16p11.2 haploinsufficiency reduces mitochondrial biogenesis in brain endothelial cells and alters brain metabolism in adult mice.

Neurovascular abnormalities in mouse models of 16p11.2 deletion autism syndrome are reminiscent of alterations reported in murine models of glucose transporter deficiency, including reduced brain angiogenesis and behavioral alterations. Yet, whether cerebrovascular alterations in 16p11.2df/+ mice affect brain metabolism is unknown. Here, we report that anesthetized 16p11.2df/+ mice display elevated brain glucose uptake, a phenomenon recapitulated in mice with endothelial-specific 16p11.2 haplodeficiency. Awake 16p11.2df/+ mice display attenuated relative fluctuations of extracellular brain glucose following systemic glucose administration. Targeted metabolomics on cerebral cortex extracts reveals enhanced metabolic responses to systemic glucose in 16p11.2df/+ mice that also display reduced mitochondria number in brain endothelial cells. This is not associated with changes in mitochondria fusion or fission proteins, but 16p11.2df/+ brain endothelial cells lack the splice variant NT-PGC-1α, suggesting defective mitochondrial biogenesis. We propose that altered brain metabolism in 16p11.2df/+ mice is compensatory to endothelial dysfunction, shedding light on previously unknown adaptative responses.

Metabolomic profiles in relation to benchmark polycyclic aromatic compounds (PACs) and trace elements in two seabird species from Arctic Canada.

While exposure of birds to oil-related contaminants has been documented, the related adverse effects this exposure has on Arctic marine birds remain unexplored. Metabolomics can play an important role to explore biologically relevant metabolite biomarkers in relation to different stressors, even at benchmark levels of contamination. The aim of this study was to characterize the metabolomics profiles in relation to polycyclic aromatic compounds (PACs) and trace elements in the liver of two seabird species in the Canadian Arctic. In July 2018, black guillemots (Cepphus grylle) and thick-billed murres (Uria lomvia) were collected by hunters from a region where natural oil seeps occur in the seabed near Qikiqtarjuaq, Nunavut, Canada. A total of 121 metabolites were identified in liver tissue samples using reversed phase and hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry platforms to detect non-polar and polar metabolites, respectively. Sixty-nine metabolites showed excellent repeatability and linearity and were used to examine possible effects of oil-related contaminants exposure (PACs and trace elements). Metabolites including 3-hydroxy anthranilic acid, adenine, adenosine, adenosine mono-phosphate, ascorbic acid, butyrylcarnitine, cholic acid, guanosine, guanosine mono-phosphate, inosine, norepinephrine and threonine showed significant differences (more than two fold) between the two species. Elevated adenine and adenosine, along with decreased reduced/oxidized glutathione ratio, highlighted the potential for oxidative stress in murres. Lipid peroxidation and superoxide dismutase activity assays also confirmed these metabolomic findings. These results will help to characterize the baseline metabolomic profiles of Arctic seabird species with different foraging behaviour and trace element burden.

Urinary metabotype of severe asthma evidences decreased carnitine metabolism independent of oral corticosteroid treatment in the U-BIOPRED study.

INTRODUCTION: Asthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication. METHODS: Baseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12-18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods. RESULTS: A total of 90 metabolites were identified, with 40 significantly altered (p<0.05, false discovery rate <0.05) in severe asthma and 23 by OCS use. Multivariate modelling showed that observed metabotypes in healthy participants and patients with mild-to-moderate asthma differed significantly from those in patients with severe asthma (p=2.6×10-20), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10-4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings. CONCLUSIONS: This is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma.

Genome-Wide Discovery of miRNAs with Differential Expression Patterns in Responses to Salinity in the Two Contrasting Wheat Cultivars.

Salinity is a serious environmental issue. It has a substantial effect on crop yield, as many crop species are sensitive to salinity due to climate change, and it impact is continuing to increase. Plant microRNAs (miRNAs) contribute to salinity stress response in bread wheat. However, the underlying molecular mechanisms by which miRNAs confer salt tolerance in wheat are unclear. We conducted a genome-wide discovery study using Illumina high throughput sequencing and comprehensive in silico analysis to obtain insight into the underlying mechanisms by which small RNAs confer tolerance to salinity in roots of two contrasting wheat cvv., namely Suntop (salt-tolerant) and Sunmate (salt-sensitive). A total of 191 microRNAs were identified in both cultivars, consisting of 110 known miRNAs and 81 novel miRNAs; 181 miRNAs were shared between the two cultivars. The known miRNAs belonged to 35 families consisted of 23 conserved and 12 unique families. Salinity stress induced 43 and 75 miRNAs in Suntop and Sunmate, respectively. Among them, 14 and 29 known and novel miRNAs were expressed in Suntop and 37 and 38 in Sunmate. In silico analysis revealed 861 putative target mRNAs for the 75 known miRNAs and 52 putative target mRNAs for the 15 candidate novel miRNAs. Furthermore, seven miRNAs including tae-miR156, tae-miR160, tae-miR171a-b, tae-miR319, tae-miR159a-b, tae-miR9657 and novel-mir59 that regulate auxin responsive-factor, SPL, SCL6, PCF5, R2R3 MYB, and CBL-CIPK, respectively, were predicted to contribute to salt tolerance in Suntop. This information helps further our understanding of how the molecular mechanisms of salt tolerance are mediated by miRNAs and may facilitate the genetic improvement of wheat cultivars.

Genotypic Difference in the Responses to Nitrogen Fertilizer Form in Tibetan Wild and Cultivated Barley.

Nitrogen (N) availability and form have a dramatic effect on N uptake and assimilation in plants, affecting growth and development. In the previous studies, we found great differences in low-N tolerance between Tibetan wild barley accessions and cultivated barley varieties. We hypothesized that there are different responses to N forms between the two kinds of barleys. Accordingly, this study was carried out to determine the response of four barley genotypes (two wild, XZ16 and XZ179; and two cultivated, ZD9 andHua30) under 4Nforms (NO3-, NH4+, urea and glycine). The results showed significant reduction in growth parameters such as root/shoot length and biomass, as well as photosynthesis parameters and total soluble protein content under glycine treatment relative to other N treatments, for both wild and cultivated barley, however, XZ179 was least affected. Similarly, ammonium adversely affected growth parameters in both wild and cultivated barleys, with XZ179 being severely affected. On the other hand, both wild and cultivated genotypes showed higher biomass, net photosynthetic rate, chlorophyll and protein in NO3- treatment relative to other three N treatments. It may be concluded that barley undisputedly grows well under inorganic nitrogen (NO3-), however in response to the organic N wild barley prefer glycine more than cultivated barely.

Distinct Basal Metabolism in Three Mouse Models of Neurodevelopmental Disorders.

Prevalence of metabolic disturbances is higher among individuals with neurodevelopmental disorders (NDDs), yet this association has been largely overlooked. Investigation into human disease remains challenging, as a complete pathophysiological understanding relies on accurate modeling and highly controlled variables. Genetically engineered mouse models are widely used to gain insight into the biology of human NDDs, but research focus has been on behavioral and neurophysiological abnormalities. Such models not only allow for evaluating usefulness in reproducing human features, including similarities and discrepancies with rodent phenotypes, but they also represent a unique opportunity to observe and quantify novel anomalies. Here, we present the first characterization and comparison of basal metabolism in three mouse models of NDDs, namely, Down syndrome (DS; Dp(16)Yey/+ mice), 16p11.2 deletion syndrome (16pDel; 16p11.2df/+ mice), and fragile X syndrome [FXS; Fmr1 knock-out (KO) mice] and their wild-type (WT) counterparts. Using the Comprehensive Lab Animal Monitoring System (CLAMS) coupled to EchoMRI, as well as quantification of key plasma metabolites by liquid chromatography mass spectrometry (LC-MS), our in vivo study reveals that each mouse model expresses a unique metabolic signature that is sex-specific, independent of the amount of food consumed and minimally influenced by physical activity. In particular, we identify striking differences in body composition, respiratory exchange ratio (RER), caloric expenditure (CE), and concentrations of circulating plasma metabolites related to mitochondrial function. Providing novel insight into NDD-associated metabolic alterations is an essential prerequisite for future preclinical and clinical interventions.

Physiological, ultrastructural, biochemical, and molecular responses of glandless cotton to hexavalent chromium (Cr6+) exposure.

Glandless cotton can be grown to obtain cotton seeds free of toxic gossypol for use as both food and feed. However, they are not grown normally due to their lesser productivity and higher susceptibility to biotic stress. Great attention has been paid to biotic stresses rather than abiotic stresses on glandless cotton. Chromium (Cr) is a common pollutant of soil and considered a serious threat to plants due to its adverse effects on different functions. Although numerous studies are available on the toxicity of Cr6+ in various plants. However, its adverse effects and mechanism of toxicity in glandless cotton can seldom be found in the literature. This study examined the Cr6+ effect on glandless cotton in comparison to glanded cotton. Four pairs of glanded and glandless cotton near-isogenic lines (NILs) were exposed to different doses (0, 10, 50, and 100 µM/L) of Cr6+ for seven days, and biochemical, physiological, molecular, and ultrastructure changes were observed, which were significantly affected by Cr6+ at high concentrations in all NILs. The effect of Cr6+ on ionic contents shows the same trend in glanded and glandless NILs except for manganese (Mn2+) that show inhibition in glandless (ZMS-12w and Coker-312w) and enhance in the glanded NIL (ZMS-17). The gene expression of superoxide dismutase (SOD) and peroxidase (POD) revealed similar trends as enzyme activities in glandless NILs. The principal component analysis (PCA) and Agglomerative hierarchical clustering (AHC) results of all NILs from morpho-physiological traits, cluster ZMS-16, and ZMS-17 into Cr6+ sensitive group. While the glandless NILs have the potential to cope with the Cr toxicity by increasing the antioxidant enzyme activity and their gene expression. This study also revealed that Cr6+ tolerance in cotton is genotypic and has an independent mechanism in the root that not related to low gossypol.

Resemblance and Difference of Seedling Metabolic and Transporter Gene Expression in High Tolerance Wheat and Barley Cultivars in Response to Salinity Stress.

To elucidate inter-specific similarity and difference of tolerance mechanism against salinity stress between wheat and barley, high tolerant wheat cv. Suntop and sensitive cv. Sunmate and tolerant barley cv. CM72 were hydroponically grown in a greenhouse with 100 mM NaCl. Glutathione, secondary metabolites, and genes associated with Na+ transport, defense, and detoxification were examined to discriminate the species/cultivar difference in response to salinity stress. Suntop and CM72 displayed damage to a lesser extent than in Sunmate. Compared to Sunmate, both Suntop and CM72 recorded lower electrolyte leakage and reactive oxygen species (ROS) production, higher leaf relative water content, and higher activity of PAL (phenylalanine ammonia-lyase), CAD (cinnamyl alcohol dehydrogenase), PPO (polyphenol oxidase), SKDH (shikimate dehydrogenase), and more abundance of their mRNA under salinity stress. The expression of HKT1, HKT2, salt overly sensitive (SOS)1, AKT1, and NHX1 was upregulated in CM72 and Suntop, while downregulated in Sunmate. The transcription factor WRKY 10 was significantly induced in Suntop but suppressed in CM72 and Sunmate. Higher oxidized glutathione (GSSG) content was accumulated in cv. CM72 and Sunmate, but increased glutathione (GSH) content and the ratio of GSH/GSSG were observed in leaves and roots of Suntop under salinity stress. In conclusion, glutathione homeostasis and upregulation of the TaWRKY10 transcription factor played a more important role in wheat salt-tolerant cv. Suntop, which was different from barley cv. CM72 tolerance to salinity stress. This new finding could help in developing salinity tolerance in wheat and barley cultivars.

Physiological and molecular mechanisms of cobalt and copper interaction in causing phyto-toxicity to two barley genotypes differing in Co tolerance.

The combined effects of cobalt (Co) and copper (Cu) in their toxicity to plants is poorly studied although these two metals co-exist commonly in soil. In this study, a hydroponic experiment was carried out to investigate the effect of longer exposure of two barley genotypes differing in Co tolerance to the combined Co and Cu stress. The results confirmed the previous findings that Co accumulation in plant tissues was reduced by Cu presence, while Cu accumulation was stimulated by Co presence. Moreover, both single and combined treatments of Co and Cu reduced the mineral (Mn, Zn and K) uptake. Co and Cu applied alone or in combination at rate of 50 µM resulted in the significant reduction of plant growth and increase of oxidative stress (ROS and MDA), and meanwhile the capacity of scavenging active oxygen species (AOS) was enhanced, reflected by increased phytochelatin (PC) and glutathione (GSH and GSSG) content, as well as expression of the related genes (HvPCS1 and HvGR1). Yan66, a Co tolerant genotype was less affected in oxidative stress, and had higher AOS scavenging capacity in comparison with Ea52, a Co sensitive one. Among three HvSOD isoforms, only HvFeSOD expression was up-regulated in the combined treatment relative to control as well as the treatment of Co or Cu alone, while HvCuZnSOD and HvMnSOD were down-regulated and unaffected, respectively. In addition, the expressions of metal transporter genes (HvHMA2, HvHMA3 and HvHMA5) varied with genotype and metal treatments, with the extent being greater in Yan66 on the whole. The results suggest that upon longer exposure to the combined stress of Co and Cu, the greater phyto-toxicity than each element alone is associated with more Cu accumulation stimulated by Co and that, the higher regulation of transporter genes observed in Yan66 could in part explain for its higher metal tolerance ability.

Dysregulation of the Tryptophan Pathway Evidences Gender Differences in COPD.

Increased activity of indoleamine 2,3-dioxygenase (IDO) and tryptophan hydroxylase (TPH) have been reported in individuals with chronic obstructive pulmonary disease (COPD). We therefore investigated the effect of gender stratification upon the observed levels of tryptophan metabolites in COPD. Tryptophan, serotonin, kynurenine, and kynurenic acid were quantified in serum of never-smokers (n = 39), smokers (n = 40), COPD smokers (n = 27), and COPD ex-smokers (n = 11) by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The individual metabolite associations with lung function, blood, and bronchoalveolar lavage (BAL) immune-cell composition, as well as chemokine and cytokine levels, were investigated. Stratification by gender and smoking status revealed that the observed alterations in kynurenine and kynurenic acid, and to a lesser extent serotonin, were prominent in males, irrespective of COPD status (kynurenine p = 0.005, kynurenic acid p = 0.009, and serotonin p = 0.02). Inferred serum IDO activity and kynurenine levels decreased in smokers relative to never-smokers (p = 0.005 and p = 0.004, respectively). In contrast, inferred tryptophan hydroxylase (TPH) activity and serotonin levels showed an increase with smoking that reached significance with COPD (p = 0.01 and p = 0.01, respectively). Serum IDO activity correlated with blood CXC chemokine ligand 9 (CXCL9, p = 0.0009, r = 0.93) and chemokine (C-C motif) ligand 4 (CCL4.(p = 0.04, r = 0.73) in female COPD smokers. Conversely, serum serotonin levels correlated with BAL CD4+ T-cells (%) (p = 0.001, r = 0.92) and CD8+ T-cells (%) (p = 0.002, r = -0.90) in female COPD smokers, but not in male COPD smokers (p = 0.1, r = 0.46 and p = 0.1, r = -0.50, respectively). IDO- and TPH-mediated tryptophan metabolites showed gender-based associations in COPD, which were primarily driven by smoking status.

Metabolomic Analysis of Yeast and Human Cells: Latest Advances and Challenges.

Liquid chromatography-mass spectrometry (LC-MS) based nontargeted metabolomics has been applied to a wide range of biological samples and can provide information on thousands of compounds. However, reliable identification of the compounds remains a challenge affecting result interpretation. In this protocol, we describe comparable yeast cell and whole blood metabolome sample preparation for extracting similar compound groups, and we present a LC-MS method using the all ion fragmentation (AIF) approach for the purposes of increasing accuracy in metabolite annotation. Our method enables database-dependent targeted as well as nontargeted metabolomics analysis from the same data acquisition, while simultaneously improving the accuracy in metabolite identification to increase the quality of the resulting biological information.

Comparison of underivatized silica and zwitterionic sulfobetaine hydrophilic interaction liquid chromatography stationary phases for global metabolomics of human plasma.

To increase metabolome coverage in global LC-MS metabolomics, often both reversed-phase liquid chromatography (RPLC) and hydrophilic-interaction liquid chromatography (HILIC) are implemented in parallel. However, there is a lack of consensus in the literature on the best HILIC stationary phase to employ for global metabolomics of human biological fluids. The objective of this study was to compare in detail the performance of two commonly employed HILIC phases: zwitterionic sulfobetaine ZIC-HILIC stationary phase and an underivatized silica HILIC stationary phase. During method development, the effect of salt concentration in the mobile phase was also investigated, and 5 mM ammonium acetate was selected. The stationary phases were evaluated using a mixture of 37 polar standards covering a range of logP values (-10 to 3.73), molecular weights (59-776 Da), charges (15 anions, 11 cations, and 11 neutral) as well as 17 lipid standards to understand phospholipid behaviour on the two stationary phases. The criteria used for the comparison included the quality of the chromatographic peak shape, adequate analyte retention, peak separation capability, and metabolite coverage. The zwitterionic ZIC-HILIC column provided better chromatographic performance over the silica stationary phase with 14 standards achieving good quality peaks compared to the 7 with the silica column. Only 2 standards were undetected with the ZIC-HILIC column compared to the 14 undetected with the silica column. In human plasma, 1966 and 1650 metabolites were observed on the ZIC-HILIC column in positive and negative electrospray ionization (ESI) respectively. On the silica HILIC column, 1773 and 2028 metabolites were observed in positive and negative ESI respectively, showing comparable performance of the two phases. Next, the effect of adding 10 mM ammonium phosphate to the samples to improve the analyte peak shape and metabolite coverage was investigated for both ZIC-HILIC and silica HILIC. In contrast with recently reported results for pZIC-HILIC, there was no clear evidence that ammonium phosphate addition was beneficial for human plasma samples. In conclusion, ZIC-HILIC provided better chromatographic performance for polar plasma metabolomics than underivatized silica in terms of chromatographic peak shape and chromatographic resolution, while maintaining comparable metabolite coverage. The addition of ammonium phosphate to human plasma was not beneficial for either of the two stationary phases.

LC-MS-Based Metabolomics of Biofluids Using All-Ion Fragmentation (AIF) Acquisition.

The field of liquid chromatography-mass spectrometry (LC-MS)-based nontargeted metabolomics has advanced significantly and can provide information on thousands of compounds in biological samples. However, compound identification remains a major challenge, which is crucial in interpreting the biological function of metabolites. Herein, we present a LC-MS method using the all-ion fragmentation (AIF) approach in combination with a data processing method using an in-house spectral library. For the purposes of increasing accuracy in metabolite annotation, up to four criteria are used: (1) accurate mass, (2) retention time, (3) MS/MS fragments, and (4) product/precursor ion ratios. The relative standard deviation between ion ratios of a metabolite in a biofluid vs. its analytical standard is used as an additional metric for confirming metabolite identity. Furthermore, we include a scheme to distinguish co-eluting isobaric compounds. Our method enables database-dependent targeted as well as nontargeted metabolomics analysis from the same data acquisition, while simultaneously improving the accuracy in metabolite identification to increase the quality of the resulting biological information.

Adipocyte Expression of SLC19A1 Links DNA Hypermethylation to Adipose Tissue Inflammation and Insulin Resistance.

Context: Insulin resistance (IR) is promoted by a chronic low-grade inflammation in white adipose tissue (WAT). The latter might be regulated through epigenetic mechanisms such as DNA methylation. The one carbon cycle (1CC) is a central metabolic process governing DNA methylation. Objective: To identify adipocyte-expressed 1CC genes linked to WAT inflammation, IR, and their causal role. Design: Cohort study. Setting: Outpatient academic clinic. Participants: Obese and nonobese subjects. Methods: Gene expression and DNA methylation arrays were performed in subcutaneous WAT and isolated adipocytes. In in vitro differentiated human adipocytes, gene knockdown was achieved by small interfering RNA, and analyses included microarray, quantitative polymerase chain reaction, DNA methylation by enzyme-linked immunosorbent assay and pyrosequencing, protein secretion by enzyme-linked immunosorbent assay, targeted metabolomics, and luciferase reporter and thermal shift assays. Main Outcome Measures: Effects on adipocyte inflammation. Results: In adipocytes from obese individuals, global DNA hypermethylation was associated positively with gene expression of proinflammatory pathways. Among the 1CC genes, IR in vivo and proinflammatory gene expression in WAT were most strongly and inversely associated with SLC19A1, a gene encoding a membrane folate carrier. SLC19A1 knockdown in human adipocytes perturbed intracellular 1CC metabolism, induced global DNA hypermethylation, and increased expression of proinflammatory genes. Several CpG loci linked SLC19A1 to inflammation; validation studies were focused on the chemokine C-C motif chemokine ligand 2 (CCL2) in which methylation in the promoter (cg12698626) regulated CCL2 expression and CCL2 secretion through altered transcriptional activity. Conclusions: Reduced SLC19A1 expression in human adipocytes induces DNA hypermethylation, resulting in increased expression of specific proinflammatory genes, including CCL2. This constitutes an epigenetic mechanism that might link dysfunctional adipocytes to WAT inflammation and IR.

Development of a Liquid Chromatography-High Resolution Mass Spectrometry Metabolomics Method with High Specificity for Metabolite Identification Using All Ion Fragmentation Acquisition.

High-resolution mass spectrometry (HRMS)-based metabolomics approaches have made significant advances. However, metabolite identification is still a major challenge with significant bottleneck in translating metabolomics data into biological context. In the current study, a liquid chromatography (LC)-HRMS metabolomics method was developed using an all ion fragmentation (AIF) acquisition approach. To increase the specificity in metabolite annotation, four criteria were considered: (i) accurate mass (AM), (ii) retention time (RT), (iii) MS/MS spectrum, and (iv) product/precursor ion intensity ratios. We constructed an in-house mass spectral library of 408 metabolites containing AMRT and MS/MS spectra information at four collision energies. The percent relative standard deviations between ion ratios of a metabolite in an analytical standard vs sample matrix were used as an additional metric for establishing metabolite identity. A data processing method for targeted metabolite screening was then created, merging m/z, RT, MS/MS, and ion ratio information for each of the 413 metabolites. In the data processing method, the precursor ion and product ion were considered as the quantifier and qualifier ion, respectively. We also included a scheme to distinguish coeluting isobaric compounds by selecting a specific product ion as the quantifier ion instead of the precursor ion. An advantage of the current AIF approach is the concurrent collection of full scan data, enabling identification of metabolites not included in the database. Our data acquisition strategy enables a simultaneous mixture of database-dependent targeted and nontargeted metabolomics in combination with improved accuracy in metabolite identification, increasing the quality of the biological information acquired in a metabolomics experiment.

Metabolomics analysis identifies sex-associated metabotypes of oxidative stress and the autotaxin-lysoPA axis in COPD.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease and a leading cause of mortality and morbidity worldwide. The aim of this study was to investigate the sex dependency of circulating metabolic profiles in COPD.Serum from healthy never-smokers (healthy), smokers with normal lung function (smokers), and smokers with COPD (COPD; Global Initiative for Chronic Obstructive Lung Disease stages I-II/A-B) from the Karolinska COSMIC cohort (n=116) was analysed using our nontargeted liquid chromatography-high resolution mass spectrometry metabolomics platform.Pathway analyses revealed that several altered metabolites are involved in oxidative stress. Supervised multivariate modelling showed significant classification of smokers from COPD (p=2.8×10-7). Sex stratification indicated that the separation was driven by females (p=2.4×10-7) relative to males (p=4.0×10-4). Significantly altered metabolites were confirmed quantitatively using targeted metabolomics. Multivariate modelling of targeted metabolomics data confirmed enhanced metabolic dysregulation in females with COPD (p=3.0×10-3) relative to males (p=0.10). The autotaxin products lysoPA (16:0) and lysoPA (18:2) correlated with lung function (forced expiratory volume in 1 s) in males with COPD (r=0.86; p<0.0001), but not females (r=0.44; p=0.15), potentially related to observed dysregulation of the miR-29 family in the lung.These findings highlight the role of oxidative stress in COPD, and suggest that sex-enhanced dysregulation in oxidative stress, and potentially the autotaxin-lysoPA axis, are associated with disease mechanisms and/or prevalence.

Metabolomics analysis identifies different metabotypes of asthma severity.

In this study, we sought to determine whether asthma has a metabolic profile and whether this profile is related to disease severity.We characterised the serum from 22 healthy individuals and 54 asthmatics (12 mild, 20 moderate, 22 severe) using liquid chromatography-high-resolution mass spectrometry-based metabolomics. Selected metabolites were confirmed by targeted mass spectrometry assays of eicosanoids, sphingolipids and free fatty acids.We conclusively identified 66 metabolites; 15 were significantly altered with asthma (p≤0.05). Levels of dehydroepiandrosterone sulfate, cortisone, cortisol, prolylhydroxyproline, pipecolate and N-palmitoyltaurine correlated significantly (p<0.05) with inhaled corticosteroid dose, and were further shifted in individuals treated with oral corticosteroids. Oleoylethanolamide increased with asthma severity independently of steroid treatment (p<0.001). Multivariate analysis revealed two patterns: 1) a mean difference between controls and patients with mild asthma (p=0.025), and 2) a mean difference between patients with severe asthma and all other groups (p=1.7×10-4). Metabolic shifts in mild asthma, relative to controls, were associated with exogenous metabolites (e.g. dietary lipids), while those in moderate and severe asthma (e.g. oleoylethanolamide, sphingosine-1-phosphate, N-palmitoyltaurine) were postulated to be involved in activating the transient receptor potential vanilloid type 1 (TRPV1) receptor, driving TRPV1-dependent pathogenesis in asthma.Our findings suggest that asthma is characterised by a modest systemic metabolic shift in a disease severity-dependent manner, and that steroid treatment significantly affects metabolism.

Unveiling differences between patients with acute coronary syndrome with and without ST elevation through fingerprinting with CE-MS and HILIC-MS targeted analysis.

Differences in the degree and severity of Acute Coronary Syndrome, associated to differences in the electrocardiogram, together with blood tests of biomarkers classify patients for diagnosis and treatment. Cases where the electrocardiogram and/or biomarkers are not conclusive still appear, and there is a need for complementary biomarkers for routine determinations. Metabolomics approaches with blind fingerprinting could reveal differences in metabolites, which must be confirmed by means of targeted determinations. CE-MS and HILIC-MS are well suited for the determination of highly polar compounds, like those from to the intermediate metabolism, altered due to acute stress induced by myocardial infarction. Serum from patients with ST-elevated and non-ST elevated myocardial infarction was collected at intensive care and emergency units, and fingerprinted with CE-MS. Data pretreatment and analysis showed up carnitine-related compounds and amino acids differentially present in both groups. Acylcarnitines and amino acids were then quantitatively measured with HILIC-MS-QqQ. The significance of the differences and the sensitivity/specificity of each compound were individually evaluated. The ratio of free carnitine to acylcarnitines, together with the ratios of acetylcarnitine to betaine, to threonine, and to citrulline, showed high significance and area under the curve in the respective receiver operating characteristic curves. This study opens new possibilities for defining new sets of biomarkers for refining the diagnosis of the patients with difficult classification.

Metabolite fingerprinting by capillary electrophoresis-mass spectrometry.

Changes in metabolite concentrations in response to specific diseases, treatments, diets, or other factors can be used to understand the complex mechanisms that control and regulate the human body and potentially detect the onset of disease prior to the observation of symptoms in a patient. Different analytical and chemometric platforms are necessary to detect as many metabolites as possible in different biological fluids. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) is a particularly attractive, although still not common, approach for metabolomics for the detection of mainly polar and ionic metabolites. Among its main features, CE provides the capability to separate complex mixtures with high resolution and minimum sample treatment. However, the routine, automated use of CE-MS is not without challenges. In this chapter we describe a well-tested method for fingerprinting serum and urine using CE-TOF-MS. We describe below a sensitive and quite robust method for metabolomics with CE-MS including sample treatment, separation conditions, instrumental setup, and identification of 76 metabolites in the profile. Useful advice for daily practice is also included for every step of the procedure.

Analytical protocols based on LC-MS, GC-MS and CE-MS for nontargeted metabolomics of biological tissues.

Invasive, site-specific metabolite information could be better obtained from tissues. Hence, highly sensitive mass spectrometry-based metabolomics coupled with separation techniques are increasingly in demand in clinical research for tissue metabolomics application. Applying these techniques to nontargeted tissue metabolomics provides identification of distinct metabolites. These findings could help us to understand alterations at the molecular level, which can also be applied in clinical practice as screening markers for early disease diagnosis. However, tissues as solid and heterogeneous samples pose an additional analytical challenge that should be considered in obtaining broad, reproducible and representative analytical profiles. This manuscript summarizes the state of the art in tissue (human and animal) treatment (quenching, homogenization and extraction) for nontargeted metabolomics with mass spectrometry.