Unknown Metabolites Research Articles

IMet: A Network-Based Computational Tool To Assist in the Annotation of Metabolites from Tandem Mass Spectra

Structural annotation of metabolites relies mainly on tandem mass spectrometry (MS/MS) analysis. However, approximately 90% of the known metabolites reported in metabolomic databases do not have annotated spectral data from standards. This situation has fostered the development of computational tools that predict fragmentation patterns in silico and compare these to experimental MS/MS spectra. However, because such methods require the molecular structure of the detected compound to be available for the algorithm, the identification of novel metabolites in organisms relevant for biotechnological and medical applications remains a challenge. Here, we present iMet, a computational tool that facilitates structural annotation of metabolites not described in databases. iMet uses MS/MS spectra and the exact mass of an unknown metabolite to identify metabolites in a reference database that are structurally similar to the unknown metabolite. The algorithm also suggests the chemical transformation that converts the known metabolites into the unknown one. As a proxy for the structural annotation of novel metabolites, we tested 148 metabolites following a leave-one-out cross-validation procedure or by using MS/MS spectra experimentally obtained in our laboratory. We show that for 89% of the 148 metabolites at least one of the top four matches identified by iMet enables the proper annotation of the unknown metabolites. To further validate iMet, we tested 31 metabolites proposed in the 2012-16 CASMI challenges. iMet is freely available at http://imet.seeslab.net .

Top-down Metabolomic Approaches for Nitrogen-Containing Metabolites.

Streamlining the processes that reveal heteroatom-containing metabolites and their biosynthetic genes is essential in integrated metabolomics studies. These metabolites are especially targeted for their potential pharmaceutical activities. By using a Fourier-transform ion cyclotron resonance-mass spectrometry (FTICR-MS) instrument, we provide top-down targeted metabolomic analyses using ultrahigh-resolution liquid chromatography-mass spectrometry (LC-MS), high-resolution matrix-assisted laser desorption/ionization (MALDI), and high-resolution imaging mass spectrometry (IMS) with 15N labeling of nitrogen-containing metabolites. In this study, we efficiently extract known and unknown chemicals and spatial information from the medicinal plant Catharanthus roseus, which sources several cancer drugs. The ultrahigh-resolution LC-MS analysis showed that the molecular formula of 65 N-metabolites were identified using the petals, peduncles, leaves, petioles, stems, and roots of the non- and 15N-labeled Catharanthus plants. The high resolution MALDI analysis showed the molecular formula of 64 N-metabolites using the petals, leaves, and stems of the non- and 15N-labeled Catharanthus. The chemical assignments using molecular formulas stored in databases identified known and unknown metabolites. The comparative analyses using the assigned metabolites revealed that most of the organ-specific ions are derived from unknown N-metabolites. The high-resolution IMS analysis characterized the spatial accumulation patterns of 32 N-metabolites using the buds, leaves, stems, and roots in Catharanthus. The comparative analysis using the non- and 15N-labeled IMS data showed the same spatial accumulation patterns of a non- and 15N-labeled metabolite in the organs, showing that top-down analysis can be performed even in IMS analysis.

Indirect Redox Transformations of Iron, Copper, and Chromium Catalyzed by Extremely Acidophilic Bacteria.

Experiments were carried out to examine redox transformations of copper and chromium by acidophilic bacteria (Acidithiobacillus, Leptospirillum, and Acidiphilium), and also of iron (III) reduction by Acidithiobacillus spp. under aerobic conditions. Reduction of iron (III) was found with all five species of Acidithiobacillus tested, grown aerobically on elemental sulfur. Cultures maintained at pH 1.0 for protracted periods displayed increasing propensity for aerobic iron (III) reduction, which was observed with cell-free culture liquors as well as those containing bacteria. At. caldus grown on hydrogen also reduced iron (III) under aerobic conditions, confirming that the unknown metabolite(s) responsible for iron (III) reduction were not (exclusively) sulfur intermediates. Reduction of copper (II) by aerobic cultures of sulfur-grown Acidithiobacillus spp. showed similar trends to iron (III) reduction in being more pronounced as culture pH declined, and occurring in both the presence and absence of cells. Cultures of Acidithiobacillus grown anaerobically on hydrogen only reduced copper (II) when iron (III) (which was also reduced) was also included; identical results were found with Acidiphilium cryptum grown micro-aerobically on glucose. Harvested biomass of hydrogen-grown At. ferridurans oxidized iron (II) but not copper (I), and copper (I) was only oxidized by growing cultures of Acidithiobacillus spp. when iron (II) was also included. The data confirmed that oxidation and reduction of copper were both mediated by acidophilic bacteria indirectly, via iron (II) and iron (III). No oxidation of chromium (III) by acidophilic bacteria was observed even when, in the case of Leptospirillum spp., the redox potential of oxidized cultures exceeded +900 mV. Cultures of At. ferridurans and A. cryptum reduced chromium (VI), though only when iron (III) was also present, confirming an indirect mechanism and contradicting an earlier report of direct chromium reduction by A. cryptum. Measurements of redox potentials of iron, copper and chromium couples in acidic, sulfate-containing liquors showed that these differed from situations where metals are not complexed by inorganic ligands, and supported the current observations of indirect copper oxido-reduction and chromium reduction mediated by acidophilic bacteria. The implications of these results for both industrial applications of acidophiles and for exobiology are discussed.

The influence of a chronic L-carnitine administration on the plasma metabolome of male Fischer344 rats.

L-carnitine has been advertised as a fat-lowering and performance-enhancing supplement, although scientific evidence for its effectiveness is lacking. The uptake of about 1-2 g of L-carnitine per day may result in the formation of metabolites like trimethylamine-N-oxide (TMAO), which in turn may be converted to potential carcinogens or promote the development of cardiovascular diseases. To assess whether an L-carnitine supplementation changes overall metabolism or causes the formation of previously unknown metabolites, we analyzed plasma samples from Fischer344 rats originating from a previous study using a multi-platform metabolomics approach comprising LC-MS/MS and GC×GC-MS methods. Despite an intake of up to 352mg L-carnitine/kg body weight/day for 1 year, plasma concentrations of only 29 out of 359 metabolites were significantly influenced, the induced concentration changes being often comparatively small. Nevertheless, a clear dose-response relationship and a substantial concentration increase were observed for TMAO, i.e. a tenfold higher TMAO level was measured in the high-dose group when compared to the control (2.5 versus 25.0μM). Although L-carnitine supplementation did not cause large changes in the plasma metabolome, a higher risk for cardiovascular disease due to chronically elevated TMAO plasma concentrations cannot be excluded.

Metabolism of Zearalenone and Its Major Modified Forms in Pigs.

The Fusarium mycotoxin zearalenone (ZEN) can be conjugated with polar molecules, like sugars or sulfates, by plants and fungi. To date, the fate of these modified forms of ZEN has not yet been elucidated in animals. In order to investigate whether ZEN conjugates contribute to the total ZEN exposure of an individual, ZEN (10 µg/kg b.w.) and equimolar amounts of two of its plant metabolites (ZEN-14-O-β-glucoside, ZEN-16-O-β-glucoside) and of one fungal metabolite (ZEN-14-sulfate) were orally administered to four pigs as a single bolus using a repeated measures design. The concentrations of ZEN, its modified forms and its mammalian metabolites ZEN-14-glucuronide, α-zearalenol (α-ZEL) and α-ZEL-14-glucuronide in excreta were analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) based methods. The biological recovery of ZEN in urine was 26% ± 10%, the total biological recovery in excreta was 40% ± 8%. Intact ZEN-14-sulfate, ZEN-14-O-β-glucoside and ZEN-16-O-β-glucoside were neither detected in urine nor in feces. After ZEN-14-sulfate application, 19% ± 5% of the administered dose was recovered in urine. In feces, no ZEN metabolites were detected. The total biological recoveries of ZEN-14-O-β-glucoside and ZEN-16-O-β-glucoside in the form of their metabolites in urine were 19% ± 11% and 13% ± 7%, respectively. The total biological recoveries in urine and feces amounted to 48% ± 7% and 34 ± 3%. An explanation for the low biological recoveries could be extensive metabolization by intestinal bacteria to yet unknown metabolites. In summary, ZEN-14-sulfate, ZEN-14-O-β-glucoside, and ZEN-16-O-β-glucoside were completely hydrolyzed in the gastrointestinal tract of swine, thus contributing to the overall toxicity of ZEN.

Alteration of neurotransmission and skeletogenesis in sea urchin Arbacia lixula embryos exposed to copper oxide nanoparticles

The extensive use of copper oxide nanoparticles (CuO NPs) in many applications has raised concerns over their toxicity on environment and human health. Herein, the embryotoxicity of CuO NPs was assessed in the black sea urchin Arbacia lixula, an intertidal species commonly present in the Mediterranean. Fertilized eggs were exposed to 0.7, 10 and 20ppb of CuO NPs, until pluteus stage. Interferences with the normal neurotransmission pathways were observed in sea urchin embryos. In detail, evidence of cholinergic and serotoninergic systems affection was revealed by dose-dependent decreased levels of choline and N-acetyl serotonin, respectively, measured by nuclear magnetic resonance (NMR)-based metabolomics, applied for the first time to our knowledge on sea urchin embryos. The metabolic profile also highlighted a significant CuO NP dose-dependent increase of glycine, a component of matrix proteins involved in the biomineralization process, suggesting perturbed skeletogenesis accordingly to skeletal defects in spicule patterning observed previously in the same sea urchin embryos. However, the expression of skeletogenic genes, i.e. SM30 and msp130, did not differ among groups, and therefore altered primary mesenchyme cell (PMC) migration was hypothesized. Other unknown metabolites were detected from the NMR spectra, and their concentrations found to be reflective of the CuO NP exposure levels. Overall, these findings demonstrate the toxic potential of CuO NPs to interfere with neurotransmission and skeletogenesis of sea urchin embryos. The integrated use of embryotoxicity tests and metabolomics represents a highly sensitive and effective tool for assessing the impact of NPs on aquatic biota.

In vivo metabolite identification of acotiamide in rats using ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry.

Acotiamide hydrochloride (ACT) is a drug used for the treatment of functional dyspepsia. Understanding which metabolites are likely to be formed in vivo is essential for interpreting pharmacology, pharmacokinetic and toxicology data. The metabolism of ACT has been investigated using a specific and sensitive liquid chromatography positive ion electrospray ionization high-resolution tandem mass spectrometry method. In vivo samples including rat plasma, urine and feces were collected separately after dosing healthy Sprague-Dawley rats at a dose of 20 mg kg -1 ACT at different time points up to 24 h. The metabolites were enriched by optimized sample preparation involving protein precipitation using acetonitrile followed by solid-phase extraction. The mass defect filter technique was used for better detection of both predicted and unexpected drug metabolites with the majority of interference ions removed. The structural elucidation of the metabolites was performed by comparing their [M + H]+ ions and their product ions with those of the parent drug. As a result, a total of seven hitherto unknown metabolites were characterized from the biosamples. The only phase I metabolite detected was N-despropyl acotiamide, whereas six phase II glucuronide conjugate metabolites were identified.

Insights into the lifestyle of uncultured bacterial natural product factories associated with marine sponges

The as-yet uncultured filamentous bacteria "Candidatus Entotheonella factor" and "Candidatus Entotheonella gemina" live associated with the marine sponge Theonella swinhoei Y, the source of numerous unusual bioactive natural products. Belonging to the proposed candidate phylum "Tectomicrobia," Candidatus Entotheonella members are only distantly related to any cultivated organism. The Ca E. factor has been identified as the source of almost all polyketide and modified peptides families reported from the sponge host, and both Ca Entotheonella phylotypes contain numerous additional genes for as-yet unknown metabolites. Here, we provide insights into the biology of these remarkable bacteria using genomic, (meta)proteomic, and chemical methods. The data suggest a metabolic model of Ca Entotheonella as facultative anaerobic, organotrophic organisms with the ability to use methanol as an energy source. The symbionts appear to be auxotrophic for some vitamins, but have the potential to produce most amino acids as well as rare cofactors like coenzyme F420 The latter likely accounts for the strong autofluorescence of Ca Entotheonella filaments. A large expansion of protein families involved in regulation and conversion of organic molecules indicates roles in host-bacterial interaction. In addition, a massive overrepresentation of members of the luciferase-like monooxygenase superfamily points toward an important role of these proteins in Ca Entotheonella. Furthermore, we performed mass spectrometric imaging combined with fluorescence in situ hybridization to localize Ca Entotheonella and some of the bioactive natural products in the sponge tissue. These metabolic insights into a new candidate phylum offer hints on the targeted cultivation of the chemically most prolific microorganisms known from microbial dark matter.

Preliminary 1H NMR-based Metabolomics Analysis to Explore The Link Between SrtA Function and Cariogenicity of S.mutans

Objectives: Streptococcus mutans is the major pathogen associated with caries, and its adhesion to tooth surfaces is the first step in cariogenesis. Sortase A (SrtA) is a key enzyme responsible for anchoring proteins in the cell wall; deleting the gene reduces the cariogenicity of the pathogen. To gain insight into how SrtA supports cariogenicity, we performed a metabonomics study based on 1H nuclear magnetic resonance in which we compared extracellular metabolites of wild-type S. mutans UA159 and a SrtA-deficient strain. Methods: Metabolite differences between strains were identified using a combination of principal component analysis and orthogonality partial least squares discriminant analysis. Results: Several differences were identified, corresponding mostly to unknown metabolites. Some amino acids such as leucine and valine (δ0.92-1.20 ppm), lactic acid (δ1.28 ppm), oxoglutaric acid (δ3.00 ppm), and glycine (δ3.60 ppm) were found to differ between the strains. Conclusions: These results highlight the feasibility of using metabonomics for functional analysis of genes and correlation with clinical effects.

Metabolomic Profiling Defines Distinct Metabolic Signature Associated with FLT3/ITD AML

Abstract AML is a hematological disorder resulting from proliferation and expansion of malignant myeloid cells. Clinical outcome for AML remains dismal despite intensive therapy in part due to the disease heterogeneity with various cytogenetic and molecular lesions. Fms-Like Tyrosine Kinase-3 (FLT3) is a receptor tyrosine kinase expressed hematopoietic stem/progenitor cells. Activating mutations of FLT3 gene due to internal tandem duplication of the juxtamembrane domain coding sequence (FLT3/ITD) causes autonomous cellular proliferations leading to disease progression. Metabolomic profiling has been successfully utilized to identify metabolic alterations in hematological disorders. However, no studies on metabolic alterations associated with pediatric AML have been reported at this time. In this study we propose to establish the metabolomic landscape in pediatric AML patients and identify differential expression of metabolites based on FLT3/ITD status. Cellular and plasma metabolomics profile was generated from 32 matching diagnostic material from 16 patients with and without FLT3/ITD (N=8 for each cohort and each sample was run in duplicate) treated on COG-AAML0531 study. Global metabolomics profiling was performed on a Thermo Q-Exactive Oribtrap mass spectrometer with Dionex UHPLC and autosampler. All samples were analyzed in duplicate in positive and negative heated electrospray ionization with a mass resolution of 35,000 at m/z 200 as separate injections. Separation was achieved on an ACE 18-pfp 100 x 2.1 mm, 2 µm column with mobile phase A as 0.1% formic acid in water and mobile phase B as acetonitrile. This is a polar embedded stationary phase that provides comprehensive coverage, but does have some limitation is the coverage of very polar species. The flow rate was 350 µL/min with a column temperature of 25¡C. 4 µL was injected for negative ions and 2 µL for positive ions. Statistical analysis was performed using MetaboAnalyst software using all metabolites (known and unknown) as well as only the annotated metabolites. Univariate analysis was performed by volcano plot and Multivariate analysis was performed using PCA, PLS-DA and OPLS-DA. Total of 2966 plasma metabolome (779 negative and 2187 positive) and 1742 (227 negative and 1515 positive) cellular metabolome features were identified. All subsequent data analyses were normalized to the sum of metabolites for each sample. Comparison of the cellular metabolome in patients with and without FLT3/ITD identified 12 known and 135 unknown metabolites that were significantly different between two cohorts (p<0.05). Similar comparison of the plasma metabolome identified19 known and 300 unknown metabolites in the patient with and without FLT3/ITD (top results are shown in Fig.1). Orthogonal partial least squares-discriminant analysis (OPLS-DA) showed clear separation between the 2 groups (Fig.2). Some of the top known plasma metabolites (p<0.01) differentiating patients by FLT3 status include guanine, pyrimidine-2-3dicarboxylate, acetylglycine, acetyl-L-alanine, aminobutonate gaba, L-carnitine, methyl-2 oxovaleric acid, asparagine, acetyl arginine, Hydroxydecanoic acid, cysteic acid and glycocholic acid. Within leukemic cells top metabolites differentiating between FLT3 status included actyly carnitine, adenosine monophosphate, hypoxanthine, diaminohepatanedioate, guanine and sphingosine. Metaboanalyst Pathway analysis module mapped the differentiating metabolites to aminoacyl-tRNA biosynthesis, Glycerophospholipid metabolism, Cysteine and methionine metabolism, Pantothenate and CoA biosynthesis, Purine metabolism. This pilot study defines distinct metabolomics signature associated with genomic subtype of AML (FLT3/ITD). As metabolomics provides an insight into the ultimate metabolic destination of normal and malignant hematopoiesis, it has a potential to provide a unique insight into the altered metabolic pathways in AML and identify pathways and networks that might be shared by varied genomic subtypes of AML. Such data can help merge rare genomic variants based on shared metabolic signatures and more appropriately guide directed therapies. Disclosures No relevant conflicts of interest to declare.

Cellular Metabolomic Profiling in AML Cell Lines Is Linked to Differences in in Vitro Cytarabine Chemosensitivity

Background: AML is a hematological disorder resulting from proliferation and expansion of malignant myeloid cells. Expansion of malignant cells within the bone marrow and bloodstream causes severe clinical consequences. AML is a clinically challenging disease as many chemotherapeutic agents recommended in current guidelines have narrow therapeutic ranges and significant interpatient variation in treatment response. This can lead to severe therapeutic complications that are potentially fatal if concentrations are too high or failure to achieve treatment response if concentrations are too low. There is an ongoing search for predictive biomarkers to improve appropriate selection and dosing of chemotherapeutic agents. The emerging field of metabolomics has yielded promising results in the pursuit of precision medicine. However, there is still a gap in our knowledge of how metabolomics relate to treatment response to anti-leukemic agents for AML treatment. In this study, we propose to perform global metabolomics profiling of AML cell lines representing different risk groups to identify metabolic biomarkers predictive of in vitro cytotoxicity of most commonly used anti-leukemic agents.Methods: We performed global metabolomics on eight AML cell lines (MV4.11, KG-1, HL-60, Kasumi-1, AML-193, ME1, MOLM16, THP-1) using liquid chromatography high resolution mass spectrometry (LC-HRMS). Univariate and multivariate analyses were performed on the metabolite peak intensity values from LC-HRMS using MetaboAnalyst. Each cell line was treated with varying concentrations of cytarabine. Cell viability was then determined using MTT cell survival assays. Area under the cell survival curve (AUC) and IC50 values as well as correlation between metabolites and in vitro cytotoxicity were conducted using GraphPad Prism software.Results: A total of 2344 metabolites were detected in the global metabolome for the 6 cell lines. The metabolomes were filtered by removing metabolites present in fewer than 80% of cells or a signal below the cutoff threshold. Further analysis was conducted on the filtered dataset of 1634 metabolites. Since not all the metabolites detected have been successfully annotated, we performed an additional analysis with the dataset filtered to only include 197 annotated metabolites We classified cell lines into sensitive vs. resistant based on in vitro cytotoxicity. As shown in Figure 1, we observed significant differences in metabolic profiles (23 differentially expressed metabolites) between cytarabine sensitive vs. resistant cell lines. Multivariate analysis showed clear separation between sensitive and resistant groups, also shown in Figure 1.Through Spearman correlation analysis, we identified 6 unique known metabolites with significant correlation with cytotoxic AUC values for cytarabine. Of these known metabolites, one also showed significant differential expression between the sensitivity groups in the filtered dataset (Guanosine). 39 currently unknown metabolites also showed significant correlation with cytotoxic AUC values for cytarabine, with 9 metabolites also showing significant differential expression between the sensitivity groups.We expanded our correlation analysis to include an additional three commonly used chemotherapeutic agents in AML treatment regimens (clofarabine, doxorubicin and etoposide). We identified 12 unique known metabolites with significant correlation with drug sensitivity (6 for clofarabine, 6 for doxorubicin, 0 for etoposide). For unknown metabolites, 237 also showed significant correlation with cytotoxic AUC values among the four chemotherapeutic agents.Conclusion: Overall, our results demonstrate that metabolomics differences contribute towards drug resistance. In addition, it could potentially identify predictive biomarkers for chemosensitivity to various anti-leukemic drugs. Our results provide opportunity to further explore these metabolites in patient samples for association with clinical response. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interstitial Cystitis-Associated Urinary Metabolites Identified by Mass-Spectrometry Based Metabolomics Analysis.

This study on interstitial cystitis (IC) aims to identify a unique urine metabolomic profile associated with IC, which can be defined as an unpleasant sensation including pain and discomfort related to the urinary bladder, without infection or other identifiable causes. Although the burden of IC on the American public is immense in both human and financial terms, there is no clear diagnostic test for IC, but rather it is a disease of exclusion. Very little is known about the clinically useful urinary biomarkers of IC, which are desperately needed. Untargeted comprehensive metabolomic profiling was performed using gas-chromatography/mass-spectrometry to compare urine specimens of IC patients or health donors. The study profiled 200 known and 290 unknown metabolites. The majority of the thirty significantly changed metabolites before false discovery rate correction were unknown compounds. Partial least square discriminant analysis clearly separated IC patients from controls. The high number of unknown compounds hinders useful biological interpretation of such predictive models. Given that urine analyses have great potential to be adapted in clinical practice, research has to be focused on the identification of unknown compounds to uncover important clues about underlying disease mechanisms.

FluxFix: automatic isotopologue normalization for metabolic tracer analysis.

BackgroundIsotopic tracer analysis by mass spectrometry is a core technique for the study of metabolism. Isotopically labeled atoms from substrates, such as [13C]-labeled glucose, can be traced by their incorporation over time into specific metabolic products. Mass spectrometry is often used for the detection and differentiation of the isotopologues of each metabolite of interest. For meaningful interpretation, mass spectrometry data from metabolic tracer experiments must be corrected to account for the naturally occurring isotopologue distribution. The calculations required for this correction are time consuming and error prone and existing programs are often platform specific, non-intuitive, commercially licensed and/or limited in accuracy by using theoretical isotopologue distributions, which are prone to artifacts from noise or unresolved interfering signals.ResultsHere we present FluxFix (http://fluxfix.science), an application freely available on the internet that quickly and reliably transforms signal intensity values into percent mole enrichment for each isotopologue measured. ‘Unlabeled’ data, representing the measured natural isotopologue distribution for a chosen analyte, is entered by the user. This data is used to generate a correction matrix according to a well-established algorithm. The correction matrix is applied to labeled data, also entered by the user, thus generating the corrected output data. FluxFix is compatible with direct copy and paste from spreadsheet applications including Excel (Microsoft) and Google sheets and automatically adjusts to account for input data dimensions. The program is simple, easy to use, agnostic to the mass spectrometry platform, generalizable to known or unknown metabolites, and can take input data from either a theoretical natural isotopologue distribution or an experimentally measured one.ConclusionsOur freely available web-based calculator, FluxFix (http://fluxfix.science), quickly and reliably corrects metabolic tracer data for natural isotopologue abundance enabling faster, more robust and easily accessible data analysis.

Measuring fecal testosterone metabolites in spotted hyenas: Choosing the wrong assay may lead to erroneous results

ABSTRACTEnzyme-immunoassays (EIA) that detect fecal testosterone metabolites (fTM) are powerful tools to monitor gonadal activity non-invasively. However, a challenge with testosterone EIAs might be their potential for cross-reactivities with structurally similar glucocorticoid metabolites. Therefore, we aimed to verify the capability of four different testosterone EIAs to monitor fTM without reflecting changes in adrenocortical activity in spotted hyenas by analyzing fecal samples following testosterone and ACTH challenge tests. We demonstrated that none of the testosterone EIAs is appropriate to measure fTM as all of them showed substantial cross-reactivities to unknown metabolites. Our study underlines the importance of validating androgen EIAs.

Abstract 13814: DMGV is a Novel Marker of Liver Fat and Predicts Future Development of Type 2 Diabetes

Background: Unbiased “non-targeted” metabolite profiling techniques hold considerable promise for biomarker and pathway discovery, though there have been few successful applications to human disease. We aimed to uncover novel biomarkers of non-alcoholic fatty liver disease (NAFLD) and cardiometabolic disease using non-targeted metabolomic profiling in well-phenotyped human cohorts. Methods: Non-targeted metabolite profiling was performed using hydrophilic interaction chromatography on a UHPLC Q-Exactive hybrid orbitrap LC/MS system. We searched HMDB, Metlin, Chemspider, and Pubchem databases using a 5-ppm mass tolerance for potential identifications of unknown metabolites. Following unambiguous metabolite identification, a high-throughput targeted metabolite assay was generated using a triple quadrupole MS. Stepwise logistic regression was used to determine the association of metabolites with clinical traits and outcomes. Results: The non-targeted platform was applied first to a discovery cohort of 1066 Framingham Heart Study (FHS) Gen 3 participants, of whom 470 underwent a CT scan as part of a comprehensive assessment of adipose depots, including liver fat. An unknown metabolite with a mass/charge ratio of 202.1185 had the strongest association with CT-defined liver fat, which was quantified using the liver-to-phantom ratio (P=1.16E-10, adjusting for age, sex, smoking, alcohol consumption, HDL and triglyceride concentration, HOMA-IR, hypertension, and BMI). Following structural elucidation by LC-MS and the synthesis of a chemical standard, we confirmed the peak identity as α-keto-δ-(N G N G -dimethylguanidino)-valeric acid (DMGV). DMGV was significantly associated with non-alcoholic steatohepatitis (NASH), in a cohort of 36 patients with biopsy-proven NASH and 36 age, sex, and BMI-matched controls (OR=1.96, P=0.011, conditionally adjusted for age, sex, and BMI). Further, DMGV was predictive of future type 2 diabetes mellitus (T2D) in 196 cases of incident diabetes and 126 controls in the Malmo Diet and Cancer Study (OR=1.56, P=8.6E-4, adjusting for age, sex, fasting glucose, and BMI; mean follow-up 12.8 years). Conclusions: DMGV is a novel marker of NAFLD and NASH, and is a predictor of future T2D.

Abstract 12037: Metabolite Profiles Associated With Variation in Visceral Adiposity: The Multi-Ethnic Study of Atherosclerosis

Introduction: Understanding the association between serum metabolites and variation in visceral adipose tissue (VAT) content could provide novel insights into the pathogenesis and prevention of obesity and obesity-associated diseases. Methods: We analyzed data from participants in the Multi-Ethnic Study of Atherosclerosis who underwent untargeted metabolomics profiling of blood serum collected between 2000-2002 by proton nuclear magnetic resonance ( 1 H NMR) spectroscopy on a Bruker DRX600 spectrometer at 600 MHz using a standard 1-D spectrum and subsequent assessment of VAT area (cm 2 ) between 2002-2005 by computed tomography. Multivariable linear regression was used to evaluate the associations between NMR metabolic features and VAT area. Metabolites were identified with spiking and peak connectivity and with spectral library verification. A false discovery rate threshold of 1% was applied for statistical significance. Results: 1,102 participants were included with median age 63 years; 52% male; 40% white; median BMI 27 kg/m 2 . 30,590 metabolomic spectral features were evaluated. After multivariable adjustment for age, sex, race, data acquisition phase (batch 1 or 2), socioeconomic status score, smoking, physical activity, and body mass index, 2,113 spectral variables representing 14 distinct metabolites were significantly associated with VAT area ( Figure ). After additional adjustment for fasting plasma glucose or diabetes status, all metabolites except glycerol remained statistically significant. Conclusions: Using an untargeted metabolomics approach, we found a distinct BMI and glucose independent metabolite profile associated with excess visceral adiposity. These findings provide insight into the pathophysiologic and metabolic mechanisms associated with variation in VAT burden. Further identification of unknown metabolites and external validation are needed.

Untargeted multi-platform analysis of the metabolome and the non-starch polysaccharides of kiwifruit during postharvest ripening

The popularity of kiwifruit (Actinidia deliciosa) is steadily rising among consumers since their introduction in the global market 60 years ago. Kiwifruit are ripened postharvest for marketing. A good technique to follow the complex changes throughout postharvest ripening is metabolomics. So far research on the kiwifruit metabolome has been performed on kiwifruit development or on-vine ripening. This study aimed at a comprehensive description of processes during postharvest ripening of kiwifruit. Metabolic changes in kiwifruit during six well-defined stages of postharvest ripening and two non-marketable stages due to water loss were monitored using an untargeted multi-platform approach consisting of GC×GC–MS, GC–MS and NMR. In addition, postharvest cell wall polysaccharide modifications were followed using multiple chromatographic approaches. Using the multi-platform approach, it was possible to follow the process of postharvest ripening kiwifruit. Investigation of soluble metabolites in kiwifruit yielded mainly sugars, sugar-related substances and organic acids as well as other known and unknown metabolites. Sugars predominantly increased during ripening while organic acids predominantly decreased. In addition, unexpected changes in the concentration of some known and unknown metabolites were observed. Changes of the non-starch polysaccharides were dominated by a continuous loss of neutral pectic side chains, whereas minor changes were observed for homogalacturonan and hemicellulosic polysaccharides only. Overall the three applied platforms and the cell wall characterization were complementary and allowed a comprehensive description of the kiwifruit metabolome during postharvest ripening.

Untargeted Metabolomics Analysis of ABCC6-Deficient Mice Discloses an Altered Metabolic Liver Profile.

Loss-of-function mutations in the transmembrane ABCC6 transport protein cause pseudoxanthoma elasticum (PXE), an ectopic, metabolic mineralization disorder that affects the skin, eye, and vessels. ABCC6 is assumed to mediate efflux of one or several small molecule compounds from the liver cytosol to the circulation. Untargeted metabolomics using liquid chromatography-mass spectrometry was employed to inspect liver cytosolic extracts from mice with targeted disruption of the Abcc6 gene. Absence of the ABCC6 protein induced an altered profile of metabolites in the liver causing accumulation of compounds as more features were upregulated than downregulated in ABCC6-deficient mice. However, no differences of the identified metabolites in liver could be detected in plasma, whereas urine reflected some of the changes. Of note, N-acetylated amino acids and pantothenic acid (vitamin B5), which is involved in acetylation reactions, were accumulated in the liver. None of the identified metabolites seems to explain mineralization in extrahepatic tissues, but the present study now shows that abrogated ABCC6 function does cause alterations in the metabolic profile of the liver in accordance with PXE being a metabolic disease originating from liver disturbance. Further studies of these changes and the further identification of yet unknown metabolites may help to clarify the liver-related pathomechanism of PXE.

LaeA negatively regulates dothistromin production in the pine needle pathogen Dothistroma septosporum

In filamentous fungi both pathway-specific and global regulators regulate genes involved in the biosynthesis of secondary metabolites. LaeA is a global regulator that was named for its mutant phenotype, loss of aflR expression, due to its effect on the aflatoxin-pathway regulator AflR in Aspergillus spp. The pine needle pathogen Dothistroma septosporum produces a polyketide virulence factor, dothistromin, that is chemically related to aflatoxin and whose pathway genes are also regulated by an ortholog of AflR. However, dothistromin biosynthesis is distinctive because it is switched on during early (rather than late) exponential growth phase and the genes are dispersed in six loci across one chromosome instead of being clustered. It was therefore of interest to determine whether the function of the global regulator LaeA is conserved in D. septosporum. To address this question, a LaeA ortholog (DsLaeA) was identified and its function analyzed in D. septosporum. In contrast to aflatoxin production in Aspergillus spp., deletion of DsLaeA resulted in enhanced dothistromin production and increased expression of the pathway regulatory gene DsAflR. Although expression of other putative secondary metabolite genes in D. septosporum showed a range of different responses to loss of DsLaeA function, thin layer chromatography revealed increased levels of a previously unknown metabolite in DsLaeA mutants. In addition, these mutants exhibited reduced asexual sporulation, germination and hydrophobicity. Our data suggest that although the developmental regulatory role of DsLaeA is conserved, its role in the regulation of secondary metabolism differs from that of LaeA in A. nidulans and appears to be species specific. This study provides a step towards understanding fundamental differences in regulation of clustered and fragmented groups of secondary metabolite genes that may shed light on understanding functional adaptation in secondary metabolism.

Antibiotics in microbial coculture.

Today, the frequency of discovery of new antibiotics in microbial culture is significantly decreasing. The evidence from whole-genome surveys suggests that many genes involved in the synthesis of unknown metabolites do exist but are not expressed under conventional cultivation conditions. Therefore, it is urgently necessary to study the conditions that make otherwise silent genes active in microbes. Here we overview the knowledge on the antibiotic production promoted by cocultivation of multiple microbial strains. Accumulating evidence indicates that cocultivation can be an effective way to stimulate the production of substances that are not formed during pure cultivation. Characterization of the promotive factors produced by stimulator strains is expected to give clues to the development of effective cultivation conditions for drug discovery.