Untargeted Metabolomics Studies Research Articles

Xylitol is prothrombotic and associated with cardiovascular risk.

The pathways and metabolites that contribute to residual cardiovascular disease risks are unclear. Low-calorie sweeteners are widely used sugar substitutes in processed foods with presumed health benefits. Many low-calorie sweeteners are sugar alcohols that also are produced endogenously, albeit at levels over 1000-fold lower than observed following consumption as a sugar substitute. Untargeted metabolomics studies were performed on overnight fasting plasma samples in a discovery cohort (n = 1157) of sequential stable subjects undergoing elective diagnostic cardiac evaluations; subsequent stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed on an independent, non-overlapping validation cohort (n = 2149). Complementary isolated human platelet, platelet-rich plasma, whole blood, and animal model studies examined the effect of xylitol on platelet responsiveness and thrombus formation in vivo. Finally, an intervention study was performed to assess the effects of xylitol consumption on platelet function in healthy volunteers (n = 10). In initial untargeted metabolomics studies (discovery cohort), circulating levels of a polyol tentatively assigned as xylitol were associated with incident (3-year) major adverse cardiovascular event (MACE) risk. Subsequent stable isotope dilution LC-MS/MS analyses (validation cohort) specific for xylitol (and not its structural isomers) confirmed its association with incident MACE risk [third vs. first tertile adjusted hazard ratio (95% confidence interval), 1.57 (1.12-2.21), P < .01]. Complementary mechanistic studies showed xylitol-enhanced multiple indices of platelet reactivity and in vivo thrombosis formation at levels observed in fasting plasma. In interventional studies, consumption of a xylitol-sweetened drink markedly raised plasma levels and enhanced multiple functional measures of platelet responsiveness in all subjects. Xylitol is associated with incident MACE risk. Moreover, xylitol both enhanced platelet reactivity and thrombosis potential in vivo. Further studies examining the cardiovascular safety of xylitol are warranted.

Tidy-Direct-to-MS: An Open-Source Data-Processing Pipeline for Direct Mass Spectrometry-Based Metabolomics Experiments.

Direct-to-Mass Spectrometry and ambient ionization techniques can be used for biochemical fingerprinting in a fast way. Data processing is typically accomplished with vendor-provided software tools. Here, a novel, open-source functionality, entitled Tidy-Direct-to-MS, was developed for data processing of direct-to-MS data sets. It allows for fast and user-friendly processing using different modules for optional sample position detection and separation, mass-to-charge ratio drift detection and correction, consensus spectra calculation, and bracketing across sample positions as well as feature abundance calculation. The tool also provides functionality for the automated comparison of different sets of parameters, thereby assisting the user in the complex task of finding an optimal combination to maximize the total number of detected features while also checking for the detection of user-provided reference features. In addition, Tidy-Direct-to-MS has the capability for data quality review and subsequent data analysis, thereby simplifying the workflow of untargeted ambient MS-based metabolomics studies. Tidy-Direct-to-MS is implemented in the Python programming language as part of the TidyMS library and can thus be easily extended. Capabilities of Tidy-Direct-to-MS are showcased in a data set acquired in a marine metabolomics study reported in MetaboLights (MTBLS1198) using a transmission mode Direct Analysis in Real Time-Mass Spectrometry (TM-DART-MS)-based method.

MetaboAnalyst 6.0: towards a unified platform for metabolomics data processing, analysis and interpretation.

We introduce MetaboAnalyst version 6.0 as a unified platform for processing, analyzing, and interpreting data from targeted as well as untargeted metabolomics studies using liquid chromatography - mass spectrometry (LC-MS). The two main objectives in developing version 6.0 are to support tandem MS (MS2) data processing and annotation, as well as to support the analysis of data from exposomics studies and related experiments. Key features of MetaboAnalyst 6.0 include: (i) a significantly enhanced Spectra Processing module with support for MS2 data and the asari algorithm; (ii) a MS2 Peak Annotation module based on comprehensive MS2 reference databases with fragment-level annotation; (iii) a new Statistical Analysis module dedicated for handling complex study design withmultiple factors orphenotypic descriptors; (iv) a Causal Analysis module for estimating metabolite - phenotype causal relations based on two-sample Mendelian randomization,and (v) a Dose-Response Analysis module for benchmark dose calculations. In addition, we have also improved MetaboAnalyst's visualization functions, updated its compound database and metabolite sets, and significantly expanded its pathway analysis support to around 130 species. MetaboAnalyst 6.0 is freely available at https://www.metaboanalyst.ca.

LC-MS based untargeted metabolomics studies of the metabolic response of Ginkgo biloba extract on arsenism patients

Arsenic is an environmentally ubiquitous toxic metalloid. Chronic exposure to arsenic may lead to arsenicosis, while no specific therapeutic strategies are available for the arsenism patients. And Ginkgo biloba extract (GBE) exhibited protective effect in our previous study. However, the mechanisms by which GBE protects the arsenism patients remain poorly understood. A liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics analysis was used to study metabolic response in arsenism patients upon GBE intervention. In total, 39 coal-burning type of arsenism patients and 50 healthy residents were enrolled from Guizhou province of China. The intervention group (n = 39) were arsenism patients orally administered with GBE (three times per day) for continuous 90 days. Plasma samples from 50 healthy controls (HC) and 39 arsenism patients before and after GBE intervention were collected and analyzed by established LC-MS method. Statistical analysis was performed by MetaboAnalyst 5.0 to identify differential metabolites. Multivariate analysis revealed a separation in arsenism patients between before (BG) and after GBE intervention (AG) group. It was observed that 35 differential metabolites were identified between BG and AG group, and 30 of them were completely or partially reversed by GBE intervention, with 14 differential metabolites significantly up-regulated and 16 differential metabolites considerably down-regulated. These metabolites were involved in promoting immune response and anti-inflammatory functions, and alleviating oxidative stress. Taken together, these findings indicate that the GBE intervention could probably exert its protective effects by reversing disordered metabolites modulating these functions in arsenism patients, and provide insights into further exploration of mechanistic studies.

Untargeted metabolomics of buffalo urine reveals hydracyrlic acid, 3-bromo-1-propanol and benzyl serine as potential estrus biomarkers

Buffalo is a silent heat animal and doesn't show prominent signs of estrous like cattle so it becomes difficult for farmers to determine the receptivity of the animal based purely on the animal behaviour. India, having a huge population size, needs to produce more milk for the population. Successful artificial insemination greatly depends on the receptivity of the animal. Hence the present study aimed to identify the changes in the metabolome of the buffalo. GC–MS based mass spectrometric analysis was deployed for the determination of estrous by differential expression of metabolites. It was found that hydracrylic acid, 3-bromo-1-propanol and benzyl serine were significantly upregulated in the estrous phase of buffalo (p.value ≤0.05, FC ≥ 2). The pathway enrichment analysis also supported the same as pathways related to amino acid metabolism and fatty acid metabolism were up regulated along with the Warburg effect which is linked to the rapid cell proliferation which might help prepare animals to meet the energy requirement during the estrous. Further analysis of the metabolic biomarkers using ROC analysis also supported these three metabolites as probable biomarkers as they were identified with AUC values of 0.7 or greater. SignificanceThe present study focuses on the untargeted metabolomics studies of buffalo urine with special reference to the estrous phase of reproductive cycle. The estrous signals are more prominent in cattle, where animals show clear estrous signals such as mounting and discharge along with vocal signals. Buffalo is a silent heat animal and it becomes difficult for farmers to detect the estrous based on the physical and behavioral signals. Hence the present study focuses on GC–MS based untargeted metabolomics to identify differentially expressed urine metabolites. In this study, hydracrylic acid, 3-bromo-1-propanol and benzyl serine were found to be significantly upregulated in the estrous phase of buffalo (p-value ≤0.05, FC ≥ 2). Further confirmation of the metabolic biomarkers was done using Receiver operating characteristics (ROC) analysis which also supported these three metabolites as probable biomarkers as they had AUC values of 0.7 or greater. Hence, this study will be of prime importance for the people working in the area of animal metabolomics.

An inulin-type fructan CP-A from Codonopsis pilosula alleviates TNBS-induced ulcerative colitis based on serum-untargeted metabolomics.

Ulcerative colitis (UC) is an inflammatory disease with abdominal pain, diarrhea, and bloody stool as the main symptoms. Several studies have confirmed that polysaccharides are effective against UC. It is commonly accepted that the traditional benefits of Radix Codonopsis can be attributed to its polysaccharide contents, and inulin-type fructan CP-A is the main active monomer in the polysaccharide components. Herein, we established a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced UC rat model and lipopolysaccharide (LPS)-induced colonic epithelial cell model (NCM460) to investigate the effect of CP-A on UC. Untargeted metabolomics studies were conducted to identify differential metabolites using ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS) and enrich metabolic pathways in rat serum. The in vivo assays demonstrated that CP-A reduces colonic macroscopic injury, disease activity index (DAI), histopathological score, interleukin (IL)-8, and tumor necrosis factor-α (TNF-α) levels, as well as the expression of intercellular adhesion molecules. On the other hand, CP-A increases IL-10 and transforming growth factor-β (TGF-β) levels. The in vitro experiments indicated that CP-A treatment could reduce nitric oxide (NO) and IL-1β after LPS stimulation. The metabolomics results suggested that CP-A therapy for UC may be related to the mammalian target of rapamycin (mTOR) signaling pathway. The in vitro and in vivo validation of the pathway showed similar results, indicating that CP-A alleviates UC by preventing the activation of mTOR/p70S6K signaling pathway. These findings offer a fresh approach to treating UC and a theoretical foundation for the future advancement of CP-A.NEW & NOTEWORTHY We report that an inulin-type fructan from Codonopsis pilosula CP-A exhibits a therapeutic effect on experimental colitis. Its mechanism may be to alleviate intestinal inflammation by preventing the activation of mammalian target of rapamycin (mTOR)/p70S6K signaling pathway. These findings offer a fresh approach to treating ulcerative colitis (UC) and a theoretical foundation for the future advancement of CP-A.

Targeting Gut Microbiome With Prebiotic in Patients With CKD: The TarGut-CKD Study

IntroductionDisruption of gut microbiota underpins some of the metabolic alterations observed in chronic kidney disease (CKD). MethodsIn a non-randomized, open-label, 3-phase pilot trial, with repeated measures within each phase, we examined the efficacy of oligofructose-enriched inulin (p-inulin) in changing the gut microbiome and their metabolic products in 15 CKD patients. The stability of microbiome and metabolome was studied during the pre-treatment phase (8 weeks), a p-inulin treatment phase (12 weeks), and a post-treatment phase (8 weeks) of the study. ResultsStudy participants completed 373 of the 420 expected study visits (88.8%). Adherence to p-inulin was 83.4%. 16S rRNA sequencing was performed in 368 stool samples. A total of 1085 stool, urine, and plasma samples were subjected to untargeted metabolomic studies. P-inulin administration altered the composition of the gut microbiota significantly, with an increase in abundance of Bifidobacterium and Anaerostipes. Inter-subject variations in microbiome and metabolome were larger than intra-subject variation, indicating the stability of the gut microbiome within each phase of the study. Overall metabolite compositions assessed by beta diversity in urine and stool metabolic profiles were significantly different across study phases. Several specific metabolites in stool, urine and plasma were significant at FDR ≤ 0.1 over phase. Specifically, there was significant enrichment in microbial metabolites derived from saccharolysis. ConclusionResults from our study highlight the stability of the gut microbiome and the expansive effect of p-inulin on microbiome and host co-metabolism in CKD patients. Findings from this study will enable rigorous design of microbiome-based intervention trials.

Characterization of isomeric acetyl amino acids and di-acetyl amino acids by LC/MS/MS.

Acetylation of amino acids is important in the molecular biology and biochemistry because they are part of several metabolic pathways. N-acetyl amino acids can form through degradation of N-acetyl proteins or direct acetylation of amino acids by specific enzymes. Acetylation of α-amino acids can be either on the alpha -NH2 or on the side-chain functional group, where both the acetyl products are isomeric and can show different biological roles. Theoretically, all proteinogenic α-amino acids are expected to undergo acetylation and they can be a part of metabolome. Thus, it is essential to detect and identify all the possible acetylated products of α-amino acids for untargeted metabolomics studies. In this study, it is aimed to synthesize and characterize all acetylated products of natural α-amino acids. A total of 20 Nα -acetyl amino acids (1-20), six side-chain acetyl amino acids (21-26), and six diacetyl amino acids (27-32) were synthesized and characterized by liquid chromatography-electrospray ionizationtandem mass spectrometry (LC-ESI-MS/MS). The [M + H]+ ions of all the acetyl amino acids were subjected to MS/MS experiments to obtain their structural information. Apart from the expected loss of (H2 O + CO) (immonium ions), most of the acetyl amino acids specifically showed loss of H2 O and loss of a ketene (C2 H2 O) from [M+H]+ ions. The side-chain acetyl amino acids showed a clear-cut structure specific fragment ions that enabled easy differentiation from their isomeric Nα -acetyl amino acids. The other isomeric/isobaric acetyl amino acids could also be easily distinguished by their MS/MS spectra. The MS/MS of immonium ions of the acetyl amino acids were also studied, and they included characteristic products reflecting the structures of parent Nα -acetyl and side-chain acetyl amino acids.

Study on the potential mechanism of Qingxin Lianzi Yin Decoction on renoprotection in db/db mice via network pharmacology and metabolomics

BackgroundDiabetic nephropathy (DN) was one of the most popular and most significant microvascular complications of diabetes mellitus. Qingxin Lianzi Yin Decoction (QXLZY) was a traditional Chinese classical formula, suitable for chronic urinary system diseases. QXLZY had good clinical efficacy in early DN, but the underlying molecular mechanism remained unrevealed. PurposeThis study aimed to establish the content determination method of QXLZY index components and explore the mechanism of QXLZY on DN by network pharmacology and metabolomics studies. MethodsFirstly, the content determination methods of QXLZY were established with calycosin-7-O-β-d-glucoside, acteoside, baicalin and glycyrrhizic acid as index components. Secondly, pharmacological experiments of QXLZY were evaluated using db/db mice. UHPLC-LTQ-Orbitrap MS was used to carry out untargeted urine metabolomics, serum metabolomics, and kidney metabolomics studies. Thirdly, employing network pharmacology, key components and targets were analyzed. Finally, targeted metabolomics studies were performed on the endogenous constituents in biological samples for validation based on untargeted metabolomics results. ResultsA method for the simultaneous determination of multiple index components in QXLZY was established, which passed the comprehensive methodological verification. It was simple, feasible, and scientific. The QXLZY treatment alleviated kidney injury of db/db mice, included the degree of histopathological damage and the level of urinary microalbumin/creatinine ratio. Untargeted metabolomics studies had identified metabolic dysfunction in pathways associated with amino acid metabolism in db/db mice. Treatment with QXLZY could reverse metabolite abnormalities and influence the pathways related to energy metabolism and amino acid metabolism. It had been found that pathways with a high degree were involved in signal transduction, prominently on amino acids metabolism and lipid metabolism, analyzed by network pharmacology. Disorders of amino acid metabolism did occur in db/db mice. QXLZY could revert the levels of metabolites, such as quinolinic acid, arginine, and asparagine. ConclusionThis study was the first time to demonstrate that QXLZY alleviated diabetes-induced pathological changes in the kidneys of db/db mice by correcting disturbances in amino acid metabolism. This work could provide a new experimental basis and theoretical guidance for the rational application of QXLZY on DN, exploring the new pharmacological effect of traditional Chinese medicine, and promoting in-depth research and development.

Altered metabolic profiles and targets relevant to the protective effect of acteoside on diabetic nephropathy in db/db mice based on metabolomics and network pharmacology studies

Ethnopharmacological relevanceDiabetic nephropathy (DN) was a major cause of end-stage renal failure and a common microvascular complication in patients with diabetes mellitus (DM). Acteoside (ACT) was the main ingredient extracted from the leaves of Rehmannia glutinosa, which had the functions of entering the lung, moisturizing the skin and relieving itching, nourishing yin and tonifying the kidney, cooling blood, and stopping bleeding. ACT had attracted worldwide interest because of its therapeutic effects on DM and its complications. Aim of the studyTo clarify the metabolic profiles and targets of ACT in db/db mice based on metabolomics and network pharmacology studies. Materials and methodsDb/db mice were used to observe the biochemical indices and histopathological changes in the kidney to evaluate the pharmacological effects of ACT on DN. Untargeted metabolomics studies were performed to investigate by UHPLC-LTQ-Orbitrap MS on urine, serum, and kidney samples. The key targets and pathways were analyzed by network pharmacology. For the pathways enriched by untargeted metabolomics, targeted metabolomics by UHPLC-QQQ-MS/MS was performed in kidney samples for validation. Sensitive biomarkers in kidney samples were evaluated. The effect of ACT on the improvement of DN from the perspective of metabolism of small molecules in vivo was described. ResultsACT could delay the progression of DN and improve the degree of histopathological damage to the kidney. The pathways were focused on amino acid metabolism by untargeted metabolomics. Through network pharmacology analysis, the effect pathways were related to signal transduction, carbohydrate, lipid, amino acid metabolism and mainly affected the endocrine and immune systems. Amino acid metabolism was disturbed in the kidney of db/db mice, which could be callback by ACT, such as tryptophan, glutamine, cysteine, leucine, threonine, proline, phenylalanine, histidine, serine, arginine, asparagine by targeted metabolomics. ConclusionsIn conclusion, this study provided strong support for ACT on DN treatment in clinics. Meanwhile, the Rehmannia glutinosa was used fully to raise the income level of farmers economically, while achieving the social benefit of empowering rural revitalization.

Optimization Strategies for Mass Spectrometry-Based Untargeted Metabolomics Analysis of Small Polar Molecules in Human Plasma.

The untargeted approach to mass spectrometry-based metabolomics has a wide potential to investigate health and disease states, identify new biomarkers for diseases, and elucidate metabolic pathways. All this holds great promise for many applications in biological and chemical research. However, the complexity of instrumental parameters on advanced hybrid mass spectrometers can make the optimization of the analytical method immensely challenging. Here, we report a strategy to optimize the selected settings of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for untargeted metabolomics studies of human plasma, as a sample matrix. Specifically, we evaluated the effects of the reconstitution solvent in the sample preparation procedure, the injection volume employed, and different mass spectrometry-related operating parameters including mass range, the number of data-dependent fragmentation scans, collision energy mode, duration of dynamic exclusion time, and mass resolution settings on the metabolomics data quality and output. This study highlights key instrumental variables influencing the detection of metabolites along with suggested settings for the IQ-X tribrid system and proposes a new methodological framework to ensure increased metabolome coverage.

Detection and Quantification of Ammonia as the Ammonium Cation in Human Saliva by 1H NMR: A Promising Probe for Health Status Monitoring, with Special Reference to Cancer.

Ammonia (NH3) has been shown to be a key biomarker for a wide variety of diseases, such as hepatic and chronic kidney diseases (CKD), and cancers. It also has relevance to the oral health research area, and, hence, its determination in appropriate biofluids and tissues is of much importance. However, since it contains exchangeable >N-H protons, its analysis via 1H NMR spectroscopy, which is a widely employed technique in untargeted metabolomic studies, is rendered complicated. In this study, we focused on the 1H NMR analysis of this biomarker in less invasively collected human saliva samples, and we successfully identified and quantified it as ammonium cation (NH4+) in post-collection acidulated forms of this biofluid using both the standard calibration curve and standard addition method (SAM) approaches. For this purpose, n = 27 whole mouth saliva (WMS) samples were provided by healthy human participants, and all donors were required to follow a fasting/oral environment abstention period of 8 h prior to collection. Following acidification (pH 2.00), diluted WMS supernatant samples treated with 10% (v/v) D2O underwent 1H NMR analysis (600 MHz). The acquired results demonstrated that NH4+ can be reliably determined in these supernatants via integration of the central line of its characteristic 1:1:1 intensity triplet resonance (complete spectral range δ = 6.97-7.21 ppm). Experiments performed also demonstrated that any urease-catalysed NH3 generation occurring post-sampling in WMS samples did not affect the results acquired during the usual timespan of laboratory processing required prior to analysis. Further experiments demonstrated that oral mouth-rinsing episodes conducted prior to sample collection, as reported in previous studies, gave rise to major decreases in salivary NH4+ levels thereafter, which renormalised to only 50-60% of their basal control concentrations at the 180-min post-rinsing time point. Therefore, the WMS sample collection method employed significantly affected the absolute levels of this analyte. The LLOD was 60 μmol/L with 128 scans. The mean ± SD salivary NH4+ concentration of WMS supernatants was 11.4 ± 4.5 mmol/L. The potential extension of these analytical strategies to the screening of other metabolites with exchangeable 1H nuclei is discussed, as is their relevance to the monitoring of human disorders involving the excessive generation and/or uptake of cellular/tissue material, or altered homeostasis, in NH3.

Extracellular mechanical forces and ferroptosis in triple negative breast cancer (TNBC): Targeting of aurora kinase A (AURKA).

e13086 Background: The mechanics of tumor microenvironment, primarily induced by extracellular matrix (ECM), function to regulate cell biological behaviors. However, it is unclear if and how triple negative breast cancer (TNBC) cells adapt their biological characteristics to variable extracellular mechanical forces. Methods: TNBC tissues of different stiffness from patients were harvested for untargeted metabolomics to investigate the potential effects of mechanical forces on metabolism. TNBC cells were cultured within gels of different type I collagen (Col-I) densities, which were the major component of ECM, to simulate the effects of extracellular mechanical forces on cells. The metabolic changes of TNBC cells were also examined by using untargeted metabolomics. TNBC cells bathed in different Col-I densities were injected subcutaneously into the breast pad of nude mice. Ferroptosis indicators were evaluated between different groups of mechanical force both in vitro and in vivo. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were carried out to examine the expression of ferroptosis-related genes and proteins between different groups of mechanical force. Results: Untargeted metabolomics studies showed significant differences in ferroptosis-related metabolites between TNBC tissues with different stiffness, which were also verified in TNBC cells subjected to different extracellular mechanical forces. In vitro experiment suggested that TNBC cells cultured within high Col-I density (strong extracellular forces) exhibited higher levels of lipid peroxidation, reactive oxygen accumulation, mitochondria membrane potential and iron concentration, and lower levels of total glutathione and reduced glutathione concentration, compared with low Col-I density (weak extracellular forces). In addition, mitochondria in high Col-I density group appeared to have volume decrease, fracture and fuzzy cristae. The above results indicated that extracellular mechanical force significantly induced ferroptosis in TNBC. RT-qPCR and western blot of ferroptosis-related genes and protein showed that both mRNA and protein levels of aurora kinase A (AURKA) were significantly decreased in TNBC cells with strong extracellular mechanical force. In addition, overexpression of AURKA inhibited ferroptosis caused by mechanical forces. Furthermore, this cause and effect among mechanical force, AURKA and ferroptosis was also evident in vivo experiments. Conclusions: Our data clarified the role of extracellular mechanical forces in TNBC and showed that the extracellular mechanical forces induced ferroptosis through decreasing the expression of AURKA.

Instrumental Drift in Untargeted Metabolomics: Optimizing Data Quality with Intrastudy QC Samples.

Untargeted metabolomics is an important tool in studying health and disease and is employed in fields such as biomarker discovery and drug development, as well as precision medicine. Although significant technical advances were made in the field of mass-spectrometry driven metabolomics, instrumental drifts, such as fluctuations in retention time and signal intensity, remain a challenge, particularly in large untargeted metabolomics studies. Therefore, it is crucial to consider these variations during data processing to ensure high-quality data. Here, we will provide recommendations for an optimal data processing workflow using intrastudy quality control (QC) samples that identifies errors resulting from instrumental drifts, such as shifts in retention time and metabolite intensities. Furthermore, we provide an in-depth comparison of the performance of three popular batch-effect correction methods of different complexity. By using different evaluation metrics based on QC samples and a machine learning approach based on biological samples, the performance of the batch-effect correction methods were evaluated. Here, the method TIGER demonstrated the overall best performance by reducing the relative standard deviation of the QCs and dispersion-ratio the most, as well as demonstrating the highest area under the receiver operating characteristic with three different probabilistic classifiers (Logistic regression, Random Forest, and Support Vector Machine). In summary, our recommendations will help to generate high-quality data that are suitable for further downstream processing, leading to more accurate and meaningful insights into the underlying biological processes.

Evaluation of extraction methods for untargeted metabolomic studies for future applications in zebrafish larvae infection models

Sample preparation in untargeted metabolomics should allow reproducible extractions of as many molecules as possible. Thus, optimizing sample preparation is crucial. This study compared six different extraction procedures to find the most suitable for extracting zebrafish larvae in the context of an infection model. Two one-phase extractions employing methanol (I) and a single miscible phase of methanol/acetonitrile/water (II) and two two-phase methods using phase separation between chloroform and methanol/water combinations (III and IV) were tested. Additional bead homogenization was used for methods III and IV (III_B and IV_B). Nine internal standards and 59 molecules of interest (MoInt) related to mycobacterial infection were used for method evaluation. Two-phase methods (III and IV) led to a lower feature count, higher peak areas of MoInt, especially amino acids, and higher coefficients of variation in comparison to one-phase extractions. Adding bead homogenization increased feature count, peak areas, and CVs. Extraction I showed higher peak areas and lower CVs than extraction II, thus being the most suited one-phase method. Extraction III and IV showed similar results, with III being easier to execute and less prone to imprecisions. Thus, for future applications in zebrafish larvae metabolomics and infection models, extractions I and III might be chosen.

Human Archaeological Dentin as Source of Polar and Less Polar Metabolites for Untargeted Metabolomic Research: The Case of Yersinia pestis.

Metabolomic approaches, such as in clinical applications of living individuals, have shown potential use for solving questions regarding the past when applied to archaeological material. Here, we study for the first time the potential of this Omic approach as applied to metabolites extracted from archaeological human dentin. Dentin obtained from micro sampling the dental pulp of teeth of victims and non-victims of Yersinia pestis (plague) from a 6th century Cambridgeshire site are used to evaluate the potential use of such unique material for untargeted metabolomic studies on disease state through liquid chromatography hyphenated to high-resolution mass spectrometry (LC-HRMS). Results show that small molecules of both likely endogenous and exogenous sources are preserved for a range of polar and less polar/apolar metabolites in archaeological dentin; however, untargeted metabolomic profiles show no clear differentiation between healthy and infected individuals in the small sample analysed (n = 20). This study discusses the potential of dentin as a source of small molecules for metabolomic assays and highlights: (1) the need for follow up research to optimise sampling protocols, (2) the requirements of studies with larger sample numbers and (3) the necessity of more databases to amplify the positive results achievable with this Omic technique in the archaeological sciences.

Combination of peak-picking and binning for NMR-based untargeted metabonomics study

In NMR-based untargeted metabolomic studies, 1H NMR spectra are usually divided into equal bins/buckets to diminish the effects of peak shift caused by sample status or instrument instability, and to reduce the number of variables used as input for the multivariate statistical analysis. It was noticed that the peaks near bin boundaries may cause significant changes in integral values of adjacent bins, and the weaker peak may be obscured if it is allocated in the same bin with intense peaks. Several efforts have been taken to improve the performance of binning. Here we propose an alternative method, named P-Bin, based on the combination of the classic peak-picking and binning procedures. The location of each peak defined by peak-picking is used as the center of the individual bin. P-Bin is expected to keep all spectral information associated with the peaks and significantly reduce the data size as the spectral regions without peaks are not considered. In addition, both peak-picking and binning are routine procedures, making P-Bin easy to be implemented. To verify the performance, two sets of experimental data from human plasma and Ganoderma lucidum (G. lucidum) extracts were processed using the conventional binning method and the proposed method, before the principal component analysis (PCA) and the orthogonal projection to latent structures discriminant analysis (OPLS-DA). The results indicate that the proposed method has improved both the clustering performance of PCA score plots and the interpretability of OPLS-DA loading plots, and P-Bin could be an improved version of data preparation for metabonomic study.

Anti-oxidative and anti-aging effects of mannoprotein-rich yeast cell wall enzymatic hydrolysate by modulating gut microbiota and metabolites in Caenorhabditis elegans

In this study, antioxidant and anti-aging studies were carried out by mannoprotein-rich yeast cell wall enzymatic hydrolysate (MYH) obtained by enzymatic hydrolysis of yeast cell wall through the Caenorhabditis elegans (C. elegans) model. It was found that MYH could improve the lifespan and anti-stress ability of C. elegans by increasing the activity of antioxidant enzymes such as T-SOD, GSH-PX and CAT, and reducing the levels of MDA, ROS and apoptosis. At the same time, through the verification expression of corresponding mRNA, it was found that MYH exerted antioxidant and anti-aging activities by up-regulating the translation of MTL-1, DAF-16, SKN-1 and SOD-3 mRNA, and down-regulating the translation of AGE-1 and DAF-2 mRNA. In addition, it was found that MYH could improve the composition and distribution of the gut microbiota of C. elegans, and significantly improve the level of metabolites through the sequencing of gut microbiota and untargeted metabolomic studies. It has contributed to studying the antioxidant and anti-aging activities of microorganisms such as yeast through the level of gut microbiota and metabolites and the development of related functional foods.

Model-driven data curation pipeline for LC–MS-based untargeted metabolomics

There is still no community consensus regarding strategies for data quality review in liquid chromatography mass spectrometry (LC-MS)-based untargeted metabolomics. Assessing the analytical robustness of data, which is relevant for inter-laboratory comparisons and reproducibility, remains a challenge despite the wide variety of tools available for data processing. The aim of this study was to provide a model to describe the sources of variation in LC-MS-based untargeted metabolomics measurements, to use it to build a comprehensive curation pipeline, and to provide quality assessment tools for data quality review. Human serum samples (n=392) were analyzed by ultraperformance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) using an untargeted metabolomics approach. The pipeline and tools used to process this dataset were implemented as part of the open source, publicly available TidyMS Python-based package. The model was applied to understand data curation practices used by the metabolomics community. Sources of variation, which are often overlooked in untargeted metabolomic studies, were identified in the analysis. New tools were used to characterize certain types of variations. The developed pipeline allowed confirming data robustness by comparing the experimental results with expected values predicted by the model. New quality control practices were introduced to assess the analytical quality of data.

The artificial sweetener erythritol and cardiovascular event risk.

Artificial sweeteners are widely used sugar substitutes, but little is known about their long-term effects on cardiometabolic disease risks. Here we examined the commonly used sugar substitute erythritol and atherothrombotic disease risk. In initial untargeted metabolomics studies in patients undergoing cardiac risk assessment (n = 1,157; discovery cohort, NCT00590200 ), circulating levels of multiple polyol sweeteners, especially erythritol, were associated with incident (3 year) risk for major adverse cardiovascular events (MACE; includes death or nonfatal myocardial infarction or stroke). Subsequent targeted metabolomics analyses in independent US (n = 2,149, NCT00590200 ) and European (n = 833, DRKS00020915 ) validation cohorts of stable patients undergoing elective cardiac evaluation confirmed this association (fourth versus first quartile adjusted hazard ratio (95% confidence interval), 1.80 (1.18-2.77) and 2.21 (1.20-4.07), respectively). At physiological levels, erythritol enhanced platelet reactivity in vitro and thrombosis formation in vivo. Finally, in a prospective pilot intervention study ( NCT04731363 ), erythritol ingestion in healthy volunteers (n = 8) induced marked and sustained (>2 d) increases in plasma erythritol levels well above thresholds associated with heightened platelet reactivity and thrombosis potential in in vitro and in vivo studies. Our findings reveal that erythritol is both associated with incident MACE risk and fosters enhanced thrombosis. Studies assessing the long-term safety of erythritol are warranted.