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Background: Live single-cell metabolomic studies encounter inherent difficulties attributed to the limited sample volume, minimal compound quantity, and insufficient sensitivity in the Mass Spectrometry (MS) method used to obtain single-cell data. However, understanding cellular heterogeneity, functional diversity, and metabolic processes within individual cells is essential. Exploring how individual cells respond to stimuli, including drugs, environmental changes, or signaling molecules, offers insights into biology, oncology, and drug discovery. Efficient release of cell contents (lysis) is vital for accurate metabolite detection at the single-cell level. Despite this, traditional approaches in live single cell metabolomics methods do not emphasize efficient lysis to prevent sample dilution. Instead, current live single cell metabolomics methods use direct infusion to introduce the cell into the mass spectrometry without prior chromatographic separation or a lysis step, which adversely affects sensitivity and metabolic coverage., Results: To address this, we developed an integrated single-cell electrical lysis and nano spray (SCEL-nS) platform coupled to an Orbitrap MS capable of efficiently lysing a single cell after being sampled with specially manufactured micropipettes. Lysis efficiency was validated by comparing live cell stain fluorescent intensities of intact and electrically lysed cells through microscopy imaging. The SCEL-nS platform successfully induced the breakdown of a single cell, significantly reducing the live cell stain's fluorescent intensity indicating cell membrane breakdown. Additionally, SCEL-nS was validated by measuring single cells spiked with the anti-cancer drug tamoxifen by MS. SCEL-nS use resulted in statistically significant increase in the peak measured by the method compared to the traditional non-lysis method., Significance: Overall, our results demonstrate that the newly incorporated SCEL-nS platform achieved higher sensitivities compared to traditional live single cell analysis methods., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Vascular ageing is associated with increased arterial stiffness and cardiovascular mortality that might be linked to altered vascular energy metabolism. The aim of this study was to establish a Seahorse XFe96 Analyzer-based methodology for the reliable, functional assessment of mitochondrial respiration and glycolysis in single murine aortic rings and to validate this functional assay by characterising alterations in vascular energy metabolism in aged mice. Healthy young and old C57BL/6 mice were used for the analyses. An optimised setup consisting of the Seahorse XFe96 Analyzer and Seahorse Spheroid Microplates was applied for the mitochondrial stress test and the glycolysis stress test on the isolated murine aortic rings, supplemented with analysis of NAD content in the aorta. To confirm the age-dependent stiffness of the vasculature, pulse wave velocity was measured in vivo. In addition, the activity of vascular nitric oxide synthase and vascular wall morphology were analysed ex vivo. The vascular ageing phenotype in old mice was confirmed by increased aortic stiffness, vascular wall remodelling, and nitric oxide synthase activity impairment. The rings of the aorta taken from old mice showed changes in vascular energy metabolism, including impaired spare respiratory capacity, maximal respiration, glycolysis, and glycolytic capacity, as well as a fall in the NAD pool. In conclusion, optimised Seahorse XFe96-based analysis to study energy metabolism in single aortic rings of murine aorta revealed a robust impairment of functional vascular respiratory and glycolytic capacity in old mice linked to NAD deficiency that coincided with age-related aortic wall remodelling and stiffness., (© 2024. The Author(s).)

Background: Isoprostanes and prostaglandins are biomarkers for oxidative stress and inflammation. Their role in Alzheimer's disease (AD) pathophysiology is yet unknown. In the current study, we aim to identify the association of isoprostanes and prostaglandins with the Amyloid, Tau, Neurodegeneration (ATN) biomarkers (Aβ-42, p-tau, and t-tau) of AD pathophysiology in mild cognitive impairment (MCI) subjects., Methods: Targeted metabolomics profiling was performed using liquid chromatography-mass spectrometry (LCMS) in 147 paired plasma-CSF samples from the Ace Alzheimer Center Barcelona and 58 CSF samples of MCI patients from the Mannheim/Heidelberg cohort. Linear regression was used to evaluate the association of metabolites with CSF levels of ATN biomarkers in the overall sample and stratified by Aβ-42 pathology and APOE genotype. We further evaluated the role of metabolites in MCI to AD dementia progression., Results: Increased CSF levels of PGF2α, 8,12-iso-iPF2α VI, and 5-iPF2α VI were significantly associated (False discovery rate (FDR) < 0.05) with higher p-tau levels. Additionally, 8,12-iso-iPF2α VI was associated with increased total tau levels in CSF. In MCI due to AD, PGF2α was associated with both p-tau and total tau, whereases 8,12-iso-iPF2α VI was specifically associated with p-tau levels. In APOE stratified analysis, association of PGF2α with p-tau and t-tau was observed in only APOE ε4 carriers while 5-iPF2α VI showed association with both p-tau and t-tau in APOE ε33 carriers. CSF levels of 8,12- iso-iPF2α VI showed association with p-tau and t-tau in APOE ε33/APOE ε4 carriers and with t-tau in APOE ε3 carriers. None of the metabolites showed evidence of association with MCI to AD progression., Conclusions: Oxidative stress (8,12-iso-iPF2α VI) and inflammatory (PGF2α) biomarkers are correlated with biomarkers of AD pathology during the prodromal stage of AD and relation of PGF2α with tau pathology markers may be influenced by APOE genotype., (© 2024. The Author(s).)

Photoactivated chemotherapy agents form a new branch of physically targeted anticancer agents with potentially lower systemic side effects for patients. On the other hand, limited information exists on the intracellular interactions between the photoreleased metal cage and the photoreleased anticancer inhibitor. In this work, we report a new biological study of the known photoactivated compound Ru-STF31 in the glioblastoma cancer cell line, U87MG. Ru-STF31 targets nicotinamide phosphoribosyltransferase (NAMPT), an enzyme overexpressed in U87MG. Ru-STF31 is activated by red light irradiation and releases two photoproducts: the ruthenium cage and the cytotoxic inhibitor STF31 . This study shows that Ru-STF31 can significantly decrease intracellular NAD + levels in both normoxic (21% O 2 ) and hypoxic (1% O 2 ) U87MG cells. Strikingly, NAD + depletion by light activation of Ru-STF31 in hypoxic U87MG cells could not be rescued by the addition of extracellular NAD + . Our data suggest an oxygen-dependent active role of the ruthenium photocage released by light activation.

Since its first appearance, severe acute respiratory syndrome coronavirus 2 quickly spread around the world and the lack of adequate PCR testing capacities, especially during the early pandemic, led the scientific community to explore new approaches such as mass spectrometry (MS). We developed a proteomics workflow to target several tryptic peptides of the nucleocapsid protein. A highly selective multiple reaction monitoring-cubed (MRM 3 ) strategy provided a sensitivity increase in comparison to conventional MRM acquisition. Our MRM 3 approach was first tested on an Amsterdam public health cohort (alpha-variant, 760 participants) detecting viral nucleocapsid protein peptides from nasopharyngeal swabs samples presenting a cycle threshold value down to 35 with sensitivity and specificity of 94.2% and 100.0%, without immunopurification. A second iteration of the MS-diagnostic test, able to analyze more than 400 samples per day, was clinically validated on a Leiden-Rijswijk public health cohort (delta-variant, 2536 participants) achieving 99.9% specificity and 93.1% sensitivity for patients with cycle threshold values up to 35. In this manuscript, we also developed and brought the first proof of the concept of viral variant monitoring in a complex matrix using targeted MS., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Signaling lipids (SLs) play a crucial role in various signaling pathways, featuring diverse lipid backbone structures. Emerging evidence showing the biological significance and biomedical values of SLs has strongly spurred the advancement of analytical approaches aimed at profiling SLs. Nevertheless, the dramatic differences in endogenous abundances across lipid classes as well as multiple isomers within the same lipid class makes the development of a generic analytical method challenging. A better analytical method that combines comprehensive coverage and high sensitivity is needed to enable us to gain a deeper understanding of the biochemistry of these molecules in health and disease. In this study, we developed a fast and comprehensive targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for profiling SLs. The platform enables analyses of 260 metabolites covering oxylipins (isoprostanes, prostaglandins and other oxidized lipids), free fatty acids, lysophospholipids, sphingoid bases (C16, C18), platelet activating factors (C16, C18), endocannabinoids and bile acids. Various validation parameters including linearity, limit of detection, limit of quantification, extraction recovery, matrix effect, intra-day and inter-day precision were used to characterize this method. Metabolite quantitation was successfully achieved in both NIST Standard Reference Material for human plasma (NIST SRM 1950) and pooled human plasma, with 109 and 144 metabolites quantitated. The quantitation results in NIST SRM 1950 plasma demonstrated good correlations with certified or previously reported values in published literature. This study introduced quantitative data for 37 SLs for the first time. Metabolite concentrations measured in NIST SRM 1950 will serve as essential reference data for facilitating interlaboratory comparisons. The methodology established here will be the cornerstone for in-depth profiling of signaling lipids across diverse biological samples and contexts., 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 B.V. All rights reserved.)

Hepatocytes are responsible for maintaining a stable blood glucose concentration during periods of nutrient scarcity. The breakdown of glycogen and de novo synthesis of glucose are crucial metabolic pathways deeply interlinked with lipid metabolism. Alterations in these pathways are often associated with metabolic diseases with serious clinical implications. Studying energy metabolism in human cells is challenging. Primary hepatocytes are still considered the golden standard for in vitro studies and have been instrumental in elucidating key aspects of energy metabolism found in vivo. As a result of several limitations posed by using primary cells, a multitude of alternative hepatocyte cellular models emerged as potential substitutes. Yet, there remains a lack of clarity regarding the precise applications for which these models accurately reflect the metabolic competence of primary hepatocytes. In this study, we compared primary hepatocytes, stem cell-derived hepatocytes, adult donor-derived liver organoids, immortalized Upcyte-hepatocytes and the hepatoma cell line HepG2s in their response to a glucose production challenge. We observed the highest net glucose production in primary hepatocytes, followed by organoids, stem-cell derived hepatocytes, Upcyte-hepatocytes and HepG2s. Glucogenic gene induction was observed in all tested models, as indicated by an increase in G6PC and PCK1 expression. Lipidomic analysis revealed considerable differences across the models, with organoids showing the closest similarity to primary hepatocytes in the common lipidome, comprising 347 lipid species across 19 classes. Changes in lipid profiles as a result of the glucose production challenge showed a variety of, and in some cases opposite, trends when compared to primary hepatocytes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Flavio Bonanini and Dorota Kurek are employees of Mimetas BV, which markets advanced in vitro system for drug development. The authors declare they have no additional conflict of interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The severity of COVID-19 is linked to an imbalanced immune response. The dysregulated metabolism of small molecules and bioactive lipids has also been associated with disease severity. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyze over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). This is the third publication in a series, and it reports the results of comprehensive lipidome profiling using targeted LC-MS/MS. We identified 1076 lipid features across 25 subclasses, including glycerophospholipids, sterols, glycerolipids, and sphingolipids, among which 531 lipid features were dramatically changed in the plasma of intensive care unit (ICU) patients compared to patients in the ward. Patients in the ICU showed 1.3-57-fold increases in ceramides, (lyso-)glycerophospholipids, diglycerides, triglycerides, and plasmagen phosphoethanolamines, and 1.3-2-fold lower levels of a cyclic lysophosphatidic acid, sphingosine-1-phosphates, sphingomyelins, arachidonic acid-containing phospholipids, lactosylceramide, and cholesterol esters compared to patients in the ward. Specifically, phosphatidylinositols (PIs) showed strong fatty acid saturation-dependent behavior, with saturated fatty acid (SFA)- and monosaturated fatty acid (MUFA)-derived PI decreasing and polystaturated (PUFA)-derived PI increasing. We also found ~4000 significant Spearman correlations between lipids and multiple clinical markers of immune response with |R| ≥ 0.35 and FDR corrected Q < 0.05. Except for lysophosphatidic acid, lysophospholipids were positively associated with the CD4 fraction of T cells, and the cytokines IL-8 and IL-18. In contrast, sphingosine-1-phosphates were negatively correlated with innate immune markers such as CRP and IL-6. Further indications of metabolic changes in moderate COVID-19 disease were demonstrated in recovering ward patients compared to those at the start of hospitalization, where 99 lipid species were altered (6 increased by 30-62%; 93 decreased by 1.3-2.8-fold). Overall, these findings support and expand on early reports that dysregulated lipid metabolism is involved in COVID-19.

The evolving field of multi-omics combines data and provides methods for simultaneous analysis across several omics levels. Here, we integrated genomics (transmitted and non-transmitted polygenic scores [PGSs]), epigenomics, and metabolomics data in a multi-omics framework to identify biomarkers for Attention-Deficit/Hyperactivity Disorder (ADHD) and investigated the connections among the three omics levels. We first trained single- and next multi-omics models to differentiate between cases and controls in 596 twins (cases = 14.8%) from the Netherlands Twin Register (NTR) demonstrating reasonable in-sample prediction through cross-validation. The multi-omics model selected 30 PGSs, 143 CpGs, and 90 metabolites. We confirmed previous associations of ADHD with glucocorticoid exposure and the transmembrane protein family TMEM, show that the DNA methylation of the MAD1L1 gene associated with ADHD has a relation with parental smoking behavior, and present novel findings including associations between indirect genetic effects and CpGs of the STAP2 gene. However, out-of-sample prediction in NTR participants (N = 258, cases = 14.3%) and in a clinical sample (N = 145, cases = 51%) did not perform well (range misclassification was [0.40, 0.57]). The results highlighted connections between omics levels, with the strongest connections between non-transmitted PGSs, CpGs, and amino acid levels and show that multi-omics designs considering interrelated omics levels can help unravel the complex biology underlying ADHD., (© 2023 The Authors. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics published by Wiley Periodicals LLC.)

The increasing prevalence of IgE-mediated cow's milk allergy (CMA) in childhood is a worldwide health concern. There is a growing awareness that the gut microbiome (GM) might play an important role in CMA development. Therefore, treatment with probiotics and prebiotics has gained popularity. This systematic review provides an overview of the alterations of the GM, metabolome, and immune response in CMA children and animal models, including post-treatment modifications. MEDLINE, PubMed, Scopus, and Web of Science were searched for studies on GM in CMA-diagnosed children, published before 1 March 2023. A total of 21 articles (13 on children and 8 on animal models) were included. The studies suggest that the GM, characterized by an enrichment of the Clostridia class and reductions in the Lactobacillales order and Bifidobacterium genus, is associated with CMA in early life. Additionally, reduced levels of short-chain fatty acids (SCFAs) and altered amino acid metabolism were reported in CMA children. Commonly used probiotic strains belong to the Bifidobacterium and Lactobacillus genera. However, only Bifidobacterium levels were consistently upregulated after the intervention, while alterations of other bacteria taxa remain inconclusive. These interventions appear to contribute to the restoration of SCFAs and amino acid metabolism balance. Mouse models indicate that these interventions tend to restore the T h 2/T h 1 balance, increase the T reg response, and/or silence the overall pro- and anti-inflammatory cytokine response. Overall, this systematic review highlights the need for multi-omics-related research in CMA children to gain a mechanistic understanding of this disease and to develop effective treatments and preventive strategies., (© 2024 The Authors. Pediatric Allergy and Immunology published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Acyl-CoAs play a significant role in numerous physiological and metabolic processes making it important to assess their concentration levels for evaluating metabolic health. Considering the important role of acyl-CoAs, it is crucial to develop an analytical method that can analyze these compounds. Due to the structural variations of acyl-CoAs, multiple analytical methods are often required for comprehensive analysis of these compounds, which increases complexity and the analysis time. In this study, we have developed a method using a zwitterionic HILIC column that enables the coverage of free CoA and short- to long-chain acyl-CoA species in one analytical run. Initially, we developed the method using an LC-QTOF instrument for the identification of acyl-CoA species and optimizing their chromatography. Later, a targeted HILIC-MS/MS method was created in scheduled multiple reaction monitoring mode using a QTRAP MS detector. The performance of the method was evaluated based on various parameters such as linearity, precision, recovery and matrix effect. This method was applied to identify the difference in acyl-CoA profiles in HepG2 cells cultured in different conditions. Our findings revealed an increase in levels of acetyl-CoA, medium- and long-chain acyl-CoA while a decrease in the profiles of free CoA in the starved state, indicating a clear alteration in the fatty acid oxidation process., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Fatty acid oxidation disorders (FAOD) are inborn errors of metabolism that occur due to deficiency of specific enzyme activities and transporter proteins involved in the mitochondrial metabolism of fatty acids, causing a deficiency in ATP production. The identification of suitable biomarkers plays a crucial role in predicting the future risk of disease and monitoring responses to therapies. Acyl-CoAs are directly involved in the steps of fatty acid oxidation and are the primary biomarkers associated with FAOD. However, acyl-CoAs are not used as diagnostic biomarkers in hospitals and clinics as they are present intracellularly with low endogenous levels. Additionally, the analytical method development of acyl-CoAs is quite challenging due to diverse physicochemical properties and instability. Hence, secondary biomarkers such as acylcarnitines are used for the identification of FAOD. In this review, the focus is on the analytical techniques that have evolved over the years for the identification and quantitation of acyl-CoAs. Among these techniques, liquid chromatography-mass spectrometry clearly has an advantage in terms of sensitivity and selectivity. Stable isotope labeling by essential nutrients in cell culture (SILEC) enables the generation of labeled internal standards. Each acyl-CoA species has a distinct pattern of instability and degradation, and the use of appropriately matched internal standards can compensate for such issues. Although significant progress has been made in measuring acyl-CoAs, more efforts are needed for bringing these technical advancements to hospitals and clinics. This review also highlights the difficulties involved in the routine use of acyl-CoAs as a diagnostic biomarker and some of the measures that can be adopted by clinics and hospitals for overcoming these limitations., Competing Interests: Declaration of Competing Interest None, (Copyright © 2023. Published by Elsevier Inc.)

The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R 2 ) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts., 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 © 2023. Published by Elsevier B.V.)

Objective: Impaired amine metabolism has been associated with the etiology of migraine, that is, why patients continue to get migraine attacks. However, evidence from cerebrospinal fluid (CSF) is lacking. Here, we evaluated individual amine levels, global amine profiles, and amine pathways in CSF and plasma of interictal migraine patients and healthy controls., Methods: CSF and plasma were sampled between 8:30 am and 1:00 pm, randomly and interchangeably over the time span to avoid any diurnal and seasonal influences, from healthy volunteers and interictal migraine patients, matched for age, sex, and sampling time. The study was approved by the local medical ethics committee. Individual amines (n = 31), global amine profiles, and specific amine pathways were analyzed using a validated ultraperformance liquid chromatography mass spectrometry platform., Results: We analyzed n = 99 participants with migraine with aura, n = 98 with migraine without aura, and n = 96 healthy volunteers. Univariate analysis with Bonferroni correction indicated that CSF L-arginine was reduced in migraine with aura (10.4%, p < 0.001) and without aura (5.0%, p = 0.03). False discovery rate-corrected CSF L-phenylalanine was also lower in migraine with aura (6.9%, p = 0.011) and without aura (8.1%, p = 0.001), p = 0.088 after Bonferroni correction. Multivariate analysis revealed that CSF global amine profiles were similar for both types of migraine (p = 0.64), but distinct from controls (p = 0.009). Global profile analyses were similar in plasma. The strongest associated pathways with migraine were related to L-arginine metabolism., Interpretation: L-Arginine was decreased in the CSF (but not in plasma) of interictal patients with migraine with or without aura, and associated pathways were altered. This suggests that dysfunction of nitric oxide signaling is involved in susceptibility to getting migraine attacks. ANN NEUROL 2023;93:715-728., (© 2022 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Alzheimer's disease (AD) is an aging-related neurodegenerative disease, leading to the progressive loss of memory and other cognitive functions. As there is still no cure for AD, the growth in the number of susceptible individuals represents a major emerging threat to public health. Currently, the pathogenesis and etiology of AD remain poorly understood, while no efficient treatments are available to slow down the degenerative effects of AD. Metabolomics allows the study of biochemical alterations in pathological processes which may be involved in AD progression and to discover new therapeutic targets. In this review, we summarized and analyzed the results from studies on metabolomics analysis performed in biological samples of AD subjects and AD animal models. Then this information was analyzed by using MetaboAnalyst to find the disturbed pathways among different sample types in human and animal models at different disease stages. We discuss the underlying biochemical mechanisms involved, and the extent to which they could impact the specific hallmarks of AD. Then we identify gaps and challenges and provide recommendations for future metabolomics approaches to better understand AD pathogenesis.

This study introduces and illustrates the potential of an integrated multi-omics approach in investigating the underlying biology of complex traits such as childhood aggressive behavior. In 645 twins (cases = 42%), we trained single- and integrative multi-omics models to identify biomarkers for subclinical aggression and investigated the connections among these biomarkers. Our data comprised transmitted and two non-transmitted polygenic scores (PGSs) for 15 traits, 78,772 CpGs, and 90 metabolites. The single-omics models selected 31 PGSs, 1614 CpGs, and 90 metabolites, and the multi-omics model comprised 44 PGSs, 746 CpGs, and 90 metabolites. The predictive accuracy for these models in the test (N = 277, cases = 42%) and independent clinical data (N = 142, cases = 45%) ranged from 43 to 57%. We observed strong connections between DNA methylation, amino acids, and parental non-transmitted PGSs for ADHD, Autism Spectrum Disorder, intelligence, smoking initiation, and self-reported health. Aggression-related omics traits link to known and novel risk factors, including inflammation, carcinogens, and smoking., (© 2022. The Author(s).)

Trained sensory panels are regularly used to rate food products but do not allow for data-driven approaches to steer food product development. This study evaluated the potential of a molecular-based strategy by analyzing 27 tomato soups that were enhanced with yeast-derived flavor products using a sensory panel as well as LC-MS and GC-MS profiling. These data sets were used to build prediction models for 26 different sensory attributes using partial least squares analysis. We found driving separation factors between the tomato soups and metabolites predicting different flavors. Many metabolites were putatively identified as dipeptides and sulfur-containing modified amino acids, which are scientifically described as related to umami or having "garlic-like" and "onion-like" attributes. Proposed identities of high-impact sensory markers (methionyl-proline and asparagine-leucine) were verified using MS/MS. The overall results highlighted the strength of combining sensory data and metabolomics platforms to find new information related to flavor perception in a complex food matrix.

Background: Fatty acid-derived lipid mediators including oxylipins, endocannabinoids (eCBs), and their analogues, have emerged as key metabolites in the inflammatory and immune response to physiological stressors., Methods: This report was based on a sub-study and secondary analyses the ACTIBATE single-center unblinded randomized controlled trial (ClinicalTrials.gov ID: NCT02365129). The study was performed in the Sport and Health University Research Institute and the Virgen de las Nieves University Hospital of the University of Granada. Eligible participants were young, sedentary adults with no chronic diseases. Here, we performed both an acute endurance and resistance exercise sub-studies (n.ß=.ß14 and 17 respectively), and a 24-week supervised exercise intervention, combining endurance and resistance exercise training at moderate-intensity (MOD-EX) or vigorous-intensity (VIG-EX) exercise groups, in young sedentary adults. Randomization was performed by unrestricted randomization. Plasma levels of oxylipins, eCBs, and their analogues were measured using liquid chromatography-tandem mass spectrometry., Findings: Both endurance and resistance exercise increased by.ß+50% the plasma levels of dihomo-..-linolenic acid and arachidonic acid (AA) omega-6 derived oxylipins, as well as eicosapentaenoic acid and docosahexaenoic acid omega-3 derived after 3 and 120.ßmin of the bout of exercise (all .. 2 .ß....ß0.219 and P.ß..±.ß0.039). These exercise modalities also increased the levels of anandamide and eCBs analogues (+25%). 145 young sedentary adults were assigned to a control (CON, n.ß=.ß54), a MOD-EX (n.ß=.ß48) or a VIG-EX (n.ß=.ß43). 102 participants were included in the final long-term analyses (CON, n.ß=.ß36; MOD-EX, n.ß=.ß33; and VIG-EX, n.ß=.ß33) of the trial. After 24-week of supervised exercise, MOD-EX decreased plasma levels of omega-6 oxylipins, concretely linoleic acid (LA) and adrenic acid derived oxylipins, and the eCBs analogues OEA and LEA in comparison to the CON (all P.ß..±.ß0.021). VIG-EX decreased LA-derived oxylipins and LEA compared to CON. No relevant adverse events were recorded., Interpretation: Endurance and resistance exercises acutely increased plasma levels of oxylipins, eCBs, and their analogues, whereas 24 weeks of exercise training decreased fasting plasma levels of omega-6 oxylipins, and eCBs analogues in young, sedentary adults., Funding: See Acknowledgments section., Competing Interests: Declaration of interests None., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

The metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or muscle biopsies, is desired. Several existing metabolomics platforms that include diverse classes of signaling lipids, energy metabolites, and amino acids and amines would be informative for suspected biochemical pathways involved in sarcopenia. The sample limitation requires an optimized sample preparation method with minimal losses during isolation and handling and maximal accuracy and reproducibility. Here, two developed sample preparation methods, BuOH-MTBE-Water (BMW) and BuOH-MTBE-More-Water (BMMW), were evaluated and compared with previously reported methods, Bligh-Dyer (BD) and BuOH-MTBE-Citrate (BMC), for their suitability for these classes. The most optimal extraction was found to be the BMMW method, with the highest extraction recovery of 63% for the signaling lipids and 81% for polar metabolites, and an acceptable matrix effect (close to 1.0) for all metabolites of interest. The BMMW method was applied on muscle tissues as small as 5 mg (dry weight) from the well-characterized, prematurely aging, DNA repair-deficient Ercc1 ∆/- mouse mutant exhibiting multiple-morbidities, including sarcopenia. We successfully detected 109 lipids and 62 polar targeted metabolites. We further investigated whether fast muscle tissue isolation is necessary for mouse sarcopenia studies. A muscle isolation procedure involving 15 min at room temperature revealed a subset of metabolites to be unstable; hence, fast sample isolation is critical, especially for more oxidative muscles. Therefore, BMMW and fast muscle tissue isolation are recommended for future sarcopenia studies. This research provides a sensitive sample preparation method for the simultaneous extraction of non-polar and polar metabolites from limited amounts of muscle tissue, supplies a stable mouse muscle tissue collection method, and methodologically supports future metabolomic mechanistic studies of sarcopenia.

Introduction: The leptin signaling pathway plays an important role as a key regulator of glucose homeostasis, metabolism control and systemic inflammatory responses. However, the metabolic effects of leptin on infectious diseases, for example tuberculosis (TB), are still little known., Objectives: In this study, we aim to investigate the role of leptin on metabolism in the absence and presence of mycobacterial infection in zebrafish larvae and mice., Methods: Metabolites in entire zebrafish larvae and the blood of mice were studied using high-resolution magic-angle-spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy and mass spectrometry, respectively. For transcriptome studies of zebrafish larvae, deep RNA sequencing was used., Results: The results show that leptin mutation leads to a similar metabolic syndrome as caused by mycobacterial infection in the two species, characterized by the decrease of 11 amine metabolites. In both species, this metabolic syndrome was not aggravated further when the leptin mutant was infected by mycobacteria. Therefore, we conclude that leptin and mycobacterial infection are both impacting metabolism non-synergistically. In addition, we studied the transcriptomes of lepb ibl54 mutant zebrafish larvae and wild type (WT) siblings after mycobacterial infection. These studies showed that mycobacteria induced a very distinct transcriptome signature in the lepb ibl54 mutant zebrafish compared to WT sibling control larvae. Furthermore, lepb ibl55 Tg (pck1:luc1) zebrafish line was constructed and confirmed this difference in transcriptional responses., Conclusions: Leptin mutation and TB lead non-synergistically to a similar metabolic syndrome. Moreover, different transcriptomic responses in the lepb ibl54   mutant and TB can lead to the similar metabolic end states., (© 2022. The Author(s).)

COVID-19 is characterised by a dysregulated immune response, that involves signalling lipids acting as mediators of the inflammatory process along the innate and adaptive phases. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). The second publication in a series reports the results of quantitative LC-MS/MS profiling of 63 small lipids including oxylipins, free fatty acids, and endocannabinoids. Compared to samples taken from ward patients, intensive care unit (ICU) patients had 2−4-fold lower levels of arachidonic acid (AA) and its cyclooxygenase-derived prostanoids, as well as lipoxygenase derivatives, exhibiting negative correlations with inflammation markers. The same derivatives showed 2−5-fold increases in recovering ward patients, in paired comparison to early hospitalisation. In contrast, ICU patients showed elevated levels of oxylipins derived from poly-unsaturated fatty acids (PUFA) by non-enzymatic peroxidation or activity of soluble epoxide hydrolase (sEH), and these oxylipins positively correlated with markers of macrophage activation. The deficiency in AA enzymatic products and the lack of elevated intermediates of pro-resolving mediating lipids may result from the preference of alternative metabolic conversions rather than diminished stores of PUFA precursors. Supporting this, ICU patients showed 2-to-11-fold higher levels of linoleic acid (LA) and the corresponding fatty acyl glycerols of AA and LA, all strongly correlated with multiple markers of excessive immune response. Our results suggest that the altered oxylipin metabolism disrupts the expected shift from innate immune response to resolution of inflammation.

The COVID-19 pandemic raised a need to characterise the biochemical response to SARS-CoV-2 infection and find biological markers to identify therapeutic targets. In support of these aims, we applied a range of LC-MS platforms to analyse over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). The first publication in a series reports the results of quantitative LC-MS/MS profiling of 56 amino acids and derivatives. A comparison between samples taken from ICU and ward patients revealed a notable increase in ten post-translationally modified amino acids that correlated with markers indicative of an excessive immune response: TNF-alpha, neutrophils, markers for macrophage, and leukocyte activation. Severe patients also had increased kynurenine, positively correlated with CRP and cytokines that induce its production. ICU and ward patients with high IL-6 showed decreased levels of 22 immune-supporting and anti-oxidative amino acids and derivatives (e.g., glutathione, GABA). These negatively correlated with CRP and IL-6 and positively correlated with markers indicative of adaptive immune activation. Including corresponding alterations in convalescing ward patients, the overall metabolic picture of severe COVID-19 reflected enhanced metabolic demands to maintain cell proliferation and redox balance, alongside increased inflammation and oxidative stress.

Variation in metabolite levels reflects individual differences in genetic and environmental factors. Here, we investigated the role of these factors in urinary metabolomics data in children. We examined the effects of sex and age on 86 metabolites, as measured on three metabolomics platforms that target amines, organic acids, and steroid hormones. Next, we estimated their heritability in a twin cohort of 1300 twins (age range: 5.7-12.9 years). We observed associations between age and 50 metabolites and between sex and 21 metabolites. The monozygotic (MZ) and dizygotic (DZ) correlations for the urinary metabolites indicated a role for non-additive genetic factors for 50 amines, 13 organic acids, and 6 steroids. The average broad-sense heritability for these amines, organic acids, and steroids was 0.49 (range: 0.25-0.64), 0.50 (range: 0.33-0.62), and 0.64 (range: 0.43-0.81), respectively. For 6 amines, 7 organic acids, and 4 steroids the twin correlations indicated a role for shared environmental factors and the average narrow-sense heritability was 0.50 (range: 0.37-0.68), 0.50 (range; 0.23-0.61), and 0.47 (range: 0.32-0.70) for these amines, organic acids, and steroids. We conclude that urinary metabolites in children have substantial heritability, with similar estimates for amines and organic acids, and higher estimates for steroid hormones.

Introduction: The metabolomics quality assurance and quality control consortium (mQACC) is enabling the identification, development, prioritization, and promotion of suitable reference materials (RMs) to be used in quality assurance (QA) and quality control (QC) for untargeted metabolomics research., Objectives: This review aims to highlight current RMs, and methodologies used within untargeted metabolomics and lipidomics communities to ensure standardization of results obtained from data analysis, interpretation and cross-study, and cross-laboratory comparisons. The essence of the aims is also applicable to other 'omics areas that generate high dimensional data., Results: The potential for game-changing biochemical discoveries through mass spectrometry-based (MS) untargeted metabolomics and lipidomics are predicated on the evolution of more confident qualitative (and eventually quantitative) results from research laboratories. RMs are thus critical QC tools to be able to assure standardization, comparability, repeatability and reproducibility for untargeted data analysis, interpretation, to compare data within and across studies and across multiple laboratories. Standard operating procedures (SOPs) that promote, describe and exemplify the use of RMs will also improve QC for the metabolomics and lipidomics communities., Conclusions: The application of RMs described in this review may significantly improve data quality to support metabolomics and lipidomics research. The continued development and deployment of new RMs, together with interlaboratory studies and educational outreach and training, will further promote sound QA practices in the community., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

This study aimed to investigate the effects of different exercise training programs on fasting plasma levels of oxylipins, endocannabinoids (eCBs), and eCBs-like molecules in middle-aged sedentary adults. A 12-week randomized controlled trial was conducted using a parallel group design. Sixty-five middle-aged adults (40-65 years old) were randomly assigned to: (a) no exercise (control group), (b) concurrent training based on international physical activity recommendations (PAR group), (c) high-intensity interval training (HIIT group), and (d) HIIT together with whole-body electromyostimulation (HIIT + EMS group). Plasma levels of oxylipins, eCBs, and eCBs-like molecules were determined in plasma samples before and after the intervention using targeted lipidomics. Body composition was assessed through dual-energy X-ray absorptiometry, and dietary intake through a food frequency questionnaire and three nonconsecutive 24-hr recalls. The physical activity recommendations, HIIT, and HIIT-EMS groups showed decreased plasma levels of omega-6 and omega-3-derived oxylipins, and eCBs and eCBs-like molecules after 12 weeks (all Δ ≤ -0.12; all p < .05). Importantly, after Bonferroni post hoc corrections, the differences in plasma levels of omega-6 and omega-3 oxylipins were not statistically significant compared with the control group (all p > .05). However, after post hoc corrections, plasma levels of anandamide and oleoylethanolamide were increased in the physical activity recommendations group compared with the control group (anandamide: Δ = 0.05 vs. -0.09; oleoylethanolamide: Δ = -0.12 vs. 0.013, all p ≤ .049). In conclusion, this study reports that a 12-week exercise training intervention, independent of the modality applied, does not modify fasting plasma levels of omega-6 and omega-3 oxylipins, eCBs, and eCBs-like molecules in middle-aged sedentary adults.

The majority of disease modifying therapies for multiple sclerosis (MS) reduce inflammation, but do no't target remyelination. Development of remyelinating therapies will benefit from a method to quantify myelin kinetics in patients with MS. We labeled myelin in vivo with deuterium, and modeled kinetics of myelin breakdown products β-galactosylceramide (β-GalC) and N-Octadecanoyl-sulfatide (NO-Sulf). Five patients with MS received 120 ml 70% D 2 O daily for 70 days and were compared with six healthy subjects who previously received the same procedure. Mass spectrometry and compartmental modeling were used to quantify the turnover rate of β-GalC and NO-Sulf in cerebrospinal fluid (CSF). Turnover rate constants of the fractions of β-GalC and NO-Sulf with non-negligible turnover were 0.00186 and 0.00714, respectively, in both healthy subjects and patients with MS. The turnover half-life of β-GalC and NO-Sulf was calculated as 373 days and 96.5 days, respectively. The effect of MS on the NO-Sulf (49.4% lower fraction with non-negligible turnover) was more pronounced compared to the effect on β-GalC turnover (18.3% lower fraction with non-negligible turnover). Kinetics of myelin breakdown products in the CSF are different in patients with MS compared with healthy subjects. This may be caused by slower myelin production in these patients, by a higher level of degradation of a more stable component of myelin, or, most likely, by a combination of these two processes. Labeling myelin breakdown products is a useful method that can be used to quantify myelin turnover in patients with progressive MS and can therefore be used in proof-of-concept studies with remyelination therapies., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Polar hydrophilic metabolites have been identified as important actors in many biochemical pathways. Despite continuous improvement and refinement of hydrophilic interaction liquid chromatography (HILIC) platforms, its application in global polar metabolomics has been underutilized. In this study, we aimed to systematically evaluate polar stationary phases for untargeted metabolomics by using HILIC columns (neutral and zwitterionic) that have been exploited widely in targeted approaches. To do so, high-resolution mass spectrometry was applied to thoroughly investigate selectivity, repeatability and matrix effect at three pH conditions for 9 classes of polar compounds using 54 authentic standards and plasma matrix. The column performance for utilization in untargeted metabolomics was assessed using plasma samples with diverse phenotypes. Our results indicate that the ZIC-c HILIC column operated at neutral pH exhibited several advantages, including superior performance for different classes of compounds, better isomer separation, repeatability and high metabolic coverage. Regardless of the column type, the retention of inorganic ions in plasma leads to extensive adduct formation and co-elution with analytes, which results in ion-suppression as part of the overall plasma matrix effect. In ZIC-c HILIC, the sodium chloride ion effect was particularly observed for amino acids and amine classes. Successful performance of HILIC for separation of plasma samples with different phenotypes highlights this mode of separation as a valuable approach in global profiling of plasma sample and discovering the metabolic changes associated with health and disease.

Objective: Omega-6 and omega-3 oxylipins are known to play a role in inflammation and cardiometabolic diseases in preclinical models. The associations between plasma levels of omega-6 and omega-3 polyunsaturated fatty acid-derived oxylipins and body composition and cardiometabolic risk factors in young adults were assessed., Methods: Body composition, brown adipose tissue, traditional serum cardiometabolic risk factors, inflammatory markers, and a panel of 83 oxylipins were analyzed in 133 young adults (age 22.1[SD 2.2] years, 67% women)., Results: Plasma levels of four omega-6 oxylipins (15-HeTrE, 5-HETE, 14,15-EpETrE, and the oxidative stress-derived 8,12-iso-iPF 2α -VI) correlated positively with adiposity, prevalence of metabolic syndrome, fatty liver index, and homeostatic model assessment of insulin resistance index and lipid parameters. By contrast, the plasma levels of three omega-3 oxylipins (14,15-DiHETE, 17,18-DiHETE, and 19,20-DiHDPA) were negatively correlated with adiposity, prevalence of metabolic syndrome, fatty liver index, homeostatic model assessment of insulin resistance index, and lipid parameters. The panel of seven oxylipins predicted adiposity better than traditional inflammatory markers such as interferon gamma or tumor necrosis factor-alpha. Pathway analyses revealed that individuals with obesity had higher plasma levels of omega-6 and lower plasma levels of omega-3 oxylipins than normal-weight individuals., Conclusion: Plasma levels of seven omega-6 and omega-3 oxylipins may have utility as early markers of cardiometabolic risk in young adults., (© 2021 The Authors. Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society (TOS).)

The simultaneous analysis of a broad range of polar ionogenic metabolites using capillary electrophoresis-mass spectrometry (CE-MS) can be challenging, as two different analytical methods are often required, that is, one for cations and one for anions. Even though CE-MS has shown to be an effective method for cationic metabolite profiling, the analysis of small anionic metabolites often results in relatively low sensitivity and poor repeatability. In this work, a novel derivatization strategy based on trimethylmethaneaminophenacetyl bromide was developed to enable CE-MS analysis of carboxylic acid metabolites using normal CE polarity (i.e., cathode in the outlet) and detection by mass spectrometry in positive ionization mode. Optimization of derivatization conditions was performed using a response surface methodology after which the optimized method (incubation time 50 min, temperature 90°C, and pH 10) was used for the analysis of carboxylic acid metabolites in extracts from HepG2 cells. For selected metabolites, detection limits were down to 8.2 nM, and intraday relative standard deviation values for replicates (n = 3) for peak areas were below 21.5%. Metabolites related to glycolysis, tricarboxylic acid cycle, and anaerobic respiration pathways were quantified in 250,000 cell lysates, and could still be detected in extracts from only 25,000 HepG2 cell lysates (∼70 cell lysates injected)., Competing Interests: The authors have declared no conflict of interest., (© 2021 The Authors. Analytical Science Advances published by Wiley‐VCH GmbH.)

Normothermic machine perfusion (NMP) is emerging as a novel preservation strategy. During NMP, the organ is maintained in a metabolically active state that may not only provide superior organ preservation, but that also facilitates viability testing before transplantation, and ex situ resuscitation of marginal kidney grafts. Although the prevailing perfusion protocols for renal NMP are refined from initial pioneering studies concerning short periods of NMP, it could be argued that these protocols are not optimally tailored to address the putatively compromised metabolic plasticity of marginal donor grafts (i.e., in the context of viability testing and/or preservation), or to meet the metabolic prerequisites associated with prolonged perfusions and the required anabolic state in the context of organ regeneration. Herein, we provide a theoretical framework for the metabolic requirements for renal NMP. Aspects are discussed along the lines of carbohydrates, fatty acids, amino acids, and micronutrients required for optimal NMP of an isolated kidney. In addition, considerations for monitoring aspects of metabolic status during NMP are discussed., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Black soldier fly (Hermetia illucens; BSF) larvae as dietary protein source have the ability to deliver nutrients and could possess functional properties that positively support animal productivity and health. More knowledge, however, is needed to assess the impact of feeding a BSF based diet on gut and animal health. Sixteen post-weaned male pigs were randomly assigned to two groups and fed for three weeks with iso-caloric and iso-proteinaceous experimental diets prepared with either soybean meal (SBM) as reference protein source or with BSF as single source of dietary protein. At the end of the trial, the pigs were sacrificed to collect relevant digesta, gut tissue and blood samples to study changes induced by the dietary treatments using ~ omics based analyses. Inclusion of BSF in the diet supports the development of the intestinal microbiome that could positively influence intestinal health. By amine metabolite analysis, we identified two metabolites i.e. sarcosine and methionine sulfoxide, in plasma that serve as markers for the ingestion of insect based ingredients. BSF seems to possess functional properties indicated by the appearance of alpha-aminobutyric acid and taurine in blood plasma of pigs that are known to induce health beneficial effects., (© 2021. The Author(s).)

Background: Leptin plays a critical role in the regulation of metabolic homeostasis. However, the molecular mechanism and cross talks between leptin and metabolic pathways leading to metabolic homeostasis across different species are not clear. This study aims to explore the effects of leptin in mice and zebrafish larvae by integration of metabolomics and transcriptomics. Different metabolomic approaches including mass spectrometry, nuclear magnetic resonance (NMR) and high-resolution magic-angle-spinning NMR spectrometry were used to investigate the metabolic changes caused by leptin deficiency in mutant ob/ob adult mice and lepb -/- zebrafish larvae. For transcriptome studies, deep RNA sequencing was used., Results: Thirteen metabolites were identified as common biomarkers discriminating ob/ob mice and lepb -/- zebrafish larvae from their respective wild type controls: alanine, citrulline, ethanolamine, glutamine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, putrescine, serine and threonine. Moreover, we also observed that glucose and lipid levels were increased in lepb -/- zebrafish larvae compared to the lepb +/+ group. Deep sequencing showed that many genes involved in proteolysis and arachidonic acid metabolism were dysregulated in ob/ob mice heads and lepb mutant zebrafish larvae compared to their wild type controls, respectively., Conclusions: Leptin deficiency leads to highly similar metabolic alterations in metabolites in both mice and zebrafish larvae. These metabolic changes show similar features as observed during progression of tuberculosis in human patients, mice and zebrafish larvae. In addition, by studying the transcriptome, we found similar changes in gene regulation related to proteolysis and arachidonic acid metabolism in these two different in vivo models.

Gut microbiota and their metabolic products are increasingly being recognized as important modulators of human health. The fecal metabolome provides a functional readout of the interactions between human metabolism and the gut microbiota in health and disease. Due to the high complexity of the fecal matrix, sample preparation often introduces technical variation, which must be minimized to accurately detect and quantify gut bacterial metabolites. Here, we tested six different representative extraction methods (single-phase and liquid-liquid extractions) and compared differences due to fecal amount, extraction solvent type and solvent pH. Our results indicate that a minimum fecal (wet) amount of 0.50 g is needed to accurately represent the complex texture of feces. The MTBE method (MTBE/methanol/water, 3.6/2.8/3.5, v / v / v ) outperformed the other extraction methods, reflected by the highest extraction efficiency for 11 different classes of compounds, the highest number of extracted features (97% of the total identified features in different extracts), repeatability (CV < 35%) and extraction recovery (≥70%). Importantly, optimization of the solvent volume of each step to the initial dried fecal material (µL/mg feces) offers a major step towards standardization, which enables confident assessment of the contributions of gut bacterial metabolites to human health.

Diagnosis of microbial disease etiology in community-acquired pneumonia (CAP) remains challenging. We undertook a large-scale metabolomics study of serum samples in hospitalized CAP patients to determine if host-response associated metabolites can enable diagnosis of microbial etiology, with a specific focus on discrimination between the major CAP pathogen groups S. pneumoniae, atypical bacteria, and respiratory viruses. Targeted metabolomic profiling of serum samples was performed for three groups of hospitalized CAP patients with confirmed microbial etiologies: S. pneumoniae (n = 48), atypical bacteria (n = 47), or viral infections (n = 30). A wide range of 347 metabolites was targeted, including amines, acylcarnitines, organic acids, and lipids. Single discriminating metabolites were selected using Student's T-test and their predictive performance was analyzed using logistic regression. Elastic net regression models were employed to discover metabolite signatures with predictive value for discrimination between pathogen groups. Metabolites to discriminate S. pneumoniae or viral pathogens from the other groups showed poor predictive capability, whereas discrimination of atypical pathogens from the other groups was found to be possible. Classification of atypical pathogens using elastic net regression models was associated with a predictive performance of 61% sensitivity, 86% specificity, and an AUC of 0.81. Targeted profiling of the host metabolic response revealed metabolites that can support diagnosis of microbial etiology in CAP patients with atypical bacterial pathogens compared to patients with S. pneumoniae or viral infections., Competing Interests: The authors have declared that no competing interests exist.

The unambiguous identification of unknown compounds is of utmost importance in the field of metabolomics. However, current identification workflows often suffer from error-sensitive methodologies, which may lead to incorrect structure annotations of small molecules. Therefore, we have developed a comprehensive identification workflow including two highly complementary techniques, i.e. liquid chromatography (LC) combined with mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), and used it to identify five taste-related retention time and m/z features in soy sauce. An off-line directed two-dimensional separation was performed in order to purify the features prior to the identification. Fractions collected during the first dimension separation (reversed phase low pH) were evaluated for the presence of remaining impurities next to the features of interest. Based on the separation between the feature and impurities, the most orthogonal second dimension chromatography (hydrophilic interaction chromatography or reversed phase high pH) was selected for further purification. Unknown compounds down to tens of micromolar concentrations were tentatively annotated by MS and structurally confirmed by MS and NMR. The mass (0.4-4.2 μg) and purity of the isolated compounds were sufficient for the acquisition of one and two-dimensional NMR spectra. The use of a directed two-dimensional chromatography allowed for a fractionation that was tailored to each feature and remaining impurities. This makes the fractionation more widely applicable to different sample matrices than one-dimensional or fixed two-dimensional chromatography. Five proline-based 2,5-diketopiperazines were successfully identified in soy sauce. These cyclic dipeptides might contribute to taste by giving a bitter flavour or indirectly enhancing umami flavour., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: John van Duynhoven and Doris Jacobs are employed by a company that manufactures and markets food products. Ulrich Braumann is employed by a company that markets and manufactures analytical equipment. The other authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

The interaction disorder between gut microbiota and its host has been documented in different non-communicable diseases (NCDs) such as metabolic syndrome, neurodegenerative disease, and autoimmune disease. The majority of these altered interactions arise through metabolic cross-talk between gut microbiota and host immune system, inducing a low-grade chronic inflammation that characterizes all NCDs. In this review, we discuss the contribution of bacterial metabolites to immune signaling pathways involved in NCDs. We then review recent advances that aid to rationally design microbial therapeutics. A deeper understanding of these intersections between host and gut microbiota metabolism using metabolomics-based system biology platform promises to reveal the fundamental mechanisms that drive metabolic predispositions to disease and suggest new avenues to use microbial therapeutic opportunities for NCDs treatment and prevention. Abbreviations : NCDs: non-communicable disease, IBD: inflammatory bowel disease, IL: interleukin, T2D: type 2 diabetes, SCFAs: short-chain fatty acids, HDAC: histone deacetylases, GPCR: G-protein coupled receptors, 5-HT: 5-hydroxytryptamine receptor signaling, DCs: dendritic cells, IECs: intestinal epithelial cells, T-reg: T regulatory cell, NF-κB: nuclear factor κB, TNF-α: tumor necrosis factor alpha, Th: T helper cell, CNS: central nervous system, ECs: enterochromaffin cells, NSAIDs: non-steroidal anti-inflammatory drugs, AhR: aryl hydrocarbon receptor, IDO: indoleamine 2,3-dioxygenase, QUIN: quinolinic acid, PC: phosphatidylcholine, TMA: trimethylamine, TMAO: trimethylamine N -oxide, CVD: cardiovascular disease, NASH: nonalcoholic steatohepatitis, BAs: bile acids, FXR: farnesoid X receptor, CDCA: chenodeoxycholic acid, DCA: deoxycholic acid, LCA: lithocholic acid, UDCA: ursodeoxycholic acid, CB: cannabinoid receptor, COBRA: constraint-based reconstruction and analysis.

A popular fragmentation technique for non-targeted analysis is called data-independent acquisition (DIA), because it provides fragmentation data for all analytes in a specific mass range. In this work, we demonstrated the strengths and weaknesses of DIA. Two types of chromatography (fractionation/3 min and hydrophilic interaction liquid chromatography (HILIC)/18 min) and three DIA protocols (variable sequential window acquisition of all theoretical mass spectra (SWATH), fixed SWATH and MS ALL ) were used to evaluate the performance of DIA. Our results show that fast chromatography and MS ALL often results in product ion overlap and complex MS/MS spectra, which reduces the quantitative and qualitative power of these DIA protocols. The combination of SWATH and HILIC allowed for the correct identification of 20 metabolites using the NIST library. After SWATH window customization (i.e., variable SWATH), we were able to quantify ten structural isomers with a mean accuracy of 103% (91-113%). The robustness of the variable SWATH and HILIC method was demonstrated by the accurate quantification of these structural isomers in 10 highly diverse blood samples. Since the combination of variable SWATH and HILIC results in good quantitative and qualitative fragmentation data, it is promising for both targeted and untargeted platforms. This should decrease the number of platforms needed in metabolomics and increase the value of a single analysis.

Delayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury in experimental models, all clinical interventions have failed. This explorative clinical evaluation examined possible metabolic origins of clinical ischemia reperfusion injury combining data from 18 pre- and post-reperfusion tissue biopsies with 36 sequential arteriovenous blood samplings over the graft in three study groups. These groups included living and deceased donor grafts with and without delayed graft function. Group allocation was based on clinical outcome. Magic angle NMR was used for tissue analysis and mass spectrometry-based platforms were used for plasma analysis. All kidneys were functional at one-year. Integration of metabolomic data identified a discriminatory profile to recognize future delayed graft function. This profile was characterized by post-reperfusion ATP/GTP catabolism (significantly impaired phosphocreatine recovery and significant persistent (hypo)xanthine production) and significant ongoing tissue damage. Failing high-energy phosphate recovery occurred despite activated glycolysis, fatty-acid oxidation, glutaminolysis and autophagia, and related to a defect at the level of the oxoglutarate dehydrogenase complex in the Krebs cycle. Clinical delayed graft function due to ischemia reperfusion injury associated with a post-reperfusion metabolic collapse. Thus, efforts to quench delayed graft function due to ischemia reperfusion injury should focus on conserving metabolic competence, either by preserving the integrity of the Krebs cycle and/or by recruiting metabolic salvage pathways., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Metabolomics is emerging as an important field in life sciences. However, a weakness of current mass spectrometry (MS) based metabolomics platforms is the time-consuming analysis and the occurrence of severe matrix effects in complex mixtures. To overcome this problem, we have developed an automated and fast fractionation module coupled online to MS. The fractionation is realized by the implementation of three consecutive high performance solid-phase extraction columns consisting of a reversed phase, mixed-mode anion exchange, and mixed-mode cation exchange sorbent chemistry. The different chemistries resulted in an efficient interaction with a wide range of metabolites based on polarity, charge, and allocation of important matrix interferences like salts and phospholipids. The use of short columns and direct solvent switches allowed for fast screening (3 min per polarity). In total, 50 commonly reported diagnostic or explorative biomarkers were validated with a limit of quantification that was comparable with conventional LC-MS(/MS). In comparison with a flow injection analysis without fractionation, ion suppression decreased from 89% to 25%, and the sensitivity was 21 times higher. The validated method was used to investigate the effects of circadian rhythm and food intake on several metabolite classes. The significant diurnal changes that were observed stress the importance of standardized sampling times and fasting states when metabolite biomarkers are used. Our method demonstrates a fast approach for global profiling of the metabolome. This brings metabolomics one step closer to implementation into the clinic.

Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms. Ten of these metabolites were common between the three different organisms comprising: methionine, asparagine, cysteine, threonine, serine, tryptophan, leucine, citrulline, ethanolamine and phenylalanine. The metabolomic changes of zebrafish larvae after infection were confirmed by nuclear magnetic resonance spectroscopy. Our study identified common biomarkers for tuberculosis disease in humans, mice and zebrafish, showing across species conservation of metabolic reprogramming processes as a result of disease. Apparently, the mechanisms underlying these processes are independent of environmental, developmental and vertebrate evolutionary factors. The zebrafish larval model is highly suited to further investigate the mechanism of metabolic reprogramming and the connection with wasting syndrome due to infection by mycobacteria.

Metabolites are small molecules involved in cellular metabolism where they act as reaction substrates or products. The term 'metabolomics' refers to the comprehensive study of these molecules. The concentrations of metabolites in biological tissues are under genetic control, but this is limited by environmental factors such as diet. In adult mono- and dizygotic twin pairs, we estimated the contribution of genetic and shared environmental influences on metabolite levels by structural equation modeling and tested whether the familial resemblance for metabolite levels is mainly explained by genetic or by environmental factors that are shared by family members. Metabolites were measured across three platforms: two based on proton nuclear magnetic resonance techniques and one employing mass spectrometry. These three platforms comprised 237 single metabolic traits of several chemical classes. For the three platforms, metabolites were assessed in 1407, 1037 and 1116 twin pairs, respectively. We carried out power calculations to establish what percentage of shared environmental variance could be detected given these sample sizes. Our study did not find evidence for a systematic contribution of shared environment, defined as the influence of growing up together in the same household, on metabolites assessed in adulthood. Significant heritability was observed for nearly all 237 metabolites; significant contribution of the shared environment was limited to 6 metabolites. The top quartile of the heritability distribution was populated by 5 of the 11 investigated chemical classes. In this quartile, metabolites of the class lipoprotein were significantly overrepresented, whereas metabolites of classes glycerophospholipids and glycerolipids were significantly underrepresented.

Metabolomics has been applied to diagnose diseases, predict disease progression, and design therapeutic strategies in various areas of medicine. However, it remains to be applied to the ocular surface diseases, where biological samples are often of limited quantities. We successfully performed proof-of-concept metabolomics assessment of volume-limited cytology samples from a clinical form of chronic inflammatory cicatrizing conjunctivitis, i.e., ocular MMP and discovered metabolic changes of signaling lipid mediators upon disease onset and progression. The metabolomics assessment revealed active oxylipins, lysophospholipids, fatty acids, and endocannabinoids alterations, from which potential biomarkers linked to inflammatory processes were identified. Possible underlying mechanisms such as dysregulated enzyme activities (e.g., lipoxygenases, cytochrome P450, and phospholipases) were suggested which may be considered as potential therapeutic targets in future studies. KEY MESSAGES: Metabolic profile of the ocular surface can be measured using impression cytology samples. Metabolomics analysis of ocular pemphigoid is presented for the first time. The metabolomics assessment of OCP patients revealed active oxylipins, lysophospholipids, fatty acids, and endocannabinoids alterations. Several oxylipins are identified as diagnostic biomarkers for OCP.

Biomarkers are of interest as potential diagnostic and predictive instruments in personalized medicine. We present the first urinary metabolomics biomarker study of childhood aggression. We aim to examine the association of urinary metabolites and neurotransmitter ratios involved in key metabolic and neurotransmitter pathways in a large cohort of twins ( N = 1,347) and clinic-referred children ( N = 183) with an average age of 9.7 years. This study is part of ACTION (Aggression in Children: Unraveling gene-environment interplay to inform Treatment and InterventiON strategies), in which we developed a standardized protocol for large-scale collection of urine samples in children. Our analytical design consisted of three phases: a discovery phase in twins scoring low or high on aggression ( N = 783); a replication phase in twin pairs discordant for aggression ( N = 378); and a validation phase in clinical cases and matched twin controls ( N = 367). In the discovery phase, 6 biomarkers were significantly associated with childhood aggression, of which the association of O-phosphoserine (β = 0.36; SE = 0.09; p = 0.004), and gamma-L-glutamyl-L-alanine (β = 0.32; SE = 0.09; p = 0.01) remained significant after multiple testing. Although non-significant, the directions of effect were congruent between the discovery and replication analyses for six biomarkers and two neurotransmitter ratios and the concentrations of 6 amines differed between low and high aggressive twins. In the validation analyses, the top biomarkers and neurotransmitter ratios, with congruent directions of effect, showed no significant associations with childhood aggression. We find suggestive evidence for associations of childhood aggression with metabolic dysregulation of neurotransmission, oxidative stress, and energy metabolism. Although replication is required, our findings provide starting points to investigate causal and pleiotropic effects of these dysregulations on childhood aggression., (Copyright © 2020 Hagenbeek, Roetman, Pool, Kluft, Harms, van Dongen, Colins, Talens, van Beijsterveldt, Vandenbosch, de Zeeuw, Déjean, Fanos, Ehli, Davies, Hottenga, Hankemeier, Bartels, Vermeiren and Boomsma.)

Metabolomics examines the small molecules involved in cellular metabolism. Approximately 50% of total phenotypic differences in metabolite levels is due to genetic variance, but heritability estimates differ across metabolite classes. We perform a review of all genome-wide association and (exome-) sequencing studies published between November 2008 and October 2018, and identify >800 class-specific metabolite loci associated with metabolite levels. In a twin-family cohort (N = 5117), these metabolite loci are leveraged to simultaneously estimate total heritability (h 2 total ), and the proportion of heritability captured by known metabolite loci (h 2 Metabolite-hits ) for 309 lipids and 52 organic acids. Our study reveals significant differences in h 2 Metabolite-hits among different classes of lipids and organic acids. Furthermore, phosphatidylcholines with a high degree of unsaturation have higher h 2 Metabolite-hits estimates than phosphatidylcholines with low degrees of unsaturation. This study highlights the importance of common genetic variants for metabolite levels, and elucidates the genetic architecture of metabolite classes.