Untargeted Gas Chromatography-mass Spectrometry Research Articles

Targeted and untargeted gas chromatography-mass spectrometry analysis of honey samples for determination of migrants from plastic packages

Plastic food packages usually contain additives which may migrate from the package into the food and then be ingested by the consumer, representing a risk for their health. In this study, targeted and untargeted analysis by gas chromatography-mass spectrometry (GC–MS) is proposed to monitor any contaminants of this type in honey. The application of dispersive liquid–liquid microextraction (DLLME) as a preconcentration technique allowed very low detection limits to be reached for all the substances. Fifteen target compounds, including styrene, phthalates, fatty acids, alkylphenols and bisphenol A, were quantified. Untargeted analyses were also carried out, allowing other migrants in the honey samples to be identified, such as two phthalates, four acids, three esters, one aldehyde, one hydrocarbon and two alkyl phenol compounds. The proposed method was seen to be a useful approach for the quantification and identification of potential migrants from plastics in challenging samples such as honey.

UV-B Radiation Largely Promoted the Transformation of Primary Metabolites to Phenols in Astragalus mongholicus Seedlings.

Ultraviolet-B (UV-B) radiation (280–320 nm) may induce photobiological stress in plants, activate the plant defense system, and induce changes of metabolites. In our previous work, we found that between the two Astragalus varieties prescribed by the Chinese Pharmacopoeia, Astragalus mongholicus has better tolerance to UV-B. Thus, it is necessary to study the metabolic strategy of Astragalus under UV-B radiation further. In the present study, we used untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-mass spectrometry (LC-MS techniques) to investigate the profiles of primary and secondary metabolic. The profiles revealed the metabolic response of Astragalus to UV-B radiation. We then used real-time polymerase chain reaction (RT-PCR) to obtain the transcription level of relevant genes under UV-B radiation (UV-B supplemented in the field, λmax = 313 nm, 30 W, lamp-leaf distance = 60 cm, 40 min·day−1), which annotated the responsive mechanism of phenolic metabolism in roots. Our results indicated that supplemental UV-B radiation induced a stronger shift from carbon assimilation to carbon accumulation. The flux through the phenylpropanoids pathway increased due to the mobilization of carbon reserves. The response of metabolism was observed to be significantly tissue-specific upon the UV-B radiation treatment. Among phenolic compounds, C6C1 carbon compounds (phenolic acids in leaves) and C6C3C6 carbon compounds (flavones in leaves and isoflavones in roots) increased at the expense of C6C3 carbon compounds. Verification experiments show that the response of phenolics in roots to UV-B is activated by upregulation of relevant genes rather than phenylalanine. Overall, this study reveals the tissues-specific alteration and mechanism of primary and secondary metabolic strategy in response to UV-B radiation.

Metabolomic Investigations of the Temporal Effects of Exposure to Pharmaceuticals and Personal Care Products and Their Mixture in the Eastern Oyster (Crassostrea virginica).

The eastern oyster (Crassostrea virginica) supports a large aquaculture industry and is a keystone species along the Atlantic seaboard. Native oysters are routinely exposed to a complex mixture of contaminants that increasingly includes pharmaceuticals and personal care products (PPCPs). Unfortunately, the biological effects of chemical mixtures on oysters are poorly understood. Untargeted gas chromatography-mass spectrometry metabolomics was utilized to quantify the response of oysters exposed to fluoxetine, N,N-diethyl-meta-toluamide, 17α-ethynylestradiol, diphenhydramine, and their mixture. Oysters were exposed to 1 µg/L of each chemical or mixture for 10 d, followed by an 8-d depuration period. Adductor muscle (n = 14/treatment) was sampled at days 0, 1, 5, 10, and 18. Trajectory analysis illustrated that metabolic effects and class separation of the treatments varied at each time point and that, overall, the oysters were only able to partially recover from these exposures post-depuration. Altered metabolites were associated with cellular energetics (i.e., Krebs cycle intermediates), as well as amino acid metabolism and fatty acids. Exposure to these PPCPs also affected metabolic pathways associated with anaerobic metabolism, osmotic stress, and oxidative stress, in addition to the physiological effects of each chemical's postulated mechanism of action. Following depuration, fewer metabolites were altered, but none of the treatments returned them to their initial control values, indicating that metabolic disruptions were long-lasting. Interestingly, the mixture did not directly cluster with individual treatments in the scores plot from partial least squares discriminant analysis, and many of its affected metabolic pathways were not well predicted from the individual treatments. The present study highlights the utility of untargeted metabolomics in developing exposure biomarkers for compounds with different modes of action in bivalves. Environ Toxicol Chem 2020;39:419-436. © 2019 SETAC.

Untargeted gas chromatography–mass spectrometry analysis and evaluation of antimicrobial and antioxidant activity of Zingiber nimmonii (J. Graham) Dalzell rhizome extracts

Background: Zingiber nimmonii (J. Graham) Dalzell (syn. Z. cernuum) endemic to peninsular India have been documented to be in use since ancient times in several traditional systems of medicine. Z. nimmonii with ethnomedicinal applications constitute a rich source of secondary metabolites toward identifying potential bioactive constituents with antioxidant and antimicrobial properties. Objective: Rhizomes of Z. nimmonii (J. Graham) Dalzell, endemic to the Western Ghats, were analyzed for bioactivity and phytochemical composition. Materials and Methods: Polyphenolic contents, namely total phenolics (TPs), total flavonoids (TFs) and total tannin (TT), were determined and expressed using gallic acid (GA), catechin (C) and tannic acid (TA) as standards. Antibacterial and antifungal activities were evaluated against two Gram-positive, three Gram-negative bacteria and three fungi by agar well diffusion method. Antioxidant activity was determined by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and Ferric Reducing Antioxidant Power (FRAP) assay, and volatile metabolite composition was determined by gas chromatography–mass spectrometry (GC-MS) analysis. Results: THE highest TF and TT contents were detected in ethyl acetate (557.64 ± 41.74 mg CE. 100 g-1 DW) and isopropanol (63.995 ± 2.062 mg TA equivalent. 100 g-1 DW) extract, respectively. High TP content in isopropanol extract (112.80 ± 10.99 mg GA equivalent. 100 g-1 DW) contributed to antimicrobial activity against Gram-positive Staphylococcus aureus (16.3 ± 0.6 mm) and antifungal activity against Aspergillus flavus (11.7 ± 0.6 mm). Methanol extracts showed high antioxidant activity as determined by DPPH (67.4 ± 12.5 μg/ml) and FRAP (127.8 ± 12.4 μg/ml) assays. Major bioactive phytochemical constituents in Z. nimmonii rhizome following GC-MS analysis included heptanediamide, N, N'-di-benzoyloxy (arachidonic acid inhibitor), n-hexadecanoic acid (antibacterial, cytotoxic, and antioxidant), and oleic acid (antitumor). Conclusion: The present study demonstrates potential of Z. nimmonii rhizomes as a rich source of secondary metabolites which can be exploited toward developing anti-infective formulations and free radical quenchers.

Impact of Diabetic Stress Conditions on Renal Cell Metabolome.

Diabetic kidney disease is a major complication in diabetes mellitus, and the most common reason for end-stage renal disease. Patients suffering from diabetes mellitus encounter glomerular damage by basement membrane thickening, and develop albuminuria. Subsequently, albuminuria can deteriorate the tubular function and impair the renal outcome. The impact of diabetic stress conditions on the metabolome was investigated by untargeted gas chromatography–mass spectrometry (GC-MS) analyses. The results were validated by qPCR analyses. In total, four cell lines were tested, representing the glomerulus, proximal nephron tubule, and collecting duct. Both murine and human cell lines were used. In podocytes, proximal tubular and collecting duct cells, high glucose concentrations led to global metabolic alterations in amino acid metabolism and the polyol pathway. Albumin overload led to the further activation of the latter pathway in human proximal tubular cells. In the proximal tubular cells, aldo-keto reductase was concordantly increased by glucose, and partially increased by albumin overload. Here, the combinatorial impact of two stressful agents in diabetes on the metabolome of kidney cells was investigated, revealing effects of glucose and albumin on polyol metabolism in human proximal tubular cells. This study shows the importance of including highly concentrated albumin in in vitro studies for mimicking diabetic kidney disease.

Blood plasma metabolic profiling of pregnant women with antenatal depressive symptoms

Antenatal depression affects ~9–19% of pregnant women and can exert persistent adverse effects on both mother and child. There is a need for a deeper understanding of antenatal depression mechanisms and the development of tools for reliable diagnosis and early identification of women at high risk. As the use of untargeted blood metabolomics in the investigation of psychiatric and neurological diseases has increased substantially, the main objective of this study was to investigate whether untargeted gas chromatography–mass spectrometry (GC–MS) plasma metabolomics in 45 women in late pregnancy, residing in Uppsala, Sweden, could indicate metabolic differences between women with and without depressive symptoms. Furthermore, seasonal differences in the metabolic profiles were explored. When comparing the profiles of cases with controls, independently of season, no differences were observed. However, seasonal differences were observed in the metabolic profiles of control samples, suggesting a favorable cardiometabolic profile in the summer vs. winter, as indicated by lower glucose and sugar acid concentrations and lactate to pyruvate ratio, and higher abundance of arginine and phosphate. Similar differences were identified between cases and controls among summer pregnancies, indicating an association between a stressed metabolism and depressive symptoms. No depression-specific differences were apparent among depressed and non-depressed women, in the winter pregnancies; this could be attributed to an already stressed metabolism due to the winter living conditions. Our results provide new insights into the pathophysiology of antenatal depression, and warrant further investigation of the use of metabolomics in antenatal depression in larger cohorts.

Reduced Glycolysis Contributed to Inhibition of Testis Spermatogenesis in Rats After Chronic Methamphetamine Exposure.

BackgroundPrevious reports suggested that methamphetamine (METH) exposure could lead to inhibition of rat testis spermatogenesis. Glycolysis and glucose metabolism as well as oxidative stress have been implicated in testis spermatogenesis. Here we explored the underlying mechanism of local metabolism and glycolysis of testis after METH exposure.Material/MethodsMETH was intraperitoneally injected into rats with different doses and duration of METH exposure to establish short-term and chronic exposure models. The serum 8-hydroxy-2 deoxyguanosine (8-OHdG) level of rats was detected by enzyme-linked immunosorbent assay. Untargeted gas chromatography-mass spectrometry analysis was applied to identify differential metabolites and metabolic signature. The mRNA expression of hypoxia inducible factor 1α (HIF1α), glucose transporter 1 (GLUT1), hexokinase 1 (HK1) and lactate dehydrogenase C (LDHC) in rat testes were detected by polymerase chain reaction. Further, we determined the 4 proteins with western blotting and immunohistochemistry.ResultsDecreased testes index and sperm counts were showed in the chronic METH group. The metabolome revealed that the main differential metabolites impacted were associated with glycolysis and glucose metabolism. The mRNA and protein expression of GLUT1, HK1, and LDHC were reduced in the chronic METH group but elevated in the short-term METH group, whereas HIF1α was upregulated in the short-term METH group but remained at baseline in the chronic METH group.ConclusionsOverall, glucose metabolism was regulated by HIF1α after short-term METH exposure. Reduced glycolysis in the testis led to impaired spermatogenesis after chronic METH exposure.

Metabolites fingerprint leaf extract of Bekkai plant, Albertisia papuana Becc as natural food seasonings using by Dayak Ethnics Community In North Kalimantan, Indonesia

Bekkai plant, Albertisia papuana is one of the unique and valuable germplasm because it is utilized by Dayak Ethnics North Kalimantan community as natural flavoring food and medical treatment. There is little data about their phytocompounds characteristic. The objective of this research was to describe the metabolites fingerprint leaf extract of A.papuana which be consumed as natural food seasonings. Ethanol leaf extracts from Dayak community originated from Tanjung Selor, North Kalimantan were analyzed using untargeted Gas Chromatography-Mass Spectrometry (GCMS). The investigation led to the identification of 22 constituents, was dominated by Capsaicin (10.06%),Capsaicin(6.41%),Capsaicin (21.33%), Dihydrocapsaicin (2.16%) and Squalene 7.69%,2,5-Furandione,3-(dodecenyl)dihydro (5.88%), 2-Octadecyl-propane-1,3-dio l(5.55%), Phytol(5.14%), Citronellylformate(3.85%), A cembratrienol (3.93%), P=Ethyguaicol(3.39%), 4,22-Stigmastadiene -3-one (3.00%), Alpha limone diepoxide (2.79%), 2,6,6-Trimethylbicylo(3.1.1) heptane(1.00%), Pentadecyl bromide (2.28%), Hexadecanoic acid (2.21%),Z-10-Tetradecen-1-olacetate(2.03%),Heptamethylene dibromide(2.00%),7-Oxabicyclo[4.1.0]heptane,2,26-trimethyl-1-(3-methyl-1,3butadienyl)-5-m ethyllene(1.78%),4,4-Dimethyl-oct-5-enal(1.64%),4,4-Dimethylcholestan-3-one(1.86%),17-Pentatriacontene (1.12%).

ADAP-GC 4.0: Application of Clustering-Assisted Multivariate Curve Resolution to Spectral Deconvolution of Gas Chromatography-Mass Spectrometry Metabolomics Data.

We report a multivariate curve resolution (MCR)-based spectral deconvolution workflow for untargeted gas chromatography-mass spectrometry metabolomics. As an essential step in preprocessing such data, spectral deconvolution computationally separates ions that are in the same mass spectrum but belong to coeluting compounds that are not resolved completely by chromatography. As a result of this computational separation, spectral deconvolution produces pure fragmentation mass spectra. Traditionally, spectral deconvolution has been achieved by using a model peak approach. We describe the fundamental differences between the model peak-based and the MCR-based spectral deconvolution and report ADAP-GC 4.0 that employs the latter approach while overcoming the associated computational complexity. ADAP-GC 4.0 has been evaluated using GC-TOF data sets from a 27-standards mixture at different dilutions and urine with the mixture spiked in, and GC Orbitrap data sets from mixtures of different standards. It produced the average matching scores 960, 959, and 926 respectively. Moreover, its performance has been compared against MS-DIAL, eRah, and ADAP-GC 3.2, and ADAP-GC 4.0 demonstrated a higher number of matched compounds and up to 6% increase of the average matching score.

Itaconic acid inhibits growth of a pathogenic marine Vibrio strain: A metabolomics approach

The antimicrobial role of itaconic acid (ITA) has been recently discovered in mammalian cells. In our previous studies, we discovered that marine molluscs biosynthesise substantial quantities of ITA when exposed to marine pathogens, but its antimicrobial function to Vibrio bacteria is currently unknown. Thus, in this study, we used an untargeted gas chromatography–mass spectrometry (GC-MS) platform to identify metabolic changes of Vibrio sp. DO1 (V. corallyliticus/neptunius-like isolate) caused by ITA exposure. Vibrio sp. DO1 was cultured in Luria-Bertani broth supplemented with 3 mM sodium acetate and with different concentrations of ITA (0, 3 and 6 mM) for 24 h. The results showed that ITA completely inhibited Vibrio sp. growth at 6 mM and partially inhibited the bacterial growth at 3 mM. A principal component analysis (PCA) revealed a clear separation between metabolite profiles of Vibrio sp. DO1 in the 3 mM ITA treatment and the control, which were different in 25 metabolites. Among the altered metabolites, the accumulation of glyoxylic acid and other metabolites in glyoxylate cycle (cis-aconitic acid, isocitric acid and fumaric acid) together with the increase of isocitrate lyase (ICL) activity in the 3 mM ITA treatment compared to the control suggest that ITA inhibited Vibrio sp. growth via disruption of central carbon metabolism.

Metabolic disturbances in synovial fluid are involved in the onset of synovitis in heifers with acute ruminal acidosis

Acute ruminal acidosis (ARA) is the result of increased intake of highly fermentable carbohydrates, which frequently occurs in dairy cattle and is associated with aseptic polysynovitis. To characterise the metabolic changes in the joints of animals with ARA, we performed an untargeted gas chromatography–mass spectrometry (GC-MS)-based metabolomic analysis of synovial fluid. Seven heifers were challenged with an intraruminal oligofructose overload (13 g/kg of body weight [BW]) dissolved in water. Synovial fluid samples were collected at 0, 9 and 24 h post-overload. Metabolome analysis revealed the presence of 67 metabolites. At 9 h post-overload, glyceric acid, cellobiose, fructose and lactic acid were all increased, whereas at 24 h, sorbitol, lactic acid and fructose levels were all increased >10-fold. At 24 h, citric acid and threonine levels were significantly reduced. We detected increased L- and D-lactate, and the presence of interleukin-6 (IL-6) in synovial fluid. Furthermore, using bovine fibroblast-like synoviocytes, we observed that D-lactate induces IL-6 synthesis. Our results suggest that ARA produces severe metabolomic changes in synovial fluid, including disturbances in starch and sucrose metabolism, and increased lactate levels. These changes were observed prior to the appearance of synovitis, suggesting a potential role in the onset of polysynovitis.

An arbuscular mycorrhizal fungus and a root pathogen induce different volatiles emitted by Medicago truncatula roots

Plants are in permanent contact with various microorganisms and are always impacted by them. To better understand the first steps of a plant’s recognition of soil-borne microorganisms, the early release of volatile organic compounds (VOCs) emitted from roots of Medicago truncatula in response to the symbiont Rhizophagus irregularis or the pathogenic oomycete Aphanomyces euteiches was analysed. More than 90 compounds were released from roots as detected by an untargeted gas chromatography-mass spectrometry approach. Principal component analyses clearly distinguished untreated roots from roots treated with either R. irregularis or A. euteiches. Several VOCs were found to be emitted specifically in response to each of the microorganisms. Limonene was specifically emitted from wild-type roots after contact with R. irregularis spores but not from roots of the mycorrhiza-deficient mutant does not make infections3. The application of limonene to mycorrhizal roots, however, did not affect the mycorrhization rate. Inoculation of roots with A. euteiches zoospores resulted in the specific emission of several sesquiterpenes, such as nerolidol, viridiflorol and nerolidol-epoxyacetate but application of nerolidol to zoospores of A. euteiches did not affect their vitality. Therefore, plants discriminate between different microorganisms at early stages of their interaction and respond differently to the level of root-emitted volatiles.

Hundred Fifty Years of Herbarium Collections Provide a Reliable Resource of Volatile Terpenoid Profiles Showing Strong Species Effect in Four Medicinal Species of Salvia Across the Mediterranean.

Herbarium samples are increasingly being recognized for their potential in answering a wide range of research questions. However, the suitability of herbarium samples for chemical analysis is largely unexplored as they are thought to be too degraded. The aim of this study was to explore terpenoid profiles across time and geographic space for four medicinal species of Salvia across the Mediterranean to assess the suitability of using herbarium specimens in chemical analyses. Herbarium samples of Salvia aethiopis, S. multicaulis, S. officinalis, and S. sclarea collected over 150 years across the Mediterranean were compared to modern samples using both targeted and untargeted gas chromatography-mass spectrometry analysis of terpene profiles. There was no effect of collection year on chemical composition, although the total concentration of the 20 assessed standards and two individual standards significantly decreased over time. Instead, chemical profiles were defined by species, with strong species effects identified on both the targeted and untargeted chemical composition. Geographic variation was a factor in regulating the untargeted chemical compositions, suggesting some underlying environmental effects. However, there was no effect of sample altitude on either the targeted or untargeted chemical compositions. Chemical composition of four Salvia species are predominantly defined by species, and there was a substantially smaller effect of year of sampling. Given these results herbarium collections may well represent a considerably underused resource for chemical analyses that can benefit biodiversity and other studies.

Olive oil quality classification and measurement of its organoleptic attributes by untargeted GC–MS and multivariate statistical-based approach

The capabilities of dynamic headspace entrainment followed by thermal desorption in combination with gas chromatography (GC) coupled to single quadrupole mass spectrometry (MS) have been tested for the determination of volatile components of olive oil. This technique has shown a great potential for olive oil quality classification by using an untargeted approach. The data processing strategy consisted of three different steps: component detection from GC–MS data using novel data treatment software PARADISe, a multivariate analysis using EZ-Info, and the creation of the statistical models. The great number of compounds determined enabled not only the development of a quality classification method as a complementary tool to the official established method “PANEL TEST” but also a correlation between these compounds and different types of defect. Classification method was finally validated using blind samples. An accuracy of 85% in oil classification was obtained, with 100% of extra virgin samples correctly classified.

The Microbiome and Metabolome of Preterm Infant Stool Are Personalized and Not Driven by Health Outcomes, Including Necrotizing Enterocolitis and Late-Onset Sepsis.

The assembly and development of the gut microbiome in infants have important consequences for immediate and long-term health. Preterm infants represent an abnormal case for bacterial colonization because of early exposure to bacteria and frequent use of antibiotics. To better understand the assembly of the gut microbiota in preterm infants, fecal samples were collected from 32 very low birth weight preterm infants over the first 6weeks of life. Infant health outcomes included health, late-onset sepsis, and necrotizing enterocolitis (NEC). We characterized bacterial compositions by 16S rRNA gene sequencing and metabolomes by untargeted gas chromatography-mass spectrometry. Preterm infant fecal samples lacked beneficial Bifidobacterium spp. and were dominated by Enterobacteriaceae, Enterococcus, and Staphylococcus organisms due to nearly uniform antibiotic administration. Most of the variance between the microbial community compositions could be attributed to the baby from which the sample derived (permutational multivariate analysis of variance [PERMANOVA] R2 = 0.48, P < 0.001), while clinical status (health, NEC, or late-onset sepsis) and overlapping times in the neonatal intensive care unit (NICU) did not explain a significant amount of variation in bacterial composition. Fecal metabolomes were also found to be unique to the individual (PERMANOVA R2 = 0.43, P < 0.001) and weakly associated with bacterial composition (Mantel statistic r = 0.23 ± 0.05, P < 0.05). No measured metabolites were found to be associated with necrotizing enterocolitis, late-onset sepsis, or a healthy outcome. Overall, preterm infant gut microbial communities were personalized and reflected antibiotic usage.IMPORTANCE Preterm infants face health problems likely related to microbial exposures, including sepsis and necrotizing enterocolitis. However, the role of the gut microbiome in preterm infant health is poorly understood. Microbial colonization differs from that of healthy term babies because it occurs in the NICU and is often perturbed by antibiotics. We measured bacterial compositions and metabolomic profiles of 77 fecal samples from 32 preterm infants to investigate the differences between microbiomes in health and disease. Rather than finding microbial signatures of disease, we found that both the preterm infant microbiome and the metabolome were personalized and that the preterm infant gut microbiome is enriched in microbes that commonly dominate in the presence of antibiotics. These results contribute to the growing knowledge of the preterm infant microbiome and emphasize that a personalized view will be important to disentangle the health consequences of the preterm infant microbiome.

Discrimination between the human prostate normal and cancer cell exometabolome by GC-MS

Serum prostate-specific antigen (PSA) is currently the most used biomarker in clinical practice for prostate cancer (PCa) detection. However, this biomarker has several drawbacks. In this work, an untargeted gas chromatography-mass spectrometry (GC-MS)-based metabolomic profiling of PCa cells was performed to prove the concept that metabolic alterations might differentiate PCa cell lines from normal prostate cell line. For that, we assessed the differences in volatile organic compounds (VOCs) profile in the extracellular medium (exometabolome) of four PCa cell lines and one normal prostate cell line at two pH values (pH 2 and 7) by GC-MS. Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal prostate cells. The most altered metabolites included ketones, aldehydes and organic acids. Among these, we highlight pentadecane-2-one and decanoic acid, which were significantly increased in PCa compared to normal cells, and cyclohexanone, 4-methylheptan-2-one, 2-methylpentane-1,3-diol, 4-methylbenzaldehyde, 1-(3,5-dimethylfuran-2-yl)ethanone, methyl benzoate and nonanoic acid, which were significantly decreased in PCa cells. The PCa volatilome was markedly influenced by the VOCs extraction pH, though the discriminant capability was similar. Overall, our data suggest that VOCs monitoring has the potential to be used as a PCa screening methodology.

Metabolic characterization of directly reprogrammed renal tubular epithelial cells (iRECs)

Fibroblasts can be directly reprogrammed to induced renal tubular epithelial cells (iRECs) using four transcription factors. These engineered cells may be used for disease modeling, cell replacement therapy or drug and toxicity testing. Direct reprogramming induces drastic changes in the transcriptional landscape, protein expression, morphological and functional properties of cells. However, how the metabolome is changed by reprogramming and to what degree it resembles the target cell type remains unknown. Using untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-MS, we characterized the metabolome of mouse embryonic fibroblasts (MEFs), iRECs, mIMCD-3 cells, and whole kidneys. Metabolic fingerprinting can distinguish each cell type reliably, revealing iRECs are most similar to mIMCD-3 cells and clearly separate from MEFs used for reprogramming. Treatment with the cytotoxic drug cisplatin induced typical changes in the metabolic profile of iRECs commonly occurring in acute renal injury. Interestingly, metabolites in the medium of iRECs, but not of mIMCD-3 cells or fibroblast could distinguish treated and non-treated cells by cluster analysis. In conclusion, direct reprogramming of fibroblasts into renal tubular epithelial cells strongly influences the metabolome of engineered cells, suggesting that metabolic profiling may aid in establishing iRECs as in vitro models for nephrotoxicity testing in the future.

Metabolic responses of Eucalyptus species to different temperature regimes.

Species and hybrids of Eucalyptus are the world's most widely planted hardwood trees. They are cultivated across a wide range of latitudes and therefore environmental conditions. In this context, comprehensive metabolomics approaches have been used to assess how different temperature regimes may affect the metabolism of three species of Eucalyptus, E. dunnii, E. grandis and E. pellita. Young plants were grown for 53 d in the greenhouse and then transferred to growth chambers at 10°C, 20°C or 30°C for another 7 d. In all three species the leaf chlorophyll content was positively correlated to temperature, and in E. pellita the highest temperature also resulted in a significant increase in stem biomass. Comprehensive metabolomics was performed using untargeted gas chromatography mass spectrometry (GC‐MS) and liquid chromatography (LC)‐MS. This approach enabled the comparison of the relative abundance of 88 polar primary metabolites from GC‐MS and 625 semi‐polar secondary metabolites from LC‐MS. Using principal components analysis, a major effect of temperature was observed in each species which was larger than that resulting from the genetic background. Compounds mostly affected by temperature treatment were subsequently selected using partial least squares discriminant analysis and were further identified. These putative annotations indicated that soluble sugars and several polyphenols, including tannins, triterpenes and alkaloids were mostly influenced.

Untargeted GC-MS Metabolomics.

Untargeted metabolomics refers to the high-throughput analysis of the metabolic state of a biological system (e.g., tissue, biological fluid, cell culture) based on the concentration profile of all measurable free low molecular weight metabolites. Gas chromatography-mass spectrometry (GC-MS), being a highly sensitive and high-throughput analytical platform, has been proven a useful tool for untargeted studies of primary metabolism in a variety of applications. As an omic analysis, GC-MS metabolomics is a multistep procedure; thus, standardization of an untargeted GC-MS metabolomics protocol requires the integrated optimization of pre-analytical, analytical, and computational steps. The main difference of GC-MS metabolomics compared to other metabolomics analytical platforms, including liquid chromatography-MS, is the need for the derivatization of the metabolite extracts into volatile and thermally stable derivatives, the latter being quantified in the metabolic profiles. This analytical step requires special care in the optimization of the untargeted GC-MS metabolomics experimental protocol. Moreover, both the derivatization of the original sample and the compound fragmentation that takes place in GC-MS impose specialized GC-MS metabolomic data identification, quantification, normalization and filtering methods. In this chapter, we describe the integrated protocol of untargeted GC-MS metabolomics with both the analytical and computational steps, focusing on the GC-MS specific parts, and provide details on any sample depending differences.

Metabolomic alterations in invasive ductal carcinoma of breast: A comprehensive metabolomic study using tissue and serum samples.

Invasive ductal carcinoma (IDC) is the most common type of breast cancer and the leading cause of breast cancer related mortality. In the present study, metabolomic profiles of 72 tissue samples and 146 serum samples were analysed using targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM/MS) and untargeted gas chromatography mass spectrometry (GC-MS) approaches. Combination of univariate and multivariate statistical treatment identified significant alterations of 42 and 32 metabolites in tissue and serum samples of IDC, respectively when compared to control. Some of the metabolite changes from tissue were also reflected in serum, indicating a bi-directional interaction of metabolites in IDC. Additionally, 8 tissue metabolites and 9 serum metabolites showed progressive change from control to benign to IDC suggesting their possible role in malignant transformation. We have identified a panel of three metabolites viz. tryptophan, tyrosine, and creatine in tissue and serum, which could be useful in screening of IDC subjects from both control and benign. The metabolomic alterations in IDC showed perturbations in purine and pyrimidine metabolism, amino sugar metabolism, amino acid metabolism, fatty acid biosynthesis etc. Comprehensively, this study provides valuable insights into metabolic adaptations of IDC, which can help to identify diagnostic markers as well as potential therapeutic targets.