Metabolite Pools Research Articles

Absolute Quantification of the Central Carbon Metabolome in Eight Commonly Applied Prokaryotic and Eukaryotic Model Systems.

Absolute quantification of intracellular metabolite pools is a prerequisite for modeling and in-depth biological interpretation of metabolomics data. It is the final step of an elaborate metabolomics workflow, with challenges associated with all steps—from sampling to quantifying the physicochemically diverse metabolite pool. Chromatographic separation combined with mass spectrometric (MS) detection is the superior platform for high coverage, selective, and sensitive detection of metabolites. Herein, we apply our quantitative MS-metabolomics workflow to measure and present the central carbon metabolome of a panel of commonly applied biological model systems. The workflow includes three chromatographic methods combined with isotope dilution tandem mass spectrometry to allow for absolute quantification of 68 metabolites of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and the amino acid and (deoxy) nucleoside pools. The biological model systems; Bacillus subtilis, Saccharomyces cerevisiae, two microalgal species, and four human cell lines were all cultured in commonly applied culture media and sampled in exponential growth phase. Both literature and databases are scarce with comprehensive metabolite datasets, and existing entries range over several orders of magnitude. The workflow and metabolite panel presented herein can be employed to expand the list of reference metabolomes, as encouraged by the metabolomics community, in a continued effort to develop and refine high-quality quantitative metabolomics workflows.

Untargeted metabolomics links glutathione to bacterial cell cycle progression.

Cell cycle progression requires the coordination of cell growth, chromosome replication, and division. Consequently, a functional cell cycle must be coupled with metabolism. However, direct measurements of metabolome dynamics remained scarce, in particular in bacteria. Here, we describe an untargeted metabolomics approach with synchronized Caulobacter crescentus cells to monitor the relative abundance changes of ~400 putative metabolites as a function of the cell cycle. While the majority of metabolite pools remains homeostatic, ~14% respond to cell cycle progression. In particular, sulfur metabolism is redirected during the G1-S transition, and glutathione levels periodically change over the cell cycle with a peak in late S phase. A lack of glutathione perturbs cell size by uncoupling cell growth and division through dysregulation of KefB, a K+/H+ antiporter. Overall, we here describe the impact of the C. crescentus cell cycle progression on metabolism, and in turn relate glutathione and potassium homeostasis to timely cell division.

Influence of growing seasons on metabolic composition, and fruit quality of avocado cultivars at ‘ready-to-eat stage’

The aim of this study was to relate the untargeted and targeted metabolites to the number of days taken to reach ‘ready-to-eat stage’ ripeness based on firmness and incidence of anthracnose in five avocado cultivars (‘Hass,’ ‘Lamb Hass,’ ‘Pinkerton,’ ‘Fuerte,’ and ‘Ryan’) harvested at different growing seasons (early, mid and late season). Cultivar ‘Ryan,’ harvested at early and mid-seasons, took longer (7 days) to ripen and showed the lowest incidence of anthracnose, whilst cv. ‘Fuerte’ ripened faster (4 days) and showed the highest incidence of anthracnose. UPLC-MS, together with principal component analysis, demonstrated clear groupings of the samples according to their cultivars. Major heterogeneity in the untargeted metabolome profile was observed in cv. ‘Ryan.’ Supervised Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) approach showed a unique biomarker ([M−H]−_ 17.48 at m/z 331.23 (Laricitrin) separated cv. ‘Ryan’ from the other cultivars. Different growing seasons of cv. ‘Hass’ were separated based on unidentified markers [early ([M−H]−_17.62 at m/z 420.31), mid ([M−H]−_18.48 at m/z 325.18) and late ([M−H]−_9.90 at m/z 329.23)]. Targeted metabolites including C7D-mannoheptulose, skin epicatechin, ferulic, p-coumaric, chlorogenic, caffeic, (-)-L-chicoric, caftaric, palmitic (16:0), oleic (18:1) and linoleic (18:2) acids contents and antioxidant activities were highest in cv. ‘Ryan’ at ready-to-eat stage irrespective of the seasons. Thus, cv. ‘Ryan’ could be a useful candidate for the avocado oil industry due to its higher fatty acid composition. The higher concentration of unsaturated fatty acids and C7D-mannoheptulose at ready-to-eat stage in cv. ‘Ryan’ avocados provides health benefits. The observed reduction in anthracnose decay in cv. Ryan avocados was attributed to the higher concentration of epicatechin in the skin and the pool of phenolic metabolites in the mesocarp.

Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses.

The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep tag in Arabidopsis (Arabidopsis thaliana) exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of >90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods. Among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.

Potential of temperature- and salinity-driven shifts in diatom compatible solute concentrations to impact biogeochemical cycling within sea ice

Sea-ice algae are an important source of primary production in polar regions, yet we have limited understanding of their responses to the seasonal cycling of temperature and salinity. Using a targeted liquid chromatography-mass spectrometry-based metabolomics approach, we found that axenic cultures of the Antarctic sea-ice diatom, Nitzschia lecointei, displayed large differences in their metabolomes when grown in a matrix of conditions that included temperatures of –1 and 4°C, and salinities of 32 and 41, despite relatively small changes in growth rate. Temperature exerted a greater effect than salinity on cellular metabolite pool sizes, though the N- or S-containing compatible solutes, 2, 3-dihydroxypropane-1-sulfonate (DHPS), glycine betaine (GBT), dimethylsulfoniopropionate (DMSP), and proline responded strongly to both temperature and salinity, suggesting complexity in their control. We saw the largest (> 4-fold) response to salinity for proline. DHPS, a rarely studied but potential compatible solute, had the highest intracellular concentrations among all compatible solutes of ~85 mM. When comparing the culture findings to natural Arctic sea-ice diatom communities, we found extensive overlap in metabolite profiles, highlighting the relevance of culture-based studies to probe environmental questions. Large changes in sea-ice diatom metabolomes and compatible solutes over a seasonal cycle could be significant components of biogeochemical cycling within sea ice.

Multiplexed Profiling and Data Processing Methods to Identify Temperature-Regulated Primary Metabolites Using Gas Chromatography Coupled to Mass Spectrometry.

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of plant material that was exposed to temperature stress. The method can be combined with stable isotope labeling and tracing experiments and is equally applicable to preparations of plant material and microbial photosynthetic organisms. The described methods are modular and can be multiplexed, that is, the same sample or a paired identical backup sample can be analyzed sequentially by more than one of the described procedures. The modules include rapid sampling and metabolic inactivation protocols for samples in a wide weight range, sample extraction procedures, chemical derivatization steps that are required to make the metabolite fraction amenable to gas chromatographic analysis, routine GC-MS methods, and procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The methods have two applications: (1) The rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification by exact quantification using GC-MS protocols that are extended by internal and external standardization.

Trimethylamine-N-oxide is present in human follicular fluid and is a negative predictor of embryo quality.

STUDY QUESTIONAre levels of trimethylamine-N-oxide (TMAO) in human follicular fluid (FF) related to IVF outcomes?SUMMARY ANSWERHigher levels of TMAO are a negative predictor of oocyte fertilization and embryo quality.WHAT IS KNOWN ALREADYTMAO is a metabolic product of dietary choline and l-carnitine produced via subsequent enzymatic modifications by the intestinal microbiota and hepatocytes. TMAO promotes inflammatory and oxidative stress pathways and has been characterized as a causative biomarker for the development of cardiometabolic disease.STUDY DESIGN, SIZE, DURATIONFor the present cross-sectional study, samples (FF and plasma) from 431 modified natural cycle (MNC)-IVF cycles of 132 patients were collected prospectively between October 2014 and March 2018 in a single academic medical center.PARTICIPANTS/MATERIALS, SETTING, METHODSTMAO and its precursors (choline, l-carnitine and gamma-butyrobetaine) were measured by ultra-high-performance liquid chromatography/mass spectrometry in (i) matched FF and plasma from 63 MNC-IVF cycles, in order to compare metabolite levels in the two matrices and (ii) FF from 232 MNC-IVF cycles in which only one oocyte was retrieved at follicular puncture. The association between metabolite levels and oocyte fertilization, embryo fragmentation percentage, embryo quality and the occurrence of pregnancy was analyzed using multilevel generalized estimating equations with adjustment for patient and cycle characteristics.MAIN RESULTS AND THE ROLE OF CHANCEThe level of choline was higher in FF as compared to matched plasma (P < 0.001). Conversely, the levels of TMAO and gamma-butyrobetaine were lower in FF as compared to plasma (P = 0.001 and P = 0.075, respectively). For all metabolites, there was a positive correlation between FF and plasma levels. Finally, levels of TMAO and its gut-derived precursor gamma-butyrobetaine were lower in FF from oocytes that underwent normal fertilization (TMAO: odds ratio [OR] 0.66 [0.49–0.90], P = 0.008 per 1.0-μmol/L increase; gamma-butyrobetaine: OR 0.77 [0.60–1.00], P = 0.047 per 0.1-μmol/L increase) and developed into top-quality embryos (TMAO: OR 0.56 [0.42–0.76], P < 0.001 per 1.0-μmol/L increase; gamma-butyrobetaine: OR 0.79 [0.62–1.00], P = 0.050 per 0.1-μmol/L increase) than in FF from oocytes of suboptimal development.LIMITATIONS, REASONS FOR CAUTIONThe individual contributions of diet, gut bacteria and liver to the metabolite pools have not been quantified in this analysis.WIDER IMPLICATIONS OF THE FINDINGSMore research on the contribution of diet and the effect of gut bacteria on FF TMAO is warranted. Since TMAO integrates diet, microbiota and genetic setup of the person, our results indicate potential important clinical implications for its use as biomarker for lifestyle interventions to improve fertility.STUDY FUNDING/COMPETING INTEREST(S)No external funding was received for this project. The Department of Obstetrics and Gynecology of the University Medical Center Groningen received an unrestricted educational grant of Ferring Pharmaceutical BV, the Netherlands. The authors have no other conflicts of interest.TRIAL REGISTRATION NUMBERNetherlands Trial Register number NTR4409.

Metabolomics: A Way Forward for Crop Improvement.

Metabolomics is an emerging branch of “omics” and it involves identification and quantification of metabolites and chemical footprints of cellular regulatory processes in different biological species. The metabolome is the total metabolite pool in an organism, which can be measured to characterize genetic or environmental variations. Metabolomics plays a significant role in exploring environment–gene interactions, mutant characterization, phenotyping, identification of biomarkers, and drug discovery. Metabolomics is a promising approach to decipher various metabolic networks that are linked with biotic and abiotic stress tolerance in plants. In this context, metabolomics-assisted breeding enables efficient screening for yield and stress tolerance of crops at the metabolic level. Advanced metabolomics analytical tools, like non-destructive nuclear magnetic resonance spectroscopy (NMR), liquid chromatography mass-spectroscopy (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), and direct flow injection (DFI) mass spectrometry, have sped up metabolic profiling. Presently, integrating metabolomics with post-genomics tools has enabled efficient dissection of genetic and phenotypic association in crop plants. This review provides insight into the state-of-the-art plant metabolomics tools for crop improvement. Here, we describe the workflow of plant metabolomics research focusing on the elucidation of biotic and abiotic stress tolerance mechanisms in plants. Furthermore, the potential of metabolomics-assisted breeding for crop improvement and its future applications in speed breeding are also discussed. Mention has also been made of possible bottlenecks and future prospects of plant metabolomics.

Imbalanced Regulation of Fungal Nutrient Transports According to Phosphate Availability in a Symbiocosm Formed by Poplar, Sorghum, and Rhizophagus irregularis.

In arbuscular mycorrhizal (AM) symbiosis, key components of nutrient uptake and exchange are specialized transporters that facilitate nutrient transport across membranes. As phosphate is a nutrient and a regulator of nutrient exchanges, we investigated the effect of P availability to extraradical mycelium (ERM) on both plant and fungus transcriptomes and metabolomes in a symbiocosm system. By perturbing nutrient exchanges under the control of P, our objectives were to identify new fungal genes involved in nutrient transports, and to characterize in which extent the fungus differentially modulates its metabolism when interacting with two different plant species. We performed transportome analysis on the ERM and intraradical mycelium of the AM fungus Rhizophagus irregularis associated to Populus trichocarpa and Sorghum bicolor under high and low P availability in ERM, using quantitative RT-PCR and Illumina mRNA-sequencing. We observed that mycorrhizal symbiosis induces expression of specific phosphate and ammonium transporters in both plants. Furthermore, we identified new AM-inducible transporters and showed that a subset of phosphate transporters is regulated independently of symbiotic nutrient exchange. mRNA-Sequencing revealed that the fungal transportome was not similarly regulated in the two host plant species according to P availability. Mirroring this effect, many plant carbohydrate transporters were down-regulated in P. trichocarpa mycorrhizal root tissue. Metabolome analysis revealed further that AM root colonization led to a modification of root primary metabolism under low and high P availability and to a decrease of primary metabolite pools in general. Moreover, the down regulation of the sucrose transporters suggests that the plant limits carbohydrate long distance transport (i.e. from shoot to the mycorrhizal roots). By simultaneous uptake/reuptake of nutrients from the apoplast at the biotrophic interface, plant and fungus are both able to control reciprocal nutrient fluxes.

Applications of stable isotope-based metabolomics and fluxomics toward synthetic biology of cyanobacteria.

Unique features of cyanobacteria (e.g., photosynthesis and nitrogen fixation) make them potential candidates for production of biofuels and other value-added biochemicals. As prokaryotes, they can be readily engineered using synthetic and systems biology tools. Metabolic engineering of cyanobacteria for the synthesis of desired compounds requires in-depth knowledge of central carbon and nitrogen metabolism, pathway fluxes, and their regulation. Metabolomics and fluxomics offer the comprehensive analysis of metabolism by directly characterizing the biochemical activities of cells. This information is acquired by measuring the abundance of key metabolites and their rates of interconversion, which can be achieved by labeling cells with stable isotopes, quantifying metabolite pool sizes and isotope incorporation by gas chromatography/liquid chromatography-mass spectrometry GC/LC-MS or nuclear magnetic resonance (NMR), and mathematical modeling to estimate in vivo metabolic fluxes. Herein, we review progress that has been made to adapt metabolomics and fluxomics tools to examine model cyanobacterial species. We summarize the application of metabolic flux analysis (MFA) strategies to identify metabolic bottlenecks that can be targeted to boost cell growth, improve stress tolerance, or enhance biochemical production in cyanobacteria. Despite the advances in metabolomics, fluxomics, and other synthetic and systems biology tools during the past years, further efforts are required to increase our understanding of cyanobacterial metabolism in order to create efficient photosynthetic hosts for the production of value-added compounds. This article is categorized under: Laboratory Methods and Technologies > Metabolomics Biological Mechanisms > Metabolism Analytical and Computational Methods > Analytical Methods.

Comparative metabolomic analysis reveals the variations in taxoids and flavonoids among three Taxus species

BackgroundTrees of the genus Taxus are highly valuable medicinal plants with multiple pharmacological effects on various cancer treatments. Paclitaxel from Taxus trees is an efficient and widely used anticancer drug, however, the accumulation of taxoids and other active ingredients can vary greatly among Taxus species. In our study, the metabolomes of three Taxus species have been investigated.ResultsA total of 2246 metabolites assigned to various primary and secondary metabolic pathways were identified using an untargeted approach. Analysis of differentially accumulated metabolites identified 358 T. media-, 220 T. cuspidata-, and 169 T. mairei-specific accumulated metabolites, respectively. By searching the metabolite pool, 7 MEP pathway precursors, 11 intermediates, side chain products and derivatives of paclitaxel, and paclitaxel itself were detected. Most precursors, initiated intermediates were highly accumulated in T. mairei, and most intermediate products approaching the end point of taxol biosynthesis pathway were primarily accumulated in T. cuspidata and T. media. Our data suggested that there were higher-efficiency pathways to paclitaxel in T. cuspidata and T. media compared with in T. mairei. As an important class of active ingredients in Taxus trees, a majority of flavonoids were predominantly accumulated in T. mairei rather than T. media and T. cuspidata. The variations in several selected taxoids and flavonoids were confirmed using a targeted approach.ConclusionsSystematic correlativity analysis identifies a number of metabolites associated with paclitaxel biosynthesis, suggesting a potential negative correlation between flavonoid metabolism and taxoid accumulation. Investigation of the variations in taxoids and other active ingredients will provide us with a deeper understanding of the interspecific differential accumulation of taxoids and an opportunity to accelerate the highest-yielding species breeding and resource utilization.

Contribution of Berry Polyphenols to the Human Metabolome.

Diets rich in berries provide health benefits, however, the contribution of berry phytochemicals to the human metabolome is largely unknown. The present study aimed to establish the impact of berry phytochemicals on the human metabolome. A “systematic review strategy” was utilized to characterize the phytochemical composition of the berries most commonly consumed in the USA; (poly)phenols, primarily anthocyanins, comprised the majority of reported plant secondary metabolites. A reference standard library and tandem mass spectrometry (MS/MS) quantitative metabolomics methodology were developed and applied to serum/plasma samples from a blueberry and a strawberry intervention, revealing a diversity of benzoic, cinnamic, phenylacetic, 3-(phenyl)propanoic and hippuric acids, and benzyldehydes. 3-Phenylpropanoic, 2-hydroxybenzoic, and hippuric acid were highly abundant (mean > 1 µM). Few metabolites at concentrations above 100 nM changed significantly in either intervention. Significant intervention effects (p < 0.05) were observed for plasma/serum 2-hydroxybenzoic acid and hippuric acid in the blueberry intervention, and for 3-methoxyphenylacetic acid and 4-hydroxyphenylacetic acid in the strawberry intervention. However, significant within-group effects for change from baseline were prevalent, suggesting that high inter-individual variability precluded significant treatment effects. Berry consumption in general appears to cause a fluctuation in the pools of small molecule metabolites already present at baseline, rather than the appearance of unique berry-derived metabolites, which likely reflects the ubiquitous nature of (poly)phenols in the background diet.

Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae.ResultsThe growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production.ConclusionThese results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.

P727Metabolic and functional differences between male and female endothelial cells from umbilical cords (HUVEC) of twin pairs

Abstract Background Personalised Medicine is one of the hallmarks of future medicine. Sex and gender differences exist in the incidence, clinical manifestation and outcome of cardiovascular diseases. Gonadal hormones are thought to account for most of these sex differences. However, besides hormones, sexual dimorphisms at the cellular level may also contribute to physiological and pathophysiological cardiovascular differences between women and men. Purpose To analyse intrinsic sex differences at the cellular level, we aimed to elucidate sex-specific differences in endothelial cell migration and energy metabolism under pro-migratory conditions in male and female HUVECs. To reduce biological variability, we used HUVECS obtained from umbilical cords from twin pairs of the opposite sex. These cells are exposed in utero to the same maternal environment, and therefore represent a valuable tool to study intrinsic sex-specific differences at the cellular level. Methods Vascular endothelial growth factor (VEGF)-stimulated migration was determined with IBIDI migration chambers. Sex-specific levels of proteins were studied using proteome profiling. Cellular metabolism was measured by Seahorse and levels of intracellular metabolites were analysed using GC-MS based technology. Results Female cells showed significantly higher VEGF-induced cell migration than male HUVECs. Proteomic profiling revealed a sex-specific response to VEGF treatment. Mitochondrial respiration rate was higher in VEGF-stimulated male HUVECs compared to female cells. Whereas mean glycolytic rates did not significantly differ between sexes, the ratio of glycolysis/mitochondrial respiration after VEGF stimulation was higher in female than in male HUVECs. Female cells had higher intracellular ATP levels after serum starvation and treatment with VEGF. Under both conditions, female cells showed altered levels of metabolite pools compared to male HUVECs. Conclusions Higher intracellular ATP and metabolite levels in female cells after serum starvation and VEGF may contribute to the observed functional sexual dimorphisms, and may also point to an increased stress tolerance of female cells. The results of our study provide a strong argument to discriminate between male and female cells in in vitro experiments. Acknowledgement/Funding The work was supported by the DZHK (German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research).

Integration of flux measurements and pharmacological controls to optimize stable isotope-resolved metabolomics workflows and interpretation

Stable isotope-resolved metabolomics (SIRM) provides information regarding the relative activity of numerous metabolic pathways and the contribution of nutrients to specific metabolite pools; however, SIRM experiments can be difficult to execute, and data interpretation is challenging. Furthermore, standardization of analytical procedures and workflows remain significant obstacles for widespread reproducibility. Here, we demonstrate the workflow of a typical SIRM experiment and suggest experimental controls and measures of cross-validation that improve data interpretation. Inhibitors of glycolysis and oxidative phosphorylation as well as mitochondrial uncouplers serve as pharmacological controls, which help define metabolic flux configurations that occur under well-controlled metabolic states. We demonstrate how such controls and time course labeling experiments improve confidence in metabolite assignments as well as delineate metabolic pathway relationships. Moreover, we demonstrate how radiolabeled tracers and extracellular flux analyses integrate with SIRM to improve data interpretation. Collectively, these results show how integration of flux methodologies and use of pharmacological controls increase confidence in SIRM data and provide new biological insights.

UPLC Orbitrap MS/MS-based fingerprints of dissolved organic matter in waste leachate driven by waste age

Waste leachate is a pool of complicated metabolites from waste treatment and disposal as a global environmental problem. The recognition of dissolved organic matter (DOM) in leachate is crucial to improve leachate treatment efficiency and comprehend waste stabilization process. The present study acquired the molecular information for DOM in 22 waste leachate samples using ultra-performance liquid chromatography coupled with hybrid quadrupole Orbitrap mass spectrometry (UPLC Orbitrap MS/MS) based on two dimensions of retention time and mass-to-charge ratio. Unique mass peaks occupied more than 20% of the detected mass peaks in each leachate, implying that the molecular information for DOM could be the fingerprint of waste landfills and storage pits. Waste age and composition predominately accounted for this unique DOM. The double-bond equivalent increased and the H/C decreased with waste age. We further found that 57 precursor ion peaks and artificial matter (confirmed as N-butylbenzenesulfonamide) were significantly correlated with waste age by multiple test and non-target screening. These molecular characteristics of raw leachate were first determined to compensate for the evolution of leachate with waste age. The fingerprints of waste leachate can be further applied in environmental monitoring scenarios, e.g., tracing landfill leakage.

NMR-based metabolite studies with 15N amino acids

15N labeled amino acids are routinely used to label proteins or nucleic acids for study by NMR. However, NMR studies of 15N labeled amino acids in metabolite studies have not been pursued extensively, presumably due to line broadening present under standard experimental conditions. In this work, we show that lowering the temperature to −5 °C allows facile characterization of 15N-labeled amino acids. Further, we show that this technique can be exploited to measure 15NH3 produced in an enzyme catalyzed reaction and the transport and metabolism of individual amino acids in mammalian cell culture. With respect to 13C-labeled amino acids, 15N-labeled amino acids are less costly and enable direct characterization of nitrogen metabolism in complex biological systems by NMR. In summary, the present work significantly expands the metabolite pools and their reactions for study by NMR.

Abstract 851: NCLX Expression Attenuates Pathological Remodeling in Experimental Cardiac Hypertrophy and Non-ischemic Heart Failure

Mitochondrial calcium ( m Ca 2+ ) uptake couples acute changes in cardiomyocyte bioenergetic demand to ATP production, but in excess triggers mitochondrial permeability transition and cardiomyocyte necrosis, as occurs during cardiac injury. Despite established roles for m Ca 2+ flux in response to acute stress, the role of m Ca 2+ signaling in chronic stress is poorly defined. As m Ca 2+ regulates the TCA cycle with the potential to affect mitochondrial signaling and/or metabolite pools required for biosynthesis, we reasoned that altered m Ca 2+ homeostasis may be an essential mechanism underlying hypertrophic growth and remodeling of the myocardium. Here, we used mice with cardiac-specific overexpression (OE) of the mitochondrial Na + /Ca 2+ exchanger (NCLX), the primary mediator of m Ca 2+ efflux in the heart, to test the hypothesis that m Ca 2+ signaling contributes to cardiac remodeling during sustained hemodynamic stress. We subjected NCLX OE (TRE-NCLX x α-MHC-tTA) and control (αMHC-tTA) mice to 12-wk transverse aortic constriction (TAC) or 4-wk infusion with angiotensin II + phenylephrine (AngII/PE) as experimental models of cardiac hypertrophy and non-ischemic heart failure. Cardiac function of NCLX OE and control mice was monitored throughout these studies via echocardiography and remodeling was assessed via tissue gravimetrics, histology, and qPCR gene expression analysis. Cardiac NCLX OE preserved ejection fraction, prevented afterload-induced hypertrophy and fibrosis, and attenuated the induction of both hypertrophic ( Nppa , Nppb , Acta1 ) and fibrotic ( Postn1 , Spp1 ) gene programs in mice subjected to 12-wk TAC. Examination at 2-wk post-TAC revealed attenuated hypertrophy and blunted hypertrophic and fibrotic gene expression in NCLX OE mice. These data indicate that increased capacity for m Ca 2+ efflux mitigates TAC-induced remodeling, prior to the development of contractile dysfunction. NCLX OE similarly attenuated atrial hypertrophy and the induction of hypertrophic and fibrotic gene programs in mice infused with AngII/PE. Together, these findings support a critical role for m Ca 2+ in driving pathological remodeling in non-ischemic heart disease and point to NCLX as a potent therapeutic target for cardiovascular disease.

Quantification of the flux of tyrosine pathway metabolites during nitisinone treatment of Alkaptonuria

Nitisinone decreases homogentisic acid (HGA) in Alkaptonuria (AKU) by inhibiting the tyrosine metabolic pathway in humans. The effect of different daily doses of nitisinone on circulating and 24 h urinary excretion of phenylalanine (PA), tyrosine (TYR), hydroxyphenylpyruvate (HPPA), hydroxyphenyllactate (HPLA) and HGA in patients with AKU was studied over a four week period. Forty AKU patients, randomised into five groups of eight patients, received doses of 1, 2, 4 or 8 mg of nitisinone daily, or no drug (control). Metabolites were analysed by tandem mass spectrometry in 24 h urine and serum samples collected before and after nitisinone. Serum metabolites were corrected for total body water and the sum of 24 hr urine plus total body water metabolites of PA, TYR, HPPA, HPLA and HGA were determined. Body weight and urine urea were used to check on stability of diet and metabolism over the 4 weeks of study. The sum of quantities of urine metabolites (PA, TYR, HPPA, HPLA and HGA) were similar pre- and post-nitisinone. The sum of total body water metabolites were significantly higher post-nitisinone (p < 0.0001) at all doses. Similarly, combined 24 hr urine:total body water ratios for all analytes were significantly higher post-nitisinone, compared with pre-nitisinone baseline for all doses (p = 0.0002 – p < 0.0001). Significantly higher concentrations of metabolites from the tyrosine metabolic pathway were observed in a dose dependant manner following treatment with nitisinone and we speculate that, for the first time, experimental evidence of the metabolite pool that would otherwise be directed towards pigment formation, has been unmasked.

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13C-pathway tracing, 13C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13C-metabolic flux analysis (13C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho–cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner–Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus’ metabolism for future lignin valorization.