers have been detected by both techniques in blood stored in PVC bags. PVC is known to be an ozone resistant material, but ozonation of blood in PVC bags stimulates leaching of plasticizers into the blood.