Metabolomics 2023 workshop report: moving toward consensus on best QA/QC practices in LC-MS-based untargeted metabolomics.

INTRODUCTION: During the Metabolomics 2023 conference, the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) presented a QA/QC workshop for LC-MS-based untargeted metabolomics. OBJECTIVES: The Best Practices Working Group disseminated recent findings from community forums and discussed aspects to include in a living guidance document. METHODS: Presentations focused on reference materials, data quality review, metabolite identification/annotation and quality assurance. RESULTS: Live polling results and follow-up discussions offered a broad international perspective on QA/QC practices. CONCLUSIONS: Community input gathered from this workshop series is being used to shape the living guidance document, a continually evolving QA/QC best practices resource for metabolomics researchers.

The Lipidomic Profile Is Associated with the Dietary Pattern in Subjects with and without Diabetes Mellitus from a Mediterranean Area.

Lipid functions can be influenced by genetics, age, disease states, and lifestyle factors, particularly dietary patterns, which are crucial in diabetes management. Lipidomics is an expanding field involving the comprehensive exploration of lipids from biological samples. In this cross-sectional study, 396 participants from a Mediterranean region, including individuals with type 1 diabetes (T1D), type 2 diabetes (T2D), and non-diabetic individuals, underwent lipidomic profiling and dietary assessment. Participants completed validated food frequency questionnaires, and lipid analysis was conducted using ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC/MS). Multiple linear regression models were used to determine the association between lipid features and dietary patterns. Across all subjects, acylcarnitines (AcCa) and triglycerides (TG) displayed negative associations with the alternate Healthy Eating Index (aHEI), indicating a link between lipidomic profiles and dietary habits. Various lipid species (LS) showed positive and negative associations with dietary carbohydrates, fats, and proteins. Notably, in the interaction analysis between diabetes and the aHEI, we found some lysophosphatidylcholines (LPC) that showed a similar direction with respect to aHEI in non-diabetic individuals and T2D subjects, while an opposite direction was observed in T1D subjects. The study highlights the significant association between lipidomic profiles and dietary habits in people with and without diabetes, particularly emphasizing the role of healthy dietary choices, as reflected by the aHEI, in modulating lipid concentrations. These findings underscore the importance of dietary interventions to improve metabolic health outcomes, especially in the context of diabetes management.

Characterization of Electrospray Ionization Complexity in Untargeted Metabolomic Studies.

The annotation of metabolites detected in LC-MS-based untargeted metabolomics studies routinely applies accurate m/z of the intact metabolite (MS1) as well as chromatographic retention time and MS/MS data. Electrospray ionization and transfer of ions through the mass spectrometer can result in the generation of multiple "features" derived from the same metabolite with different m/z values but the same retention time. The complexity of the different charged and neutral adducts, in-source fragments, and charge states has not been previously and deeply characterized. In this paper, we report the first large-scale characterization using publicly available data sets derived from different research groups, instrument manufacturers, LC assays, sample types, and ion modes. 271 m/z differences relating to different metabolite feature pairs were reported, and 209 were annotated. The results show a wide range of different features being observed with only a core 32 m/z differences reported in >50% of the data sets investigated. There were no patterns reporting specific m/z differences that were observed in relation to ion mode, instrument manufacturer, LC assay type, and mammalian sample type, although some m/z differences were related to study group (mammal, microbe, plant) and mobile phase composition. The results provide the metabolomics community with recommendations of adducts, in-source fragments, and charge states to apply in metabolite annotation workflows.

Bilateral remote ischemic conditioning in children: A two-center, double-blind, randomized controlled trial in young children undergoing cardiac surgery.

Objective: The study objective was to determine whether adequately delivered bilateral remote ischemic preconditioning is cardioprotective in young children undergoing surgery for 2 common congenital heart defects with or without cyanosis. Methods: We performed a prospective, double-blind, randomized controlled trial at 2 centers in the United Kingdom. Children aged 3 to 36 months undergoing tetralogy of Fallot repair or ventricular septal defect closure were randomized 1:1 to receive bilateral preconditioning or sham intervention. Participants were followed up until hospital discharge or 30 days. The primary outcome was area under the curve for high-sensitivity troponin-T in the first 24 hours after surgery, analyzed by intention-to-treat. Right atrial biopsies were obtained in selected participants. Results: Between October 2016 and December 2020, 120 eligible children were randomized to receive bilateral preconditioning (n = 60) or sham intervention (n = 60). The primary outcome, area under the curve for high-sensitivity troponin-T, was higher in the preconditioning group (mean: 70.0 ± 50.9 µg/L/h, n = 56) than in controls (mean: 55.6 ± 30.1 µg/L/h, n = 58) (mean difference, 13.2 µg/L/h; 95% CI, 0.5-25.8; P = .04). Subgroup analyses did not show a differential treatment effect by oxygen saturations (pinteraction = .25), but there was evidence of a differential effect by underlying defect (pinteraction = .04). Secondary outcomes and myocardial metabolism, quantified in atrial biopsies, were not different between randomized groups. Conclusions: Bilateral remote ischemic preconditioning does not attenuate myocardial injury in children undergoing surgical repair for congenital heart defects, and there was evidence of potential harm in unstented tetralogy of Fallot. The routine use of remote ischemic preconditioning cannot be recommended for myocardial protection during pediatric cardiac surgery.

Lipidome characterisation and sex-specific differences in type 1 and type 2 diabetes mellitus.

BACKGROUND: In this study, we evaluated the lipidome alterations caused by type 1 diabetes (T1D) and type 2 diabetes (T2D), by determining lipids significantly associated with diabetes overall and in both sexes, and lipids associated with the glycaemic state. METHODS: An untargeted lipidomic analysis was performed to measure the lipid profiles of 360 subjects (91 T1D, 91 T2D, 74 with prediabetes and 104 controls (CT)) without cardiovascular and/or chronic kidney disease. Ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) was conducted in two ion modes (positive and negative). We used multiple linear regression models to (1) assess the association between each lipid feature and each condition, (2) determine sex-specific differences related to diabetes, and (3) identify lipids associated with the glycaemic state by considering the prediabetes stage. The models were adjusted by sex, age, hypertension, dyslipidaemia, body mass index, glucose, smoking, systolic blood pressure, triglycerides, HDL cholesterol, LDL cholesterol, alternate Mediterranean diet score (aMED) and estimated glomerular filtration rate (eGFR); diabetes duration and glycated haemoglobin (HbA1c) were also included in the comparison between T1D and T2D. RESULTS: A total of 54 unique lipid subspecies from 15 unique lipid classes were annotated. Lysophosphatidylcholines (LPC) and ceramides (Cer) showed opposite effects in subjects with T1D and subjects with T2D, LPCs being mainly up-regulated in T1D and down-regulated in T2D, and Cer being up-regulated in T2D and down-regulated in T1D. Also, Phosphatidylcholines were clearly down-regulated in subjects with T1D. Regarding sex-specific differences, ceramides and phosphatidylcholines exhibited important diabetes-associated differences due to sex. Concerning the glycaemic state, we found a gradual increase of a panel of 1-deoxyceramides from normoglycemia to prediabetes to T2D. CONCLUSIONS: Our findings revealed an extensive disruption of lipid metabolism in both T1D and T2D. Additionally, we found sex-specific lipidome changes associated with diabetes, and lipids associated with the glycaemic state that can be linked to previously described molecular mechanisms in diabetes.

Establishing a framework for best practices for quality assurance and quality control in untargeted metabolomics.

BACKGROUND: Quality assurance (QA) and quality control (QC) practices are key tenets that facilitate study and data quality across all applications of untargeted metabolomics. These important practices will strengthen this field and accelerate its success. The Best Practices Working Group (WG) within the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) focuses on community use of QA/QC practices and protocols and aims to identify, catalogue, harmonize, and disseminate current best practices in untargeted metabolomics through community-driven activities. AIM OF REVIEW: A present goal of the Best Practices WG is to develop a working strategy, or roadmap, that guides the actions of practitioners and progress in the field. The framework in which mQACC operates promotes the harmonization and dissemination of current best QA/QC practice guidance and encourages widespread adoption of these essential QA/QC activities for liquid chromatography-mass spectrometry. KEY SCIENTIFIC CONCEPTS OF REVIEW: Community engagement and QA/QC information gathering activities have been occurring through conference workshops, virtual and in-person interactive forum discussions, and community surveys. Seven principal QC stages prioritized by internal discussions of the Best Practices WG have received participant input, feedback and discussion. We outline these stages, each involving a multitude of activities, as the framework for identifying QA/QC best practices. The ultimate planned product of these endeavors is a "living guidance" document of current QA/QC best practices for untargeted metabolomics that will grow and change with the evolution of the field.

Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.

Untargeted metabolomics is an analytical approach with numerous applications serving as an effective metabolic phenotyping platform to characterize small molecules within a biological system. Data quality can be challenging to evaluate and demonstrate in metabolomics experiments. This has driven the use of pooled quality control (QC) samples for monitoring and, if necessary, correcting for analytical variance introduced during sample preparation and data acquisition stages. Described herein is a scoping literature review detailing the use of pooled QC samples in published untargeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics studies. A literature query was performed, the list of papers was filtered, and suitable articles were randomly sampled. In total, 109 papers were each reviewed by at least five reviewers, answering predefined questions surrounding the use of pooled quality control samples. The results of the review indicate that use of pooled QC samples has been relatively widely adopted by the metabolomics community and that it is used at a similar frequency across biological taxa and sample types in both small- and large-scale studies. However, while many studies generated and analyzed pooled QC samples, relatively few reported the use of pooled QC samples to improve data quality. This demonstrates a clear opportunity for the field to more frequently utilize pooled QC samples for quality reporting, feature filtering, analytical drift correction, and metabolite annotation. Additionally, our survey approach enabled us to assess the ambiguity in the reporting of the methods used to describe the generation and use of pooled QC samples. This analysis indicates that many details of the QC framework are missing or unclear, limiting the reader's ability to determine which QC steps have been taken. Collectively, these results capture the current state of pooled QC sample usage and highlight existing strengths and deficiencies as they are applied in untargeted LC-MS metabolomics.

Metabolomics 2022 workshop report: state of QA/QC best practices in LC-MS-based untargeted metabolomics, informed through mQACC community engagement initiatives.

INTRODUCTION: The Metabolomics Quality Assurance and Quality Control Consortium (mQACC) organized a workshop during the Metabolomics 2022 conference. OBJECTIVES: The goal of the workshop was to disseminate recent findings from mQACC community-engagement efforts and to solicit feedback about a living guidance document of QA/QC best practices for untargeted LC-MS metabolomics. METHODS: Four QC-related topics were presented. RESULTS: During the discussion, participants expressed the need for detailed guidance on a broad range of QA/QC-related topics accompanied by use-cases. CONCLUSIONS: Ongoing efforts will continue to identify, catalog, harmonize, and disseminate QA/QC best practices, including outreach activities, to establish and continually update QA/QC guidelines.

Determining the role of novel metabolic pathways in driving intracranial pressure reduction after weight loss.

Idiopathic intracranial hypertension, a disease classically occurring in women with obesity, is characterized by raised intracranial pressure. Weight loss leads to the reduction in intracranial pressure. Additionally, pharmacological glucagon-like peptide-1 agonism reduces cerebrospinal fluid secretion and intracranial pressure. The potential mechanisms by which weight loss reduces intracranial pressure are unknown and were the focus of this study. Meal stimulation tests (fasted plasma sample, then samples at 15, 30, 60, 90 and 120 min following a standardized meal) were conducted pre- and post-bariatric surgery [early (2 weeks) and late (12 months)] in patients with active idiopathic intracranial hypertension. Dynamic changes in gut neuropeptides (glucagon-like peptide-1, gastric inhibitory polypeptide and ghrelin) and metabolites (untargeted ultra-high performance liquid chromatography-mass spectrometry) were evaluated. We determined the relationship between gut neuropeptides, metabolites and intracranial pressure. Eighteen idiopathic intracranial hypertension patients were included [Roux-en-Y gastric bypass (RYGB) n = 7, gastric banding n = 6 or sleeve gastrectomy n = 5]. At 2 weeks post-bariatric surgery, despite similar weight loss, RYGB had a 2-fold (50%) greater reduction in intracranial pressure compared to sleeve. Increased meal-stimulated glucagon-like peptide-1 secretion was observed after RYGB (+600%) compared to sleeve (+319%). There was no change in gastric inhibitory polypeptide and ghrelin. Dynamic changes in meal-stimulated metabolites after bariatric surgery consistently identified changes in lipid metabolites, predominantly ceramides, glycerophospholipids and lysoglycerophospholipids, which correlated with intracranial pressure. A greater number of differential lipid metabolites were observed in the RYGB cohort at 2 weeks, and these also correlated with intracranial pressure. In idiopathic intracranial hypertension, we identified novel changes in lipid metabolites and meal-stimulated glucagon-like peptide-1 levels following bariatric surgery which were associated with changes in intracranial pressure. RYGB was most effective at reducing intracranial pressure despite analogous weight loss to gastric sleeve at 2 weeks post-surgery and was associated with more pronounced changes in these metabolite pathways. We suggest that these novel perturbations in lipid metabolism and glucagon-like peptide-1 secretion are mechanistically important in driving a reduction in intracranial pressure following weight loss in patients with idiopathic intracranial hypertension. Therapeutic targeting of these pathways, for example with glucagon-like peptide-1 agonist infusion, could represent a therapeutic strategy.

Unlocking the secrets of the microbiome: exploring the dynamic microbial interplay with humans through metabolomics and their manipulation for synthetic biology applications.

Metabolomics is a powerful research discovery tool with the potential to measure hundreds to low thousands of metabolites. In this review, we discuss the application of GC-MS and LC-MS in discovery-based metabolomics research, we define metabolomics workflows and we highlight considerations that need to be addressed in order to generate robust and reproducible data. We stress that metabolomics is now routinely applied across the biological sciences to study microbiomes from relatively simple microbial systems to their complex interactions within consortia in the host and the environment and highlight this in a range of biological species and mammalian systems including humans. However, challenges do still exist that need to be overcome to maximise the potential for metabolomics to help us understanding biological systems. To demonstrate the potential of the approach we discuss the application of metabolomics in two broad research areas: (1) synthetic biology to increase the production of high-value fine chemicals and reduction in secondary by-products and (2) gut microbial interaction with the human host. While burgeoning in importance, the latter is still in its infancy and will benefit from the development of tools to detangle host-gut-microbial interactions and their impact on human health and diseases.

A proposed framework to evaluate the quality and reliability of targeted metabolomics assays from the UK Consortium on Metabolic Phenotyping (MAP/UK).

Targeted metabolite assays that measure tens or hundreds of pre-selected metabolites, typically using liquid chromatography-mass spectrometry, are increasingly being developed and applied to metabolic phenotyping studies. These are used both as standalone phenotyping methods and for the validation of putative metabolic biomarkers obtained from untargeted metabolomics studies. However, there are no widely accepted standards in the scientific community for ensuring reliability of the development and validation of targeted metabolite assays (referred to here as 'targeted metabolomics'). Most current practices attempt to adopt, with modifications, the strict guidance provided by drug regulatory authorities for analytical methods designed largely for measuring drugs and other xenobiotic analytes. Here, the regulatory guidance provided by the European Medicines Agency, US Food and Drug Administration and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use are summarized. In this Perspective, we have adapted these guidelines and propose a less onerous 'tiered' approach to evaluate the reliability of a wide range of metabolomics analyses, addressing the need for community-accepted, harmonized guidelines for tiers other than full validation. This 'fit-for-purpose' tiered approach comprises four levels-discovery, screening, qualification and validation-and is discussed in the context of a range of targeted and untargeted metabolomics assays. Issues arising with targeted multiplexed metabolomics assays, and how these might be addressed, are considered. Furthermore, guidance is provided to assist the community with selecting the appropriate degree of reliability for a series of well-defined applications of metabolomics.

Dysregulation of Amino Acid, Lipid, and Acylpyruvate Metabolism in Idiopathic Intracranial Hypertension: A Non-targeted Case Control and Longitudinal Metabolomic Study.

Background: Idiopathic intracranial hypertension (IIH) is characterized by increased intracranial pressure occurring predominantly in women with obesity. The pathogenesis is not understood. We have applied untargeted metabolomic analysis using ultrahigh-performance liquid chromatography-mass spectrometry to characterize the cerebrospinal fluid (CSF) and serum in IIH compared to control subjects. Methods and findings: Samples were collected from IIH patients (n = 66) with active disease at baseline and again at 12 months following therapeutic weight loss. Control samples were collected from gender- and weight-matched healthy controls (n = 20). We identified annotated metabolites in CSF, formylpyruvate and maleylpyruvate/fumarylpyruvate, which were present at lower concentrations in IIH compared to control subjects and returned to values observed in controls following weight loss. These metabolites showed the opposite trend in serum at baseline. Multiple amino acid metabolic pathways and lipid classes were perturbed in serum and CSF in IIH alone. Serum lipid metabolite pathways were significantly increased in IIH. Conclusions: We observed a number of differential metabolic pathways related to amino acid, lipid, and acylpyruvate metabolism, in IIH compared to controls. These pathways were associated with clinical measures and normalized with disease remission. Perturbation of these metabolic pathways provides initial understanding of disease dysregulation in IIH.

Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment.

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18-/- syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

Providing metabolomics education and training: pedagogy and considerations.

BACKGROUND: Metabolomics is a highly multidisciplinary and non-standardised research field. Metabolomics researchers must possess and apply extensive cross-disciplinary content knowledge, subjective experience-based judgement, and the associated diverse skill sets. Accordingly, appropriate educational and training initiatives are important in developing this knowledge and skills base in the metabolomics community. For these initiatives to be successful, they must consider both pedagogical best practice and metabolomics-specific contextual challenges. AIM OF REVIEW: The aim of this review is to provide consolidated pedagogical guidance for educators and trainers in metabolomics educational and training programmes. KEY SCIENTIFIC CONCEPTS OF REVIEW: In this review, we discuss the principles of pedagogical best practice as they relate to metabolomics. We then discuss the challenges and considerations in developing and delivering education and training in metabolomics. Finally, we present examples from our own teaching practice to illustrate how pedagogical best practice can be integrated into metabolomics education and training programmes.

Critical assessment of chromatographic metadata in publicly available metabolomics data repositories.

INTRODUCTION: The structural identification of metabolites represents one of the current bottlenecks in non-targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics. The Metabolomics Standard Initiative has developed a multilevel system to report confidence in metabolite identification, which involves the use of MS, MS/MS and orthogonal data. Limitations due to similar or same fragmentation pattern (e.g. isomeric compounds) can be overcome by the additional orthogonal information of the retention time (RT), since it is a system property that is different for each chromatographic setup. OBJECTIVES: In contrast to MS data, sharing of RT data is not as widespread. The quality of data and its (re-)useability depend very much on the quality of the metadata. We aimed to evaluate the coverage and quality of this metadata from public metabolomics repositories. METHODS: We acquired an overview on the current reporting of chromatographic separation conditions. For this purpose, we defined the following information as important details that have to be provided: column name and dimension, flow rate, temperature, composition of eluents and gradient. RESULTS: We found that 70% of descriptions of the chromatographic setups are incomplete (according to our definition) and an additional 10% of the descriptions contained ambiguous and/or incorrect information. Accordingly, only about 20% of the descriptions allow further (re-)use of the data, e.g. for RT prediction. Therefore, we have started to develop a unified and standardized notation for chromatographic metadata with detailed and specific description of eluents, columns and gradients. CONCLUSION: Reporting of chromatographic metadata is currently not unified. Our recommended suggestions for metadata reporting will enable more standardization and automatization in future reporting.

Quality assurance and quality control reporting in untargeted metabolic phenotyping: mQACC recommendations for analytical quality management.

BACKGROUND: Demonstrating that the data produced in metabolic phenotyping investigations (metabolomics/metabonomics) is of good quality is increasingly seen as a key factor in gaining acceptance for the results of such studies. The use of established quality control (QC) protocols, including appropriate QC samples, is an important and evolving aspect of this process. However, inadequate or incorrect reporting of the QA/QC procedures followed in the study may lead to misinterpretation or overemphasis of the findings and prevent future metanalysis of the body of work. OBJECTIVE: The aim of this guidance is to provide researchers with a framework that encourages them to describe quality assessment and quality control procedures and outcomes in mass spectrometry and nuclear magnetic resonance spectroscopy-based methods in untargeted metabolomics, with a focus on reporting on QC samples in sufficient detail for them to be understood, trusted and replicated. There is no intent to be proscriptive with regard to analytical best practices; rather, guidance for reporting QA/QC procedures is suggested. A template that can be completed as studies progress to ensure that relevant data is collected, and further documents, are provided as on-line resources. KEY REPORTING PRACTICES: Multiple topics should be considered when reporting QA/QC protocols and outcomes for metabolic phenotyping data. Coverage should include the role(s), sources, types, preparation and uses of the QC materials and samples generally employed in the generation of metabolomic data. Details such as sample matrices and sample preparation, the use of test mixtures and system suitability tests, blanks and technique-specific factors are considered and methods for reporting are discussed, including the importance of reporting the acceptance criteria for the QCs. To this end, the reporting of the QC samples and results are considered at two levels of detail: "minimal" and "best reporting practice" levels.

Suitability of Dried Blood Spots for Accelerating Veterinary Biobank Collections and Identifying Metabolomics Biomarkers With Minimal Resources.

Biomarker discovery using biobank samples collected from veterinary clinics would deliver insights into the diverse population of pets and accelerate diagnostic development. The acquisition, preparation, processing, and storage of biofluid samples in sufficient volumes and at a quality suitable for later analysis with most suitable discovery methods remain challenging. Metabolomics analysis is a valuable approach to detect health/disease phenotypes. Pre-processing changes during preparation of plasma/serum samples may induce variability that may be overcome using dried blood spots (DBSs). We report a proof of principle study by metabolite fingerprinting applying UHPLC-MS of plasma and DBSs acquired from healthy adult dogs and cats (age range 1-9 years), representing each of 4 dog breeds (Labrador retriever, Beagle, Petit Basset Griffon Vendeen, and Norfolk terrier) and the British domestic shorthair cat (n = 10 per group). Blood samples (20 and 40 µL) for DBSs were loaded onto filter paper, air-dried at room temperature (3 h), and sealed and stored (4°C for ~72 h) prior to storage at -80°C. Plasma from the same blood draw (250 µL) was prepared and stored at -80°C within 1 h of sampling. Metabolite fingerprinting of the DBSs and plasma produced similar numbers of metabolite features that had similar abilities to discriminate between biological classes and correctly assign blinded samples. These provide evidence that DBSs, sampled in a manner amenable to application in in-clinic/in-field processing, are a suitable sample for biomarker discovery using UHPLC-MS metabolomics. Further, given appropriate owner consent, the volumes tested (20-40 µL) make the acquisition of remnant blood from blood samples drawn for other reasons available for biobanking and other research activities. Together, this makes possible large-scale biobanking of veterinary samples, gaining sufficient material sooner and enabling quicker identification of biomarkers of interest.

Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached.

Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.

Acute effects of prior dietary fat ingestion on postprandial metabolic responses to protein and carbohydrate co-ingestion in overweight and obese men: A randomised crossover trial.

BACKGROUND: Obesity and insulin resistance are associated with an impaired sensitivity to anabolic stimuli such as dietary protein (anabolic resistance). Omega-3 polyunsaturated fatty acids (n-3 PUFA) may be protective against the deleterious effects of saturated fatty acids (SFA) on insulin resistance. However, the contribution of excess fat consumption to anabolic and insulin resistance and the interaction between SFA and n-3 PUFA is not well studied. AIM: The primary aim of this study was to investigate the effects of an oral fat pre-load, with or without the partial substitution of SFA with fish oil (FO)-derived n-3 PUFA, on indices of insulin and anabolic sensitivity in response to subsequent dietary protein and carbohydrate (dextrose) co-ingestion. METHODS: Eight middle-aged males with overweight or obesity (52.8 ± 2.0 yr, BMI 31.8 ± 1.4 kg·m-2) ingested either an SFA, or isoenergetic SFA and FO emulsion (FO), or water/control (Con), 4 h prior to a bolus of milk protein and dextrose. RESULTS: Lipid ingestion (in particular FO) impaired the early postprandial uptake of branched chain amino acids (BCAA) into the skeletal muscle in response to protein and dextrose, and attenuated the peak glycaemic response, but was not accompanied by differences in whole body (Matsuda Index: Con: 4.66 ± 0.89, SFA: 5.10 ± 0.94 and FO: 4.07 ± 0.59) or peripheral (forearm glucose netAUC: Con: 521.7 ± 101.7; SFA: 470.2 ± 125.5 and FO: 495.3 ± 101.6 µmol·min-1·100 g lean mass·min [t = 240-420 min]) insulin sensitivity between visits. Postprandial whole body fat oxidation was affected by visit (P = 0.024) with elevated rates in SFA and FO, relative to Con (1.85 ± 0.55; 2.19 ± 0.21 and 0.65 ± 0.35 kJ·h-1·kg-1 lean body mass, respectively), however muscle uptake of free fatty acids (FFA) was unaffected. CONCLUSION: Oral lipid preloads, consisting of SFA and FO, impair the early postprandial BCAA uptake into skeletal muscle, which occurs independent of changes in insulin sensitivity. CLINICAL TRIAL REGISTRY NUMBER: ClinicalTrials.gov Identifier NCT03146286.

Reference materials for MS-based untargeted metabolomics and lipidomics: a review by the metabolomics quality assurance and quality control consortium (mQACC).

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.