Metabolites

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Open AccessArticle

Molar-Based Targeted Metabolic Profiling of Cyanobacterial Strains with Potential for Biological Production

by Yudai Dempo, Erika Ohta, Yasumune Nakayama, Takeshi Bamba and Eiichiro Fukusaki

Metabolites 2014, 4(2), 499-516; https://doi.org/10.3390/metabo4020499 - 20 Jun 2014

Cited by 58 | Viewed by 10085

Recently, cyanobacteria have become one of the most attractive hosts for biochemical production due to its high proliferative ability and ease of genetic manipulation. Several researches aimed at biological production using modified cyanobacteria have been reported previously. However, to improve the yield of [...] Read more.

Recently, cyanobacteria have become one of the most attractive hosts for biochemical production due to its high proliferative ability and ease of genetic manipulation. Several researches aimed at biological production using modified cyanobacteria have been reported previously. However, to improve the yield of bioproducts, a thorough understanding of the intercellular metabolism of cyanobacteria is necessary. Metabolic profiling techniques have proven to be powerful tools for monitoring cellular metabolism of various organisms and can be applied to elucidate the details of cyanobacterial metabolism. In this study, we constructed a metabolic profiling method for cyanobacteria using ¹³C-labeled cell extracts as internal standards. Using this method, absolute concentrations of 84 metabolites were successfully determined in three cyanobacterial strains which are commonly used as background strains for metabolic engineering. By comparing the differences in basic metabolic potentials of the three cyanobacterial strains, we found a well-correlated relationship between intracellular energy state and growth in cyanobacteria. By integrating our results with the previously reported biological production pathways in cyanobacteria, we found putative limiting step of carbon flux. The information obtained from this study will not only help gain insights in cyanobacterial physiology but also serve as a foundation for future metabolic engineering studies using cyanobacteria. Full article

(This article belongs to the Special Issue Metabolomics and Biotechnology)

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Open AccessReview

Breath Analysis in Disease Diagnosis: Methodological Considerations and Applications

by Célia Lourenço and Claire Turner

Metabolites 2014, 4(2), 465-498; https://doi.org/10.3390/metabo4020465 - 20 Jun 2014

Cited by 209 | Viewed by 15427

Abstract

Breath analysis is a promising field with great potential for non-invasive diagnosis of a number of disease states. Analysis of the concentrations of volatile organic compounds (VOCs) in breath with an acceptable accuracy are assessed by means of using analytical techniques with high [...] Read more.

Breath analysis is a promising field with great potential for non-invasive diagnosis of a number of disease states. Analysis of the concentrations of volatile organic compounds (VOCs) in breath with an acceptable accuracy are assessed by means of using analytical techniques with high sensitivity, accuracy, precision, low response time, and low detection limit, which are desirable characteristics for the detection of VOCs in human breath. “Breath fingerprinting”, indicative of a specific clinical status, relies on the use of multivariate statistics methods with powerful in-built algorithms. The need for standardisation of sample collection and analysis is the main issue concerning breath analysis, blocking the introduction of breath tests into clinical practice. This review describes recent scientific developments in basic research and clinical applications, namely issues concerning sampling and biochemistry, highlighting the diagnostic potential of breath analysis for disease diagnosis. Several considerations that need to be taken into account in breath analysis are documented here, including the growing need for metabolomics to deal with breath profiles. Full article

(This article belongs to the Special Issue Breath Analysis in Metabolomics)

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Open AccessReview

Surveillance of C-Allocation in Microalgal Cells

by Heiko Wagner, Anne Jungandreas, Andrea Fanesi and Christian Wilhelm

Metabolites 2014, 4(2), 453-464; https://doi.org/10.3390/metabo4020453 - 19 Jun 2014

Cited by 11 | Viewed by 6619

Abstract

When microalgae are exposed to changing environmental conditions, e.g., light-dark cycles or oscillations in nutrient availability (CO₂, nitrogen, phosphate or silicate) they respond with metabolic changes in the carbon allocation pattern. Short time regulations in the time range of few seconds [...] Read more.

When microalgae are exposed to changing environmental conditions, e.g., light-dark cycles or oscillations in nutrient availability (CO₂, nitrogen, phosphate or silicate) they respond with metabolic changes in the carbon allocation pattern. Short time regulations in the time range of few seconds to minutes can be mirrored best by mass spectroscopy based metabolomics. However, these snap shots do not reflect the alterations in the carbon flow to the cellular macromolecules like protein, carbohydrate or lipid. In this review it is shown how the combination of FTIR spectroscopy and Chla-in-vivo-fluorescence based electron transport rates can reveal changes in the metabolic flux rates of carbon during a shift of the environmental conditions. The review will demonstrate in which time range FTIR spectroscopy can deliver significant information and how FTIR spectroscopy data can synergistically support metabolome analysis by mass-spectroscopy. Full article

(This article belongs to the Special Issue Metabolism in Phototrophic Prokaryotes and Algae)

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1069 KiB

Open AccessArticle

Influence of Missing Values Substitutes on Multivariate Analysis of Metabolomics Data

by Piotr S. Gromski, Yun Xu, Helen L. Kotze, Elon Correa, David I. Ellis, Emily Grace Armitage, Michael L. Turner and Royston Goodacre

Metabolites 2014, 4(2), 433-452; https://doi.org/10.3390/metabo4020433 - 16 Jun 2014

Cited by 132 | Viewed by 15555

Abstract

Missing values are known to be problematic for the analysis of gas chromatography-mass spectrometry (GC-MS) metabolomics data. Typically these values cover about 10%–20% of all data and can originate from various backgrounds, including analytical, computational, as well as biological. Currently, the most well [...] Read more.

Missing values are known to be problematic for the analysis of gas chromatography-mass spectrometry (GC-MS) metabolomics data. Typically these values cover about 10%–20% of all data and can originate from various backgrounds, including analytical, computational, as well as biological. Currently, the most well known substitute for missing values is a mean imputation. In fact, some researchers consider this aspect of data analysis in their metabolomics pipeline as so routine that they do not even mention using this replacement approach. However, this may have a significant influence on the data analysis output(s) and might be highly sensitive to the distribution of samples between different classes. Therefore, in this study we have analysed different substitutes of missing values namely: zero, mean, median, k-nearest neighbours (kNN) and random forest (RF) imputation, in terms of their influence on unsupervised and supervised learning and, thus, their impact on the final output(s) in terms of biological interpretation. These comparisons have been demonstrated both visually and computationally (classification rate) to support our findings. The results show that the selection of the replacement methods to impute missing values may have a considerable effect on the classification accuracy, if performed incorrectly this may negatively influence the biomarkers selected for an early disease diagnosis or identification of cancer related metabolites. In the case of GC-MS metabolomics data studied here our findings recommend that RF should be favored as an imputation of missing value over the other tested methods. This approach displayed excellent results in terms of classification rate for both supervised methods namely: principal components-linear discriminant analysis (PC-LDA) (98.02%) and partial least squares-discriminant analysis (PLS-DA) (97.96%) outperforming other imputation methods. Full article

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Open AccessArticle

Metabolome Consistency: Additional Parazoanthines from the Mediterranean Zoanthid Parazoanthus Axinellae

by Coralie Audoin, Vincent Cocandeau, Olivier P. Thomas, Adrien Bruschini, Serge Holderith and Grégory Genta-Jouve

Metabolites 2014, 4(2), 421-432; https://doi.org/10.3390/metabo4020421 - 30 May 2014

Cited by 24 | Viewed by 7453

Abstract

Ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry (UHPLC-MS/MS) analysis of the organic extract obtained from the Mediterranean zoanthid Parazoanthus axinellae yielded to the identification of five new parazoanthines F-J. The structures were fully determined by comparison of fragmentation patterns with [...] Read more.

Ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry (UHPLC-MS/MS) analysis of the organic extract obtained from the Mediterranean zoanthid Parazoanthus axinellae yielded to the identification of five new parazoanthines F-J. The structures were fully determined by comparison of fragmentation patterns with those of previously isolated parazoathines and MS/MS spectra simulation of in silico predicted compounds according to the metabolome consistency. The absolute configuration of the new compounds has been assigned using on-line electronic circular dichroism (UHPLC-ECD). We thus demonstrated the potential of highly sensitive hyphenated techniques to characterize the structures of a whole family of natural products within the metabolome of a marine species. Minor compounds can be characterized using these techniques thus avoiding long isolation processes that may alter the structure of the natural products. These results are also of interest to identify putative bioactive compounds present at low concentration in a complex mixture. Full article

(This article belongs to the Special Issue Cell and Tissue Metabolomics)

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Open AccessArticle

Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids

by Nobuyuki Okahashi, Shuichi Kajihata, Chikara Furusawa and Hiroshi Shimizu

Metabolites 2014, 4(2), 408-420; https://doi.org/10.3390/metabo4020408 - 30 May 2014

Cited by 20 | Viewed by 7906

Abstract

¹³C metabolic flux analysis (MFA) is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct ¹³C enrichment analysis of intracellular free amino acids (FAAs) is expected to reduce time for labeling experiments of the MFA. Measurable [...] Read more.

¹³C metabolic flux analysis (MFA) is a tool of metabolic engineering for investigation of in vivo flux distribution. A direct ¹³C enrichment analysis of intracellular free amino acids (FAAs) is expected to reduce time for labeling experiments of the MFA. Measurable FAAs should, however, vary among the MFA experiments since the pool sizes of intracellular free metabolites depend on cellular metabolic conditions. In this study, minimal ¹³C enrichment data of FAAs was investigated to perform the FAAs-based MFA. An examination of a continuous culture of Escherichia coli using ¹³C-labeled glucose showed that the time required to reach an isotopically steady state for FAAs is rather faster than that for conventional method using proteinogenic amino acids (PAAs). Considering 95% confidence intervals, it was found that the metabolic flux distribution estimated using FAAs has a similar reliability to that of the PAAs-based method. The comparative analysis identified glutamate, aspartate, alanine and phenylalanine as the common amino acids observed in E. coli under different culture conditions. The results of MFA also demonstrated that the ¹³C enrichment data of the four amino acids is required for a reliable analysis of the flux distribution. Full article

(This article belongs to the Special Issue Metabolomics and Biotechnology)

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Open AccessArticle

Adiponectin Isoforms Differentially Affect Gene Expression and the Lipidome of Primary Human Hepatocytes

by Josef Wanninger, Gerhard Liebisch, Kristina Eisinger, Markus Neumeier, Charalampos Aslanidis, Lisa Voggenreiter, Rebekka Pohl, Thomas S. Weiss, Sabrina Krautbauer and Christa Buechler

Metabolites 2014, 4(2), 394-407; https://doi.org/10.3390/metabo4020394 - 23 May 2014

Cited by 12 | Viewed by 7052

Abstract

Adiponectin (APN) exerts multiple beneficial effects in obesity and protects from liver injury. Different APN isoforms circulate in serum, and here, the effect of low molecular weight (LMW) and higher molecular weight (HMW) APN on primary human hepatocytes (PHH) has been analyzed. APN [...] Read more.

Adiponectin (APN) exerts multiple beneficial effects in obesity and protects from liver injury. Different APN isoforms circulate in serum, and here, the effect of low molecular weight (LMW) and higher molecular weight (HMW) APN on primary human hepatocytes (PHH) has been analyzed. APN is not detected in hepatocyte lysates; levels are strongly increased by HMW-APN, but not by LMW-APN, suggesting the distinct uptake/degradation of APN isoforms by PHH. Several genes with a role in fibrosis, glucose and lipid metabolism known to be regulated by HMW-APN are not affected by the LMW-isoform. Follistatin is reduced by HMW-APN and induced by LMW-APN in supernatants of PHH. Fibroblast growth factor 21 is repressed by both isoforms. Cellular triglycerides and cholesterol levels are not reduced by APN. Total phospholipids, including plasmalogens and sphingomyelins, are not changed upon APN incubation, while distinct species are either induced or repressed. Unexpectedly, total ceramide is increased by LMW-APN. Current data show that APN isoforms differentially affect hepatocyte gene expression, but do not grossly alter the hepatocyte lipidome. Full article

(This article belongs to the Special Issue Metabolomics in Complex Diseases of Man: Metabolic Syndrome)

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Open AccessArticle

Biotechnological Screening of Microalgal and Cyanobacterial Strains for Biogas Production and Antibacterial and Antifungal Effects

by Opayi Mudimu, Nataliya Rybalka, Thorsten Bauersachs, Jens Born, Thomas Friedl and Rüdiger Schulz

Metabolites 2014, 4(2), 373-393; https://doi.org/10.3390/metabo4020373 - 15 May 2014

Cited by 39 | Viewed by 10743

Abstract

Microalgae and cyanobacteria represent a valuable natural resource for the generation of a large variety of chemical substances that are of interest for medical research, can be used as additives in cosmetics and food production, or as an energy source in biogas plants. [...] Read more.

Microalgae and cyanobacteria represent a valuable natural resource for the generation of a large variety of chemical substances that are of interest for medical research, can be used as additives in cosmetics and food production, or as an energy source in biogas plants. The variety of potential agents and the use of microalgae and cyanobacteria biomass for the production of these substances are little investigated and not exploited for the market. Due to the enormous biodiversity of microalgae and cyanobacteria, they hold great promise for novel products. In this study, we investigated a large number of microalgal and cyanobacterial strains from the Culture Collection of Algae at Göttingen University (SAG) with regard to their biomass and biogas production, as well antibacterial and antifungal effects. Our results demonstrated that microalgae and cyanobacteria are able to generate a large number of economically-interesting substances in different quantities dependent on strain type. The distribution and quantity of some of these components were found to reflect phylogenetic relationships at the level of classes. In addition, between closely related species and even among multiple isolates of the same species, the productivity may be rather variable. Full article

(This article belongs to the Special Issue Metabolism in Phototrophic Prokaryotes and Algae)

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Open AccessArticle

Fast “Feast/Famine” Cycles for Studying Microbial Physiology Under Dynamic Conditions: A Case Study with Saccharomyces cerevisiae

by Camilo A. Suarez-Mendez, Andre Sousa, Joseph J. Heijnen and Aljoscha Wahl

Metabolites 2014, 4(2), 347-372; https://doi.org/10.3390/metabo4020347 - 15 May 2014

Cited by 31 | Viewed by 8406

Abstract

Microorganisms are constantly exposed to rapidly changing conditions, under natural as well as industrial production scale environments, especially due to large-scale substrate mixing limitations. In this work, we present an experimental approach based on a dynamic feast/famine regime (400 s) that leads to [...] Read more.

Microorganisms are constantly exposed to rapidly changing conditions, under natural as well as industrial production scale environments, especially due to large-scale substrate mixing limitations. In this work, we present an experimental approach based on a dynamic feast/famine regime (400 s) that leads to repetitive cycles with moderate changes in substrate availability in an aerobic glucose cultivation of Saccharomyces cerevisiae. After a few cycles, the feast/famine produced a stable and repetitive pattern with a reproducible metabolic response in time, thus providing a robust platform for studying the microorganism’s physiology under dynamic conditions. We found that the biomass yield was slightly reduced (−5%) under the feast/famine regime, while the averaged substrate and oxygen consumption as well as the carbon dioxide production rates were comparable. The dynamic response of the intracellular metabolites showed specific differences in comparison to other dynamic experiments (especially stimulus-response experiments, SRE). Remarkably, the frequently reported ATP paradox observed in single pulse experiments was not present during the repetitive perturbations applied here. We found that intracellular dynamic accumulations led to an uncoupling of the substrate uptake rate (up to 9-fold change at 20 s.) Moreover, the dynamic profiles of the intracellular metabolites obtained with the feast/famine suggest the presence of regulatory mechanisms that resulted in a delayed response. With the feast famine setup many cellular states can be measured at high frequency given the feature of reproducible cycles. The feast/famine regime is thus a versatile platform for systems biology approaches, which can help us to identify and investigate metabolite regulations under realistic conditions (e.g., large-scale bioreactors or natural environments). Full article

(This article belongs to the Special Issue Metabolomics and Biotechnology)

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Open AccessReview

MALDI Mass Spectrometry Imaging for Visualizing In Situ Metabolism of Endogenous Metabolites and Dietary Phytochemicals

by Yoshinori Fujimura and Daisuke Miura

Metabolites 2014, 4(2), 319-346; https://doi.org/10.3390/metabo4020319 - 9 May 2014

Cited by 46 | Viewed by 15790

Abstract

Understanding the spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmaceutical roles. Mass spectrometry imaging (MSI) enables determination of the distribution of ionizable molecules present in tissue sections of whole-body or single heterogeneous organ samples by direct ionization [...] Read more.

Understanding the spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmaceutical roles. Mass spectrometry imaging (MSI) enables determination of the distribution of ionizable molecules present in tissue sections of whole-body or single heterogeneous organ samples by direct ionization and detection. This emerging technique is now widely used for in situ label-free molecular imaging of endogenous or exogenous small molecules. MSI allows the simultaneous visualization of many types of molecules including a parent molecule and its metabolites. Thus, MSI has received much attention as a potential tool for pathological analysis, understanding pharmaceutical mechanisms, and biomarker discovery. On the other hand, several issues regarding the technical limitations of MSI are as of yet still unresolved. In this review, we describe the capabilities of the latest matrix-assisted laser desorption/ionization (MALDI)-MSI technology for visualizing in situ metabolism of endogenous metabolites or dietary phytochemicals (food factors), and also discuss the technical problems and new challenges, including MALDI matrix selection and metabolite identification, that need to be addressed for effective and widespread application of MSI in the diverse fields of biological, biomedical, and nutraceutical (food functionality) research. Full article

(This article belongs to the Special Issue Metabolomics and Biotechnology)

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Open AccessArticle

Short-Term Intra-Subject Variation in Exhaled Volatile Organic Compounds (VOCs) in COPD Patients and Healthy Controls and Its Effect on Disease Classification

by Christopher Phillips, Neil Mac Parthaláin, Yasir Syed, Davide Deganello, Timothy Claypole and Keir Lewis

Metabolites 2014, 4(2), 300-318; https://doi.org/10.3390/metabo4020300 - 9 May 2014

Cited by 28 | Viewed by 7775

Abstract

Exhaled volatile organic compounds (VOCs) are of interest for their potential to diagnose disease non-invasively. However, most breath VOC studies have analyzed single breath samples from an individual and assumed them to be wholly consistent representative of the person. This provided the motivation [...] Read more.

Exhaled volatile organic compounds (VOCs) are of interest for their potential to diagnose disease non-invasively. However, most breath VOC studies have analyzed single breath samples from an individual and assumed them to be wholly consistent representative of the person. This provided the motivation for an investigation of the variability of breath profiles when three breath samples are taken over a short time period (two minute intervals between samples) for 118 stable patients with Chronic Obstructive Pulmonary Disease (COPD) and 63 healthy controls and analyzed by gas chromatography and mass spectroscopy (GC/MS). The extent of the variation in VOC levels differed between COPD and healthy subjects and the patterns of variation differed for isoprene versus the bulk of other VOCs. In addition, machine learning approaches were applied to the breath data to establish whether these samples differed in their ability to discriminate COPD from healthy states and whether aggregation of multiple samples, into single data sets, could offer improved discrimination. The three breath samples gave similar classification accuracy to one another when evaluated separately (66.5% to 68.3% subjects classified correctly depending on the breath repetition used). Combining multiple breath samples into single data sets gave better discrimination (73.4% subjects classified correctly). Although accuracy is not sufficient for COPD diagnosis in a clinical setting, enhanced sampling and analysis may improve accuracy further. Variability in samples, and short-term effects of practice or exertion, need to be considered in any breath testing program to improve reliability and optimize discrimination. Full article

(This article belongs to the Special Issue Breath Analysis in Metabolomics)

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Open AccessArticle

Quantitative Metabolomics and Instationary ¹³C-Metabolic Flux Analysis Reveals Impact of Recombinant Protein Production on Trehalose and Energy Metabolism in Pichia pastoris

by Joel Jordà, Hugo Cueto Rojas, Marc Carnicer, Aljoscha Wahl, Pau Ferrer and Joan Albiol

Metabolites 2014, 4(2), 281-299; https://doi.org/10.3390/metabo4020281 - 5 May 2014

Cited by 34 | Viewed by 9413

Abstract

Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary ¹³C metabolic flux analysis (INST ¹³C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a [...] Read more.

Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary ¹³C metabolic flux analysis (INST ¹³C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol) on P. pastoris central carbon metabolism. Higher oxygen uptake and CO₂ production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by ¹³C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain. Full article

(This article belongs to the Special Issue Microbial Metabolomics)

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Open AccessArticle

Contrasting Strategies of Photosynthetic Energy Utilization Drive Lifestyle Strategies in Ecologically Important Picoeukaryotes

by Kimberly H. Halsey, Allen J. Milligan and Michael J. Behrenfeld

Metabolites 2014, 4(2), 260-280; https://doi.org/10.3390/metabo4020260 - 29 Apr 2014

Cited by 35 | Viewed by 10416

Abstract

The efficiency with which absorbed light is converted to net growth is a key property for estimating global carbon production. We previously showed that, despite considerable evolutionary distance, Dunaliella tertiolecta (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) share a common strategy of photosynthetic energy utilization [...] Read more.

The efficiency with which absorbed light is converted to net growth is a key property for estimating global carbon production. We previously showed that, despite considerable evolutionary distance, Dunaliella tertiolecta (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) share a common strategy of photosynthetic energy utilization and nearly identical light energy conversion efficiencies. These findings suggested that a single model might be appropriate for describing relationships between measures of phytoplankton production. This conclusion was further evaluated for Ostreococcus tauri RCC1558 and Micromonas pusilla RCC299 (Chlorophyta, Prasinophyceae), two picoeukaryotes with contrasting geographic distributions and swimming abilities. Nutrient-dependent photosynthetic efficiencies in O. tauri were similar to the previously studied larger algae. Specifically, absorption-normalized gross oxygen and carbon production and net carbon production were independent of nutrient limited growth rate. In contrast, all measures of photosynthetic efficiency were strongly dependent on nutrient availability in M. pusilla. This marked difference was accompanied by a diminished relationship between Chl_a:C and nutrient limited growth rate and a remarkably greater efficiency of gross-to-net energy conversion than the other organisms studied. These results suggest that the cost-benefit of decoupling pigment concentration from nutrient availability enables motile organisms to rapidly exploit more frequent encounters with micro-scale nutrient patches in open ocean environments. Full article

(This article belongs to the Special Issue Metabolism in Phototrophic Prokaryotes and Algae)

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Open AccessArticle

Metabolic Profiling of Human Peripheral Blood Mononuclear Cells: Influence of Vitamin D Status and Gender

by Magdalena Stepien, Anne P. Nugent and Lorraine Brennan

Metabolites 2014, 4(2), 248-259; https://doi.org/10.3390/metabo4020248 - 22 Apr 2014

Cited by 14 | Viewed by 6367

Abstract

Metabolic profiling of peripheral blood mononuclear cells (PBMC) could serve as a less invasive and more direct alternative to tissue biopsies or serum in metabolomic research. We conducted two exploratory independent studies in order to characterise PBMC’s metabolomic profile following short-term vitamin D3 [...] Read more.

Metabolic profiling of peripheral blood mononuclear cells (PBMC) could serve as a less invasive and more direct alternative to tissue biopsies or serum in metabolomic research. We conducted two exploratory independent studies in order to characterise PBMC’s metabolomic profile following short-term vitamin D3 supplementation and to determine gender effects. In the first study, eight healthy males and females aged 40–65 y were randomly selected for profiling of PBMCs after receiving either 15 µg of vitamin D3 or placebo for four weeks. In the second study, twenty younger healthy males and females were studied. Cell metabolites were extracted and deproteinised using methanol/chloroform/water method and analysed by GC-MS. Higher vitamin D status had no effect on the fatty acid profile of PBMCs, but inflammatory biomarkers and adipokines correlated positively with stearic acid levels. In the second study, no gender-specific metabolites were identified. Valine, leucine and aspartic acid were identified as potential BMI-sensitive amino acids. Larger studies are needed to confirm the influence of BMI on these parameters. This work clearly demonstrates the utility of metabolomics profiling of PBMCs and paves the way for future applications of metabolomics in identifying metabolic profiles of blood cells as a measure for dietary intakes or physiological status. Full article

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Open AccessArticle

Effects of Inorganic Carbon Limitation on the Metabolome of the Synechocystis sp. PCC 6803 Mutant Defective in glnB Encoding the Central Regulator PII of Cyanobacterial C/N Acclimation

by Doreen Schwarz, Isabel Orf, Joachim Kopka and Martin Hagemann

Metabolites 2014, 4(2), 232-247; https://doi.org/10.3390/metabo4020232 - 22 Apr 2014

Cited by 23 | Viewed by 8009

Abstract

Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis. Non-diazotrophic strains such as the model Synechocystis sp. PCC 6803 depend on a balanced uptake and assimilation of inorganic carbon and nitrogen sources. The internal C/N ratio is sensed via the PII protein (GlnB). We [...] Read more.

Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis. Non-diazotrophic strains such as the model Synechocystis sp. PCC 6803 depend on a balanced uptake and assimilation of inorganic carbon and nitrogen sources. The internal C/N ratio is sensed via the PII protein (GlnB). We analyzed metabolic changes of the DglnB mutant of Synechocystis sp. PCC 6803 under different CO₂ availability. The identified metabolites provided a snapshot of the central C/N metabolism. Cells of the DglnB mutant shifted to carbon-limiting conditions, i.e. a decreased C/N ratio, showed changes in intermediates of the sugar storage and particularly of the tricarboxylic acid cycle, arginine, and glutamate metabolism. The changes of the metabolome support the notion that the PII protein is primarily regulating the N-metabolism whereas the changes in C-metabolism are probably secondary effects of the PII deletion. Full article

(This article belongs to the Special Issue Metabolism in Phototrophic Prokaryotes and Algae)

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Open AccessArticle

Xanthan Gum Removal for ¹H-NMR Analysis of the Intracellular Metabolome of the Bacteria Xanthomonas axonopodis pv. citri 306

by Vanessa R. Pegos, Rafael R. Canevarolo, Aline P. Sampaio, Andrea Balan and Ana C. M. Zeri

Metabolites 2014, 4(2), 218-231; https://doi.org/10.3390/metabo4020218 - 22 Apr 2014

Cited by 4 | Viewed by 8151

Abstract

Xanthomonas is a genus of phytopathogenic bacteria, which produces a slimy, polysaccharide matrix known as xanthan gum, which involves, protects and helps the bacteria during host colonization. Although broadly used as a stabilizer and thickener in the cosmetic and food industries, xanthan gum [...] Read more.

Xanthomonas is a genus of phytopathogenic bacteria, which produces a slimy, polysaccharide matrix known as xanthan gum, which involves, protects and helps the bacteria during host colonization. Although broadly used as a stabilizer and thickener in the cosmetic and food industries, xanthan gum can be a troubling artifact in molecular investigations due to its rheological properties. In particular, a cross-reaction between reference compounds and the xanthan gum could compromise metabolic quantification by NMR spectroscopy. Aiming at an efficient gum extraction protocol, for a ¹H-NMR-based metabolic profiling study of Xanthomonas, we tested four different interventions on the broadly used methanol-chloroform extraction protocol for the intracellular metabolic contents observation. Lower limits for bacterial pellet volumes for extraction were also probed, and a strategy is illustrated with an initial analysis of X. citri’s metabolism by ¹H-NMR spectroscopy. Full article

(This article belongs to the Special Issue Sample Preparation for Metabolite Analysis)

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Open AccessReview

Rationales and Approaches for Studying Metabolism in Eukaryotic Microalgae

by Daniel Veyel, Alexander Erban, Ines Fehrle, Joachim Kopka and Michael Schroda

Metabolites 2014, 4(2), 184-217; https://doi.org/10.3390/metabo4020184 - 11 Apr 2014

Cited by 14 | Viewed by 10849

Abstract

The generation of efficient production strains is essential for the use of eukaryotic microalgae for biofuel production. Systems biology approaches including metabolite profiling on promising microalgal strains, will provide a better understanding of their metabolic networks, which is crucial for metabolic engineering efforts. [...] Read more.

The generation of efficient production strains is essential for the use of eukaryotic microalgae for biofuel production. Systems biology approaches including metabolite profiling on promising microalgal strains, will provide a better understanding of their metabolic networks, which is crucial for metabolic engineering efforts. Chlamydomonas reinhardtii represents a suited model system for this purpose. We give an overview to genetically amenable microalgal strains with the potential for biofuel production and provide a critical review of currently used protocols for metabolite profiling on Chlamydomonas. We provide our own experimental data to underpin the validity of the conclusions drawn. Full article

(This article belongs to the Special Issue Metabolism in Phototrophic Prokaryotes and Algae)

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Open AccessArticle

Stable Isotope Tracer Analysis in Isolated Mitochondria from Mammalian Systems

by Simon-Pierre Gravel, Sylvia Andrzejewski, Daina Avizonis and Julie St-Pierre

Metabolites 2014, 4(2), 166-183; https://doi.org/10.3390/metabo4020166 - 10 Apr 2014

Cited by 30 | Viewed by 11714

Abstract

Mitochondria are a focal point in metabolism, given that they play fundamental roles in catabolic, as well as anabolic reactions. Alterations in mitochondrial functions are often studied in whole cells, and metabolomics experiments using ¹³C-labeled substrates, coupled with mass isotopomer distribution analyses, [...] Read more.

Mitochondria are a focal point in metabolism, given that they play fundamental roles in catabolic, as well as anabolic reactions. Alterations in mitochondrial functions are often studied in whole cells, and metabolomics experiments using ¹³C-labeled substrates, coupled with mass isotopomer distribution analyses, represent a powerful approach to study global changes in cellular metabolic activities. However, little is known regarding the assessment of metabolic activities in isolated mitochondria using this technology. Studies on isolated mitochondria permit the evaluation of whether changes in cellular metabolic activities are due to modifications in the intrinsic properties of the mitochondria. Here, we present a streamlined approach to accurately determine ¹³C, as well as ¹²C enrichments in isolated mitochondria from mammalian tissues or cultured cells by GC/MS. We demonstrate the relevance of this experimental approach by assessing the effects of drugs perturbing mitochondrial functions on the mass isotopomer enrichment of metabolic intermediates. Furthermore, we investigate ¹³C and ¹²C enrichments in mitochondria isolated from cancer cells given the emerging role of metabolic alterations in supporting tumor growth. This original method will provide a very sensitive tool to perform metabolomics studies on isolated mitochondria. Full article

(This article belongs to the Special Issue Cancer Metabolomics)

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Application of Stable Isotope-Assisted Metabolomics for Cell Metabolism Studies

by Le You, Baichen Zhang and Yinjie J. Tang

Metabolites 2014, 4(2), 142-165; https://doi.org/10.3390/metabo4020142 - 31 Mar 2014

Cited by 38 | Viewed by 13111

Abstract

The applications of stable isotopes in metabolomics have facilitated the study of cell metabolisms. Stable isotope-assisted metabolomics requires: (1) properly designed tracer experiments; (2) stringent sampling and quenching protocols to minimize isotopic alternations; (3) efficient metabolite separations; (4) high resolution mass spectrometry to [...] Read more.

The applications of stable isotopes in metabolomics have facilitated the study of cell metabolisms. Stable isotope-assisted metabolomics requires: (1) properly designed tracer experiments; (2) stringent sampling and quenching protocols to minimize isotopic alternations; (3) efficient metabolite separations; (4) high resolution mass spectrometry to resolve overlapping peaks and background noises; and (5) data analysis methods and databases to decipher isotopic clusters over a broad m/z range (mass-to-charge ratio). This paper overviews mass spectrometry based techniques for precise determination of metabolites and their isotopologues. It also discusses applications of isotopic approaches to track substrate utilization, identify unknown metabolites and their chemical formulas, measure metabolite concentrations, determine putative metabolic pathways, and investigate microbial community populations and their carbon assimilation patterns. In addition, ¹³C-metabolite fingerprinting and metabolic models can be integrated to quantify carbon fluxes (enzyme reaction rates). The fluxome, in combination with other “omics” analyses, may give systems-level insights into regulatory mechanisms underlying gene functions. More importantly, ¹³C-tracer experiments significantly improve the potential of low-resolution gas chromatography-mass spectrometry (GC-MS) for broad-scope metabolism studies. We foresee the isotope-assisted metabolomics to be an indispensable tool in industrial biotechnology, environmental microbiology, and medical research. Full article

(This article belongs to the Special Issue Cell and Tissue Metabolomics)

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Metabolites, Volume 4, Issue 2 (June 2014) – 19 articles , Pages 142-516

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