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Sample records for nmr metabolite profiling

  1. Advances in NMR-based biofluid analysis and metabolite profiling.

    PubMed

    Zhang, Shucha; Nagana Gowda, G A; Ye, Tao; Raftery, Daniel

    2010-07-01

    Significant improvements in NMR technology and methods have propelled NMR studies to play an important role in a rapidly expanding number of applications involving the profiling of metabolites in biofluids. This review discusses recent technical advances in NMR spectroscopy based metabolite profiling methods, data processing and analysis over the last three years.

  2. NMR metabolite profiling of Greek grape marc spirits.

    PubMed

    Fotakis, Charalambos; Christodouleas, Dionysis; Kokkotou, Katerina; Zervou, Maria; Zoumpoulakis, Panagiotis; Moulos, Panagiotis; Liouni, Maria; Calokerinos, Antony

    2013-06-01

    This (1)H NMR based study profiles metabolites in Greek grape marc distillates, tsipouro and tsikoudia. Eightysix samples of indigenous and international varieties, stemming from major vine growing regions of Greece were investigated. The monitoring protocol addressed the global metabolic profile of untreated samples and accomplished the unambiguous assignment of 35 metabolites. NMR spectra were acquired by applying the robust, sensitive and rapid WET1D NMR pulse sequence, which succeeded to unveil the presence of minor compounds in a high ethanol matrix. PCA classified the samples according to their provenance, incorporating also information related to the variety, vintage year and production process within each formed regional assembly. Metabolites such as fusel alcohols, polyols, ethyl esters, mono- and di-saccharides were associated with the classification of samples. OPLS-DA ascribed to samples of common regional entity characteristic genotypic metabolites and probed to the potential influence of the vintage effect. Finally, metabolite profiling underlined the influence of the fermentation and distillation procedures.

  3. Effect of fertilizers on galanthamine and metabolite profiles in Narcissus bulbs by 1H NMR.

    PubMed

    Lubbe, Andrea; Choi, Young Hae; Vreeburg, Peter; Verpoorte, Robert

    2011-04-13

    Narcissus bulbs contain the biologically active alkaloid galanthamine, and Narcissus is being developed as a natural source of the molecule for the pharmaceutical industry. The effect of fertilizer on galanthamine production was investigated in a field study using a (1)H nuclear magnetic resonance (NMR) metabolite profiling approach. Galanthamine was quantitated and major metabolites in the bulbs were identified. The application of standard fertilization levels of nitrogen and potassium caused a significant increase in galanthamine as compared to a control. Multivariate data analysis of the (1)H NMR data revealed that applying double the standard level of nitrogen fertilizer resulted in production of more amino acids and citric acid cycle intermediates, but not more galanthamine. The results indicated that standard levels of fertilizer currently applied in The Netherlands are sufficient for optimal galanthamine accumulation in the bulbs. This study shows how (1)H NMR-based metabolic profiling can provide insight into the response of plant metabolism to agricultural practices.

  4. Rapid Etiological Classification of Meningitis by NMR Spectroscopy Based on Metabolite Profiles and Host Response

    PubMed Central

    Himmelreich, Uwe; Malik, Richard; Kühn, Till; Daniel, Heide-Marie; Somorjai, Ray L.; Dolenko, Brion; Sorrell, Tania C.

    2009-01-01

    Bacterial meningitis is an acute disease with high mortality that is reduced by early treatment. Identification of the causative microorganism by culture is sensitive but slow. Large volumes of cerebrospinal fluid (CSF) are required to maximise sensitivity and establish a provisional diagnosis. We have utilised nuclear magnetic resonance (NMR) spectroscopy to rapidly characterise the biochemical profile of CSF from normal rats and animals with pneumococcal or cryptococcal meningitis. Use of a miniaturised capillary NMR system overcame limitations caused by small CSF volumes and low metabolite concentrations. The analysis of the complex NMR spectroscopic data by a supervised statistical classification strategy included major, minor and unidentified metabolites. Reproducible spectral profiles were generated within less than three minutes, and revealed differences in the relative amounts of glucose, lactate, citrate, amino acid residues, acetate and polyols in the three groups. Contributions from microbial metabolism and inflammatory cells were evident. The computerised statistical classification strategy is based on both major metabolites and minor, partially unidentified metabolites. This data analysis proved highly specific for diagnosis (100% specificity in the final validation set), provided those with visible blood contamination were excluded from analysis; 6–8% of samples were classified as indeterminate. This proof of principle study suggests that a rapid etiologic diagnosis of meningitis is possible without prior culture. The method can be fully automated and avoids delays due to processing and selective identification of specific pathogens that are inherent in DNA-based techniques. PMID:19390697

  5. 1H-NMR metabolite profiles of different strains of Plasmodium falciparum

    PubMed Central

    Teng, Rongwei; Lehane, Adele M.; Winterberg, Markus; Shafik, Sarah H.; Summers, Robert L.; Martin, Rowena E.; van Schalkwyk, Donelly A.; Junankar, Pauline R.; Kirk, Kiaran

    2014-01-01

    Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite. PMID:25405893

  6. NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin.

    PubMed

    Jupin, M; Michiels, P J; Girard, F C; Spraul, M; Wijmenga, S S

    2014-02-01

    Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.

  7. NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin

    NASA Astrophysics Data System (ADS)

    Jupin, M.; Michiels, P. J.; Girard, F. C.; Spraul, M.; Wijmenga, S. S.

    2014-02-01

    Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.

  8. Ibuprofen metabolite profiling using a combination of SPE/column-trapping and HPLC-micro-coil NMR.

    PubMed

    Djukovic, Danijel; Appiah-Amponsah, Emmanuel; Shanaiah, Narasimhamurthy; Gowda, G A Nagana; Henry, Ian; Everly, Mike; Tobias, Brian; Raftery, Daniel

    2008-06-09

    Solid-phase extraction and column-trapping preconcentration are combined to enhance HPLC-nuclear magnetic resonance (HPLC-NMR) and applied to metabolite profiling in biological samples. Combining the two signal enhancement techniques improved the NMR signal substantially such that we were able to identify 2-hydroxyibuprofen, carboxyibuprofen, and unmetabolized ibuprofen molecules from a small urine sample after a therapeutic dose of ibuprofen. The hyphenated SPE/column-trapping method resulted in an excellent overall signal enhancement of up to 90-fold.

  9. Quantitative metabolite profiling of edible onion species by NMR and HPLC-MS.

    PubMed

    Soininen, Tuula H; Jukarainen, Niko; Auriola, Seppo O K; Julkunen-Tiitto, Riitta; Karjalainen, Reijo; Vepsäläinen, Jouko J

    2014-12-15

    Allium genus is a treasure trove of valuable bioactive compounds with potentially therapeutically important properties. This work utilises HPLC-MS and a constrained total-line-shape (CTLS) approach applied to (1)H NMR spectra to quantify metabolites present in onion species to reveal important inter-species differences. Extensive differences were detected between the sugar concentrations in onion species. Yellow onion contained the highest and red onion the lowest amounts of amino acids. The main flavonol-glucosides were quercetin 3,4'-diglucoside and quercetin 4'-glucoside. In general, the levels of flavonols were, higher in yellow onions than in red onions, and garlic and leek contained a lower amount of flavonols than the other Allium species. Our results highlight how (1)H NMR together with HPLC-MS can be useful in the quantification and the identification of the most abundant metabolites, representing an efficient means to pinpoint important functional food ingredients from Allium species.

  10. Metabolite profiling of Curcuma species grown in different regions using 1H NMR spectroscopy and multivariate analysis.

    PubMed

    Jung, Youngae; Lee, Jueun; Kim, Ho Kyoung; Moon, Byeong Cheol; Ji, Yunui; Ryu, Do Hyun; Hwang, Geum-Sook

    2012-12-07

    Curcuma is used to treat skin diseases and colic inflammatory disorders, and in insect repellants and antimicrobial and antidiabetic medications. Two Curcuma species (C. aromatica and C. longa) grown in Jeju-do and Jin-do were used in this study. Methanolic extracts were analyzed by (1)H NMR spectroscopy, and metabolite profiling coupled with multivariate analysis was applied to characterize the differences between species or origin. PCA analysis showed significantly greater differences between species than origins, and the metabolites responsible for the differences were identified. The concentrations of sugars (glucose, fructose, and sucrose) and essential oils (eucalyptol, curdione, and germacrone) were significantly different between the two species. However, the samples from Jeju-do and Jin-do were different mainly in their concentrations of organic acids (fumarate, succinate, acetate, and formate) and sugars. This study demonstrates that NMR-based metabolomics is an efficient method for fingerprinting and determining differences between Curcuma species or those grown in different regions.

  11. Influence of surfactants and humic acids on Artemia Franciscana's embryonic phospho-metabolite profile as measured by (31)P NMR.

    PubMed

    Deese, Rachel D; Weldeghiorghis, Thomas K; Haywood, Benjamin J; Cook, Robert L

    2017-05-01

    Surfactants, such as triton X-100 (Tx-100), cetylpyridinium chloride (CPC), and sodium dodecyl sulfate (SDS) are known to be toxic to Artemia Franciscana (Artemia) - an organism, frequently used to monitor the health of the aquatic environment. The phospho-metabolite profile of a living organism is often indicative of imbalances that may have been caused by environmental stressors, such as surfactants. This study utilizes in vivo(31)P NMR to monitor temporal changes in the phospho-metabolite profile of Artemia caused by Tx-100, CPC, and SDS and the ability of humic acid (HA) to mitigate the toxicity of these surfactants. It was found that, while Tx-100 does not have any effect on the phospho-metabolite profile, both CPC and SDS cause a complete retardation in growth of the phosphodiester (PDE) peak in the (31)P NMR spectrum, which is indicative of the inhibited cell replication. This growth inhibition was independently verified by the decreased guanosine triphosphate (GTP) concentration in the CPC and SDS-exposed Artemia. In addition, upon introduction of HA to the CPC and SDS-exposed Artemia, an increase of PDE peak over time is indicative of HA mitigating toxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Metabolite Profiling in Withania somnifera Roots Hydroalcoholic Extract Using LC/MS, GC/MS and NMR Spectroscopy.

    PubMed

    Trivedi, Mahendra Kumar; Panda, Parthasarathi; Sethi, Kalyan Kumar; Jana, Snehasis

    2017-03-01

    Ashwagandha (Withania somnifera) is a very well-known herbal medicine and it was well studied for its active metabolites throughout the World. Although, nearly 40 withanolides were isolated from W. somnifera root extract, still there is remaining unidentified metabolites due to very low abundance and geographical variation. Advanced separation technology with online identification by mass and nuclear magnetic resonance (NMR) are nowadays used to find out the new compounds in the crude herbal extract. This article described the metabolite profiling of ashwagandha root hydroalcoholic extract using ultra-performance liquid chromatography coupled with a positive ion electrospray ionization tandem mass spectrometry through gas chromatography mass spectrometry (GC/MS) and NMR spectroscopy. A total of 43 possible withanolides was identified and proposed their structures based on the mass of molecular and fragment ions. GC/MS and NMR analysis indicated the presence of several known withanolides including withaferin A, withanolide D, withanoside IV or VI, withanolide sulfoxide, etc. To the best of our knowledge, dihydrowithanolide D at m/z 473 (tR 7.86 min) and ixocarpalactone A at m/z 505 (tR 8.43 min) were first time identified in the ashwagandha root hydroalcoholic extract. The current study that described the identification of withanolides with summarized literature review might be helpful for designing the experiment to identify of the new chemical constituents in Withania species.

  13. Automated quantum mechanical total line shape fitting model for quantitative NMR-based profiling of human serum metabolites.

    PubMed

    Mihaleva, Velitchka V; Korhonen, Samuli-Petrus; van Duynhoven, John; Niemitz, Mathias; Vervoort, Jacques; Jacobs, Doris M

    2014-05-01

    An automated quantum mechanical total line shape (QMTLS) fitting model was implemented for quantitative nuclear magnetic resonance (NMR)-based profiling of 42 metabolites in ultrafiltrated human serum samples. Each metabolite was described by a set of chemical shifts, J-couplings, and line widths. These parameters were optimized for each metabolite in each sample by iteratively minimizing the difference between the calculated and the experimental spectrum. In total, 92.0 to 98.1 % of the signal intensities in the experimental spectrum could be explained by the calculated spectrum. The model was validated by comparison to signal integration of metabolites with isolated signals and by means of standard additions. Metabolites present at average concentration higher than 50 μM were quantified with average absolute relative error less than 10 % when using different initial parameters for the fitting procedure. Furthermore, the biological applicability of the QMTLS model was demonstrated on 287 samples from an intervention study in 37 human volunteers undergoing an exercise challenge. Our automated QMTLS model was able to cope with the large dynamic range of metabolite concentrations in serum and proved to be suitable for high-throughput analysis.

  14. Plasma and Serum Metabolite Association Networks: Comparability within and between Studies Using NMR and MS Profiling.

    PubMed

    Suarez-Diez, Maria; Adam, Jonathan; Adamski, Jerzy; Chasapi, Styliani A; Luchinat, Claudio; Peters, Annette; Prehn, Cornelia; Santucci, Claudio; Spyridonidis, Alexandros; Spyroulias, Georgios A; Tenori, Leonardo; Wang-Sattler, Rui; Saccenti, Edoardo

    2017-07-07

    Blood is one of the most used biofluids in metabolomics studies, and the serum and plasma fractions are routinely used as a proxy for blood itself. Here we investigated the association networks of an array of 29 metabolites identified and quantified via NMR in the plasma and serum samples of two cohorts of ∼1000 healthy blood donors each. A second study of 377 individuals was used to extract plasma and serum samples from the same individual on which a set of 122 metabolites were detected and quantified using FIA-MS/MS. Four different inference algorithms (ARANCE, CLR, CORR, and PCLRC) were used to obtain consensus networks. The plasma and serum networks obtained from different studies showed different topological properties with the serum network being more connected than the plasma network. On a global level, metabolite association networks from plasma and serum fractions obtained from the same blood sample of healthy people show similar topologies, and at a local level, some differences arise like in the case of amino acids.

  15. Quantitative NMR Metabolite Profiling of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Discriminates between Biofilm and Planktonic Phenotypes

    PubMed Central

    2015-01-01

    Wound bioburden in the form of colonizing biofilms is a major contributor to nonhealing wounds. Staphylococcus aureus is a Gram-positive, facultative anaerobe commonly found in chronic wounds; however, much remains unknown about the basic physiology of this opportunistic pathogen, especially with regard to the biofilm phenotype. Transcriptomic and proteomic analysis of S. aureus biofilms have suggested that S. aureus biofilms exhibit an altered metabolic state relative to the planktonic phenotype. Herein, comparisons of extracellular and intracellular metabolite profiles detected by 1H NMR were conducted for methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains grown as biofilm and planktonic cultures. Principal component analysis distinguished the biofilm phenotype from the planktonic phenotype, and factor loadings analysis identified metabolites that contributed to the statistical separation of the biofilm from the planktonic phenotype, suggesting that key features distinguishing biofilm from planktonic growth include selective amino acid uptake, lipid catabolism, butanediol fermentation, and a shift in metabolism from energy production to assembly of cell-wall components and matrix deposition. These metabolite profiles provide a basis for the development of metabolite biomarkers that distinguish between biofilm and planktonic phenotypes in S. aureus and have the potential for improved diagnostic and therapeutic use in chronic wounds. PMID:24809402

  16. Metabolite and mineral profiling of "Violetto di Niscemi" and "Spinoso di Menfi" globe artichokes by (1)H-NMR and ICP-MS.

    PubMed

    Albergamo, Ambrogina; Rotondo, Archimede; Salvo, Andrea; Pellizzeri, Vito; Bua, Daniel G; Maggio, Antonella; Cicero, Nicola; Dugo, Giacomo

    2017-05-01

    Globe artichoke has been long considered a nutraceutical food for its valuable content of bioactive compounds. However, beside a well-known polyphenol profile, poor information is available about its metabolite and mineral composition. The aim of this study was to investigate edible parts of Sicilian artichokes, 'Spinoso di Menfi' and 'Violetto di Niscemi', by (1)H NMR and ICP-MS for elucidating these compositional aspects. Although bracts and hearts of both artichokes shared a very similar metabolite pattern, 'Spinoso di Menfi' showed a higher number of metabolites, such as amino acids and polyphenols, than 'Violetto di Niscemi'. 'Spinoso di Menfi' was also marked by higher levels of macro- and microelements when compared to 'Violetto di Niscemi'. Also, artichoke heart demonstrated to accumulate higher mineral levels than bracts. (1)H NMR and ICP-MS successfully profiled metabolites and metals in such plant food, partially covering the lack of literature data about 'Spinoso di Menfi' and 'Violetto di Niscemi' artichokes.

  17. NMR-based metabonomics reveals relationship between pre-slaughter exercise stress, the plasma metabolite profile at time of slaughter, and water-holding capacity in pigs.

    PubMed

    Bertram, H C; Oksbjerg, N; Young, J F

    2010-01-01

    Nuclear magnetic resonance (NMR)-based metabonomics was applied to investigate the effects of pre-slaughter exercise stress on the plasma metabolite profile at time of slaughter. The study included a total of 40 slaughter pigs, which were exposed to one of the following treatments: No pre-slaughter stress (control treatment), pre-slaughter exercise on a treadmill and subsequently 0, 1, or 3h rest prior to slaughter. NMR-based metabonomics revealed a clear difference in the plasma metabolite profile at time of slaughter between control pigs and pigs exercised without rest, which mainly could be ascribed to increased plasma lactate due to exercise. A resting period of 1 or 3h prior to slaughter reversed the stress-induced perturbations in the plasma metabolite profile. The plasma metabolite profile at time of slaughter was highly correlated with muscle temperature 1 min post-mortem, and a correlation to WHC was also demonstrated. Lactate was found to be the metabolite of importance for the association between the plasma metabolome and pH, temperature and WHC.

  18. Metabolite profiling of Clinacanthus nutans leaves extracts obtained from different drying methods by 1H NMR-based metabolomics

    NASA Astrophysics Data System (ADS)

    Hashim, Noor Haslinda Noor; Latip, Jalifah; Khatib, Alfi

    2016-11-01

    The metabolites of Clinacanthus nutans leaves extracts and their dependence on drying process were systematically characterized using 1H nuclear magnetic resonance spectroscopy (NMR) multivariate data analysis. Principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were able to distinguish the leaves extracts obtained from different drying methods. The identified metabolites were carbohydrates, amino acid, flavonoids and sulfur glucoside compounds. The major metabolites responsible for the separation in PLS-DA loading plots were lupeol, cycloclinacosides, betulin, cerebrosides and choline. The results showed that the combination of 1H NMR spectroscopy and multivariate data analyses could act as an efficient technique to understand the C. nutans composition and its variation.

  19. Identification of Metabolites from LC-EC Profiling: GC-MS and Re-Fractionation Provide Essential Information Orthogonal to LCMS/microNMR

    PubMed Central

    Gathungu, Rose M.; Bird, Susan S.; Sheldon, Diane P.; Kautz, Roger; Vouros, Paul; Matson, Wayne R.; Kristal, Bruce S.

    2014-01-01

    HPLC-coulometric electrode-array detection (LC-EC) is a sensitive, quantitative and robust metabolomics profiling tool that complements the commonly used MS and NMR-based approaches. However, LC-EC provides little structural information. We recently demonstrated a workflow for the structural characterization of metabolites detected by LC-EC profiling, combined with LC-ESI-MS and microNMR. This methodology is now extended to include: (i) GC-EI-MS analysis to fill structural gaps left by LC-ESI-MS and NMR, and (ii) secondary fractionation of LC-collected fractions containing multiple co-eluting analytes. GC-EI-MS spectra have more informative fragment ions that are reproducible for database searches. Secondary fractionation provides enhanced metabolite characterization by reducing spectral overlap in NMR and ion-suppression in LC-ESI-MS. The need for these additional methods in the analysis of the broad chemical classes and concentration ranges found in plasma is illustrated with discussion of four specific examples, including: (i) characterization of compounds for which one or more of the detectors is insensitive (e.g., positional isomers in LC-MS, the direct detection of carboxylic groups and sulfonic groups in 1H NMR, or non-volatile species in GC-MS).; (ii) detection of labile compounds, (iii) resolution of closely eluting and/or co-eluting compounds and, (iv) the capability to harness structural similarities common in many biologically-related, LC-EC detectable compounds. PMID:24657819

  20. NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation

    SciTech Connect

    Wu, Junfang; Domellöf, Magnus; Zivkovic, Angela M.; Larsson, Göran; Öhman, Anders; Nording, Malin L.

    2016-01-15

    Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol/water (MeOH/H{sub 2}O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H{sub 2}O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. The human milk metabolomes at the early (9–24 days after delivery) and late (31–87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant. - Highlights: • 36 metabolites were simultaneously quantified in human milk by NMR. • Ultrafiltration more efficiently reduces interferences than MeOH/H{sub 2}O extraction. • Compositional changes of the human milk exist during the matured lactation stage.

  1. NMR-based metabolite profiling of human milk: A pilot study of methods for investigating compositional changes during lactation.

    PubMed

    Wu, Junfang; Domellöf, Magnus; Zivkovic, Angela M; Larsson, Göran; Öhman, Anders; Nording, Malin L

    2016-01-15

    Low-molecular-weight metabolites in human milk are gaining increasing interest in studies of infant nutrition. In the present study, the milk metabolome from a single mother was explored at different stages of lactation. Metabolites were extracted from sample aliquots using either methanol/water (MeOH/H2O) extraction or ultrafiltration. Nuclear magnetic resonance (NMR) spectroscopy was used for metabolite identification and quantification, and multi- and univariate statistical data analyses were used to detect changes over time of lactation. Compared to MeOH/H2O extraction, ultrafiltration more efficiently reduced the interference from lipid and protein resonances, thereby enabling the identification and quantification of 36 metabolites. The human milk metabolomes at the early (9-24 days after delivery) and late (31-87 days after delivery) stages of lactation were distinctly different according to multi- and univariate statistics. The late lactation stage was characterized by significantly elevated concentrations of lactose, choline, alanine, glutamate, and glutamine, as well as by reduced levels of citrate, phosphocholine, glycerophosphocholine, and N-acetylglucosamine. Our results indicate that there are significant compositional changes of the human milk metabolome also in different phases of the matured lactation stage. These findings complement temporal studies on the colostrum and transitional metabolome in providing a better understanding of the nutritional variations received by an infant.

  2. Software-assisted serum metabolite quantification using NMR.

    PubMed

    Jung, Young-Sang; Hyeon, Jin-Seong; Hwang, Geum-Sook

    2016-08-31

    The goal of metabolomics is to analyze a whole metabolome under a given set of conditions, and accurate and reliable quantitation of metabolites is crucial. Absolute concentration is more valuable than relative concentration; however, the most commonly used method in NMR-based serum metabolic profiling, bin-based and full data point peak quantification, provides relative concentration levels of metabolites and are not reliable when metabolite peaks overlap in a spectrum. In this study, we present the software-assisted serum metabolite quantification (SASMeQ) method, which allows us to identify and quantify metabolites in NMR spectra using Chenomx software. This software uses the ERETIC2 utility from TopSpin to add a digitally synthesized peak to a spectrum. The SASMeQ method will advance NMR-based serum metabolic profiling by providing an accurate and reliable method for absolute quantification that is superior to bin-based quantification. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. A NMR-based, non-targeted multistep metabolic profiling revealed L-rhamnitol as a metabolite that characterised apples from different geographic origins.

    PubMed

    Tomita, Satoru; Nemoto, Tadashi; Matsuo, Yosuke; Shoji, Toshihiko; Tanaka, Fukuyo; Nakagawa, Hiroyuki; Ono, Hiroshi; Kikuchi, Jun; Ohnishi-Kameyama, Mayumi; Sekiyama, Yasuyo

    2015-05-01

    This study utilises (1)H NMR-based metabolic profiling to characterise apples of five cultivars grown either in Japan (Fuji, Orin, and Jonagold) or New Zealand (Fuji, Jazz, and Envy). Principal component analysis (PCA) showed a clear separation between the Fuji-Orin-Jonagold class and the Jazz-Envy class, primarily corresponding to the differences in sugar signals, such as sucrose, glucose, and fructose. Multistep PCA removed the influence of dominant sugars and highlighted minor metabolites such as aspartic acid, 2-methylmalate, and an unidentified compound. These minor metabolites separated the apples into two classes according to different geographical areas. Subsequent partial least squares discriminant analysis (PLS-DA) indicated the importance of the unidentified metabolite. This metabolite was isolated using charcoal chromatography, and was identified as L-rhamnitol by 2D NMR and LC/MS analyses. The remarkable contribution of L-rhamnitol to geographic discrimination suggests that apples may be characterised according to various factors, including storage duration, cultivation method, and climate. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Identification of metabolites from liquid chromatography-coulometric array detection profiling: gas chromatography-mass spectrometry and refractionation provide essential information orthogonal to LC-MS/microNMR.

    PubMed

    Gathungu, Rose M; Bird, Susan S; Sheldon, Diane P; Kautz, Roger; Vouros, Paul; Matson, Wayne R; Kristal, Bruce S

    2014-06-01

    Liquid chromatography-coulometric array detection (LC-EC) is a sensitive, quantitative, and robust metabolomics profiling tool that complements the commonly used mass spectrometry (MS) and nuclear magnetic resonance (NMR)-based approaches. However, LC-EC provides little structural information. We recently demonstrated a workflow for the structural characterization of metabolites detected by LC-EC profiling combined with LC-electrospray ionization (ESI)-MS and microNMR. This methodology is now extended to include (i) gas chromatography (GC)-electron ionization (EI)-MS analysis to fill structural gaps left by LC-ESI-MS and NMR and (ii) secondary fractionation of LC-collected fractions containing multiple coeluting analytes. GC-EI-MS spectra have more informative fragment ions that are reproducible for database searches. Secondary fractionation provides enhanced metabolite characterization by reducing spectral overlap in NMR and ion suppression in LC-ESI-MS. The need for these additional methods in the analysis of the broad chemical classes and concentration ranges found in plasma is illustrated with discussion of four specific examples: (i) characterization of compounds for which one or more of the detectors is insensitive (e.g., positional isomers in LC-MS, the direct detection of carboxylic groups and sulfonic groups in (1)H NMR, or nonvolatile species in GC-MS), (ii) detection of labile compounds, (iii) resolution of closely eluting and/or coeluting compounds, and (iv) the capability to harness structural similarities common in many biologically related, LC-EC-detectable compounds. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. 1H NMR Based Targeted Metabolite Profiling for Understanding the Complex Relationship Connecting Oxidative Stress with Endometriosis

    PubMed Central

    Jana, Saikat K.; Srivastava, Sudha; Chakravarty, Baidyanath

    2013-01-01

    Accumulating evidence indicates the active role of oxidative stress in the development of endometriosis; however, the mechanism of reactive oxygen species generation is poorly understood. Metabonomics/metabolomics is a scientific discipline that can be used to study changes in metabolite ensembles associated with disease pathophysiology. The present study focuses on the use of proton nuclear magnetic resonance spectroscopy based targeted metabolite profiling approach to explore dysregulation in metabolites expression in women with endometriosis. Further, association of oxidative stress with the metabolite ensembles, if any, is investigated. Using multivariate statistics, partial least square discriminant analysis model was generated which could classify endometriosis patients with sensitivity and specificity of 92.83% and 100%, respectively, and with a classification rate of 96.4%. In conjunction with increased glucose metabolism, citrate and succinate were found to be elevated in endometriosis patients. Higher levels of reactive oxygen species, lipid peroxidation, and advanced oxidation protein products and lower levels of total antioxidant capacity, superoxide dismutase, catalase, and glutathione were also observed. Increased glucose metabolism and defects in the mitochondrial respiratory system are suggested to be the possible sources of excessive reactive oxygen species generation in endometriosis. PMID:23984345

  6. The Use of NMR Metabolite Profiling and in vivo Hypoglycemic Assay for Comparison of Unfractionated Aqueous Leaf Extracts of Two Ocimum Species.

    PubMed

    Casanova, Livia Marques; Espíndola-Netto, Jair Machado; Tinoco, Luzineide Wanderley; Sola-Penna, Mauro; Costa, Sônia Soares

    2016-06-01

    Ocimum basilicum and Ocimum gratissimum (Lamiaceae) are used to treat diabetes mellitus in Africa. In a previous work, we identified chicoric acid as a hypoglycemic substance in O. gratissimum. This study aims to compare the chemical metabolite profile and the hypoglycemic activity of unfractionated aqueous extracts from leaves of both Lamiaceae species. The metabolite composition of OB and OG decoctions (10% w/v) was analyzed using HPLC-DAD and NMR tools. Chicoric acid showed to be the major phenolic in both extracts, besides caftaric, caffeic, and rosmarinic acids; nevertheless, there is approximately three times more of this substance in OG. From 1D- and 2D-NMR analyses, 19 substances were identified in OB, while 12 in OG. The in vivo acute hypoglycemic activity of the extracts was assessed intraperitoneally in streptozotocin (STZ)-induced diabetic mice. The doses of 100 and 200 mg/kg of both extracts significantly reduced their glycemia, compared to controls (P < 0.05). OB was a little more effective than OG, despite the lower content of chicoric acid in OB. This result strongly suggests that components other than chicoric acid contribute to the hypoglycemic activity of the two extracts. Despite the abundance of caffeic and rosmarinic acids in OB, their hypoglycemic activity observed at 8.3 μmol/kg was low. This is the first chemical profile of crude extracts from Ocimum species by NMR. Our findings confirmed the potential of both species in DM treatment in spite of marked differences in their chemical composition. However, long-term studies are necessary in order to identify the most promising of the two species for the development of an herbal medicine.

  7. Metabolite profiling in Arabidopsis.

    PubMed

    Fiehn, Oliver

    2006-01-01

    Metabolite profiling is the multiparallel relative quantification of a mixture of compounds or compound classes using chromatography and universal detection technologies (GC-MS, LC-MS). In this respect it is an extension of classical single-target methods from which it can be distinguished by its broader view on profiling major biochemical events. This broader scope of analysis outweighs the disadvantages by making compromises in method development and the reduced accuracy for specific metabolites. This chapter exemplifies the strategies in metabolite profiling of polar compounds by gas chromatography-mass spectrometry (GC-MS). It gives experimental details on the basic steps: harvest, homogenization, extraction, fractionation, concentration, derivatization, data acquisition, raw data processing and result data tranformation.

  8. Monitoring Metabolite Profiles of Cannabis sativa L. Trichomes during Flowering Period Using 1H NMR-Based Metabolomics and Real-Time PCR.

    PubMed

    Happyana, Nizar; Kayser, Oliver

    2016-08-01

    Cannabis sativa trichomes are glandular structures predominantly responsible for the biosynthesis of cannabinoids, the biologically active compounds unique to this plant. To the best of our knowledge, most metabolomic works on C. sativa that have been reported previously focused their investigations on the flowers and leaves of this plant. In this study, (1)H NMR-based metabolomics and real-time PCR analysis were applied for monitoring the metabolite profiles of C. sativa trichomes, variety Bediol, during the last 4 weeks of the flowering period. Partial least squares discriminant analysis models successfully classified metabolites of the trichomes based on the harvest time. Δ (9)-Tetrahydrocannabinolic acid (1) and cannabidiolic acid (2) constituted the vital differential components of the organic preparations, while asparagine, glutamine, fructose, and glucose proved to be their water-extracted counterparts. According to RT-PCR analysis, gene expression levels of olivetol synthase and olivetolic acid cyclase influenced the accumulation of cannabinoids in the Cannabis trichomes during the monitoring time. Moreover, quantitative (1)H NMR and RT-PCR analysis of the Cannabis trichomes suggested that the gene regulation of cannabinoid biosynthesis in the C. sativa variety Bediol is unique when compared with other C. sativa varieties. Georg Thieme Verlag KG Stuttgart · New York.

  9. Defensive Armor of Potato Tubers: Nonpolar Metabolite Profiling, Antioxidant Assessment, and Solid-State NMR Compositional Analysis of Suberin-Enriched Wound-Healing Tissues

    PubMed Central

    Dastmalchi, Keyvan; Kallash, Linda; Wang, Isabel; Phan, Van C.; Huang, Wenlin; Serra, Olga; Stark, Ruth E.

    2016-01-01

    The cultivation, storage, and distribution of potato tubers are compromised by mechanical damage and suboptimal healing. To investigate wound-healing progress in cultivars with contrasting russeting patterns, metabolite profiles reported previously for polar tissue extracts were complemented by GC/MS measurements for nonpolar extracts and quantitative 13C NMR of interfacial solid suspensions. Potential marker compounds that distinguish cultivar type and wound-healing time point included fatty acids, fatty alcohols, alkanes, glyceryl esters, α,ω-fatty diacids, and hydroxyfatty acids. The abundant long-chain fatty acids in nonpolar extracts and solids from the smooth-skinned Yukon Gold cultivar suggested extensive suberin biopolymer formation; this hypothesis was supported by high proportions of arenes, alkenes, and carbonyl groups in the solid and among the polar markers. The absence of many potential marker classes in nonpolar Atlantic extracts and interfacial solids suggested a limited extent of suberization. Modest scavenging activities of all nonpolar extracts indicate that the majority of antioxidants produced in response to wounding are polar. PMID:26166447

  10. Defensive Armor of Potato Tubers: Nonpolar Metabolite Profiling, Antioxidant Assessment, and Solid-State NMR Compositional Analysis of Suberin-Enriched Wound-Healing Tissues.

    PubMed

    Dastmalchi, Keyvan; Kallash, Linda; Wang, Isabel; Phan, Van C; Huang, Wenlin; Serra, Olga; Stark, Ruth E

    2015-08-05

    The cultivation, storage, and distribution of potato tubers are compromised by mechanical damage and suboptimal healing. To investigate wound-healing progress in cultivars with contrasting russeting patterns, metabolite profiles reported previously for polar tissue extracts were complemented by GC/MS measurements for nonpolar extracts and quantitative (13)C NMR of interfacial solid suspensions. Potential marker compounds that distinguish cultivar type and wound-healing time point included fatty acids, fatty alcohols, alkanes, glyceryl esters, α,ω-fatty diacids, and hydroxyfatty acids. The abundant long-chain fatty acids in nonpolar extracts and solids from the smooth-skinned Yukon Gold cultivar suggested extensive suberin biopolymer formation; this hypothesis was supported by high proportions of arenes, alkenes, and carbonyl groups in the solid and among the polar markers. The absence of many potential marker classes in nonpolar Atlantic extracts and interfacial solids suggested a limited extent of suberization. Modest scavenging activities of all nonpolar extracts indicate that the majority of antioxidants produced in response to wounding are polar.

  11. Metabolite profiling of tomato (Lycopersicon esculentum) using 1H NMR spectroscopy as a tool to detect potential unintended effects following a genetic modification.

    PubMed

    Le Gall, Gwénaëlle; Colquhoun, Ian J; Davis, Adrienne L; Collins, Geoff J; Verhoeyen, Martine E

    2003-04-23

    The maize transcription factors LC and C1 were simultaneously overexpressed in tomato with the aim of producing lines with increased amounts of flavonols. The metabolite composition of these genetically modified tomatoes has been compared with that of azygous (nonmodified) controls grown side-by-side under the same conditions. It has been possible to observe metabolic changes in both types at different stages of maturity. (1)H NMR spectra showed that the levels of glutamic acid, fructose, and some nucleosides and nucleotides gradually increase from the immature to the ripe stage, whereas some amino acids such as valine and gamma-aminobutyric acid were present in higher amounts in unripe tomatoes. Apart from the significantly increased content of six main flavonoid glycosides (mainly kaempferol-3-O-rutinoside, with additional increases in kaempferol-3,7-di-O-glucoside (1), kaempferol-3-O-rutinoside-7-O-glucoside (2), kaempferol-3-O-glucoside, a dihydrokaempferol-O-hexoside (3), and naringenin-7-O-glucoside), the levels of at least 15 other metabolites were found to be different between the two types of red tomato. Among them were citric acid, sucrose, phenylalanine, and trigonelline. However, although statistically significant, these changes in mean values were relatively minor (less than 3-fold) and within the natural variation that would be observed in a field-grown crop. Nevertheless, this study clearly showed that NMR combined with chemometrics and univariate statistics can successfully trace even small differences in metabolite levels between plants and therefore represents a powerful tool to detect potential unintended effects in genetically modified crops.

  12. Toward an in Vivo Neurochemical Profile: Quantification of 18 Metabolites in Short-Echo-Time 1H NMR Spectra of the Rat Brain

    NASA Astrophysics Data System (ADS)

    Pfeuffer, Josef; Tkáč , Ivan; Provencher, Stephen W.; Gruetter, Rolf

    1999-11-01

    Localized in vivo1H NMR spectroscopy was performed with 2-ms echo time in the rat brain at 9.4 T. Frequency domain analysis with LCModel showed that the in vivo spectra can be explained by 18 metabolite model solution spectra and a highly structured background, which was attributed to resonances with fivefold shorter in vivo T1 than metabolites. The high spectral resolution (full width at half maximum approximately 0.025 ppm) and sensitivity (signal-to-noise ratio approximately 45 from a 63-μL volume, 512 scans) was used for the simultaneous measurement of the concentrations of metabolites previously difficult to quantify in 1H spectra. The strongly represented signals of N-acetylaspartate, glutamate, taurine, myo-inositol, creatine, phosphocreatine, glutamine, and lactate were quantified with Cramér-Rao lower bounds below 4%. Choline groups, phosphorylethanolamine, glucose, glutathione, γ-aminobutyric acid, N-acetylaspartylglutamate, and alanine were below 13%, whereas aspartate and scyllo-inositol were below 22%. Intra-assay variation was assessed from a time series of 3-min spectra, and the coefficient of variation was similar to the calculated Cramér-Rao lower bounds. Interassay variation was determined from 31 pooled spectra, and the coefficient of variation for total creatine was 7%. Tissue concentrations were found to be in very good agreement with neurochemical data from the literature.

  13. Metabolite profiling of a NIST Standard Reference Material for human plasma (SRM 1950): GC-MS, LC-MS, NMR, and clinical laboratory analyses, libraries, and web-based resources.

    PubMed

    Simón-Manso, Yamil; Lowenthal, Mark S; Kilpatrick, Lisa E; Sampson, Maureen L; Telu, Kelly H; Rudnick, Paul A; Mallard, W Gary; Bearden, Daniel W; Schock, Tracey B; Tchekhovskoi, Dmitrii V; Blonder, Niksa; Yan, Xinjian; Liang, Yuxue; Zheng, Yufang; Wallace, William E; Neta, Pedatsur; Phinney, Karen W; Remaley, Alan T; Stein, Stephen E

    2013-12-17

    Recent progress in metabolomics and the development of increasingly sensitive analytical techniques have renewed interest in global profiling, i.e., semiquantitative monitoring of all chemical constituents of biological fluids. In this work, we have performed global profiling of NIST SRM 1950, "Metabolites in Human Plasma", using GC-MS, LC-MS, and NMR. Metabolome coverage, difficulties, and reproducibility of the experiments on each platform are discussed. A total of 353 metabolites have been identified in this material. GC-MS provides 65 unique identifications, and most of the identifications from NMR overlap with the LC-MS identifications, except for some small sugars that are not directly found by LC-MS. Also, repeatability and intermediate precision analyses show that the SRM 1950 profiling is reproducible enough to consider this material as a good choice to distinguish between analytical and biological variability. Clinical laboratory data shows that most results are within the reference ranges for each assay. In-house computational tools have been developed or modified for MS data processing and interactive web display. All data and programs are freely available online at http://peptide.nist.gov/ and http://srmd.nist.gov/ .

  14. Metabolic responses of willow (Salix purpurea L.) leaves to mycorrhization as revealed by mass spectrometry and 1H NMR spectroscopy metabolite profiling

    PubMed Central

    Aliferis, Konstantinos A.; Chamoun, Rony; Jabaji, Suha

    2015-01-01

    The root system of most terrestrial plants form symbiotic interfaces with arbuscular mycorrhizal fungi (AMF), which are important for nutrient cycling and ecosystem sustainability. The elucidation of the undergoing changes in plants' metabolism during symbiosis is essential for understanding nutrient acquisition and for alleviation of soil stresses caused by environmental cues. Within this context, we have undertaken the task of recording the fluctuation of willow (Salix purpurea L.) leaf metabolome in response to AMF inoculation. The development of an advanced metabolomics/bioinformatics protocol employing mass spectrometry (MS) and 1H NMR analyzers combined with the in-house-built metabolite library for willow (http://willowmetabolib.research.mcgill.ca/index.html) are key components of the research. Analyses revealed that AMF inoculation of willow causes up-regulation of various biosynthetic pathways, among others, those of flavonoid, isoflavonoid, phenylpropanoid, and the chlorophyll and porphyrin pathways, which have well-established roles in plant physiology and are related to resistance against environmental stresses. The recorded fluctuation in the willow leaf metabolism is very likely to provide AMF-inoculated willows with a significant advantage compared to non-inoculated ones when they are exposed to stresses such as, high levels of soil pollutants. The discovered biomarkers of willow response to AMF inoculation and corresponding pathways could be exploited in biomarker-assisted selection of willow cultivars with superior phytoremediation capacity or genetic engineering programs. PMID:26042135

  15. rNMR: open source software for identifying and quantifying metabolites in NMR spectra

    PubMed Central

    Lewis, Ian A; Schommer, Seth C; Markley, John L

    2009-01-01

    Despite the extensive use of nuclear magnetic resonance (NMR) for metabolomics, no publicly available tools have been designed for identifying and quantifying metabolites across multiple spectra. We introduce here a new open source software tool, rNMR, which provides a simple graphics-based method for visualizing, identifying, and quantifying metabolites across multiple one- or two-dimensional NMR spectra. rNMR differs from existing software tools for NMR spectroscopy in that analyses are based on regions of interest (ROIs) rather than peak lists. ROIs contain all of the underlying NMR data within user-defined chemical shift ranges. ROIs can be inspected visually, and they support robust quantification of NMR signals. ROI-based analyses support simultaneous views of metabolite signals from up to hundreds of spectra, and ROI boundaries can be adjusted dynamically to ensure that signals corresponding to assigned atoms are analyzed consistently throughout the dataset. We describe how rNMR greatly reduces the time required for robust bioanalytical analysis of complex NMR data. An rNMR analysis yields a compact and transparent way of archiving the results from a metabolomics study so that it can be examined and evaluated by others. The rNMR website at http://rnmr.nmrfam.wisc.edu offers downloadable versions of rNMR for Windows, Macintosh, and Linux platforms along with extensive help documentation, instructional videos, and sample data. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19821464

  16. Metabolite localization in living drosophila using High Resolution Magic Angle Spinning NMR

    PubMed Central

    Sarou-Kanian, Vincent; Joudiou, Nicolas; Louat, Fanny; Yon, Maxime; Szeremeta, Frédéric; Même, Sandra; Massiot, Dominique; Decoville, Martine; Fayon, Franck; Beloeil, Jean-Claude

    2015-01-01

    We have developed new methods enabling in vivo localization and identification of metabolites through their 1H NMR signatures, in a drosophila. Metabolic profiles in localized regions were obtained using HR-MAS Slice Localized Spectroscopy and Chemical Shift Imaging at high magnetic fields. These methods enabled measurement of metabolite contents in anatomic regions of the fly, demonstrated by a decrease in β-alanine signals in the thorax of flies showing muscle degeneration. PMID:25892587

  17. Metabolite localization in living drosophila using High Resolution Magic Angle Spinning NMR.

    PubMed

    Sarou-Kanian, Vincent; Joudiou, Nicolas; Louat, Fanny; Yon, Maxime; Szeremeta, Frédéric; Même, Sandra; Massiot, Dominique; Decoville, Martine; Fayon, Franck; Beloeil, Jean-Claude

    2015-04-20

    We have developed new methods enabling in vivo localization and identification of metabolites through their (1)H NMR signatures, in a drosophila. Metabolic profiles in localized regions were obtained using HR-MAS Slice Localized Spectroscopy and Chemical Shift Imaging at high magnetic fields. These methods enabled measurement of metabolite contents in anatomic regions of the fly, demonstrated by a decrease in β-alanine signals in the thorax of flies showing muscle degeneration.

  18. NMR ANALYSIS OF MALE FATHEAD MINNOW URINARY METABOLITES: A POTENTIAL APPROACH FOR STUDYING IMPACTS OF CHEMICAL EXPOSURES

    EPA Science Inventory

    The potential for profiling endogenous metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one dimensional (1D) and two dimensional (2D) NMR spectroscopy w...

  19. NMR ANALYSIS OF MALE FATHEAD MINNOW URINARY METABOLITES: A POTENTIAL APPROACH FOR STUDYING IMPACTS OF CHEMICAL EXPOSURES

    EPA Science Inventory

    The potential for profiling endogenous metabolites in urine from male fathead minnows (Pimephales promelas) to assess chemical exposures was explored using nuclear magnetic resonance (NMR) spectroscopy. Both one dimensional (1D) and two dimensional (2D) NMR spectroscopy w...

  20. Metabolite analysis of Cannabis sativa L. by NMR spectroscopy.

    PubMed

    Flores-Sanchez, Isvett Josefina; Choi, Young Hae; Verpoorte, Robert

    2012-01-01

    NMR-based metabolomics is an analytical platform, which has been used to classify and analyze Cannabis sativa L. cell suspension cultures and plants. Diverse groups of primary and secondary metabolites were identified by comparing NMR data with reference compounds and/or by structure elucidation using ¹H-NMR, J-resolved, ¹H-¹H COSY, and ¹H-¹³C HMBC spectroscopy. The direct extraction and the extraction by indirect fractionation are two suitable methods for the C. sativa sample preparation. Quantitative analyses could be performed without requiring fractionation or isolation procedures.

  1. NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction.

    PubMed

    Jupin, Marc; Michiels, Paul J; Girard, Frederic C; Spraul, Manfred; Wijmenga, Sybren S

    2013-03-01

    Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (∼60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find

  2. NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction

    NASA Astrophysics Data System (ADS)

    Jupin, Marc; Michiels, Paul J.; Girard, Frederic C.; Spraul, Manfred; Wijmenga, Sybren S.

    2013-03-01

    Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (∼60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find

  3. Nuclear Magnetic Resonance (NMR) Spectroscopy For Metabolic Profiling of Medicinal Plants and Their Products.

    PubMed

    Kumar, Dinesh

    2016-09-02

    NMR spectroscopy has multidisciplinary applications, including excellent impact in metabolomics. The analytical capacity of NMR spectroscopy provides information for easy qualitative and quantitative assessment of both endogenous and exogenous metabolites present in biological samples. The complexity of a particular metabolite and its contribution in a biological system are critically important for understanding the functional state that governs the organism's phenotypes. This review covers historical aspects of developments in the NMR field, its applications in chemical profiling, metabolomics, and quality control of plants and their derived medicines, foods, and other products. The bottlenecks of NMR in metabolic profiling are also discussed, keeping in view the future scope and further technological interventions.

  4. Identifying metabolites related to nitrogen mineralisation using 1H NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    . T McDonald, Noeleen; Graham, Stewart; Watson, Catherine; Gordon, Alan; Lalor, Stan; Laughlin, Ronnie; Elliott, Chris; . P Wall, David

    2015-04-01

    Exploring new analysis techniques to enhance our knowledge of the various metabolites within our soil systems is imperative. Principally, this knowledge would allow us to link key metabolites with functional influences on critical nutrient processes, such as the nitrogen (N) mineralisation in soils. Currently there are few studies that utilize proton nuclear magnetic resonance spectroscopy (1H NMR) to characterize multiple metabolites within a soil sample. The aim of this research study was to examine the effectiveness of 1H NMR for isolating multiple metabolites that are related to the mineralizable N (MN) capacity across a range of 35 Irish grassland soils. Soils were measured for MN using the standard seven day anaerobic incubation (AI-7). Additionally, soils were also analysed for a range of physio-chemical properties [e.g. total N, total C, mineral N, texture and soil organic matter (SOM)]. Proton NMR analysis was carried on these soils by extracting with 40% methanol:water, lyophilizing and reconstituting in deuterium oxide and recording the NMR spectra on a 400MHz Bruker AVANCE III spectrometer. Once the NMR data were spectrally processed and analysed using multivariate statistical analysis, seven metabolites were identified as having significant relationships with MN (glucose, trimethylamine, glutamic acid, serine, aspartic acid, 4-aminohippuirc acid and citric acid). Following quantification, glucose was shown to explain the largest percentage variability in MN (72%). These outcomes suggest that sources of labile carbon are essential in regulating N mineralisation and the capacity of plant available N derived from SOM-N pools in these soils. Although, smaller in concentration, the amino acids; 4-aminohippuirc acid, glutamic acid and serine also significantly (P<0.05) explained 43%, 27% and 19% of the variability in MN, respectively. This novel study highlights the effectiveness of using 1H NMR as a practical approach to profile multiple metabolites in

  5. CHARACTERIZATION OF METABOLITES IN SMALL FISH BIOFLUIDS AND TISSUES BY NMR SPECTROSCOPY

    EPA Science Inventory

    Nuclear magnetic resonance (NMR) spectroscopy has been utilized for assessing ecotoxicity in small fish models by means of metabolomics. Two fundamental challenges of NMR-based metabolomics are the detection limit and characterization of metabolites (or NMR resonance assignments...

  6. CHARACTERIZATION OF METABOLITES IN SMALL FISH BIOFLUIDS AND TISSUES BY NMR SPECTROSCOPY

    EPA Science Inventory

    Nuclear magnetic resonance (NMR) spectroscopy has been utilized for assessing ecotoxicity in small fish models by means of metabolomics. Two fundamental challenges of NMR-based metabolomics are the detection limit and characterization of metabolites (or NMR resonance assignments...

  7. NMR-Profiles of Protein Solutions

    PubMed Central

    Pedrini, Bill; Serrano, Pedro; Mohanty, Biswaranjan; Geralt, Michael; Wüthrich, Kurt

    2014-01-01

    NMR-Profiles are quantitative one-dimensional presentations of two-dimensional [15N,1H]-correlation spectra used to monitor the quality of protein solutions prior to and during NMR structure determinations and functional studies. In our current use in structural genomics projects, a NMR-Profile is recorded at the outset of a structure determination, using a uniformly 15N-labeled micro-scale sample of the protein. We thus assess the extent to which polypeptide backbone resonance assignments can be achieved with given NMR techniques, for example, conventional triple resonance experiments or APSY-NMR. With the availability of sequence-specific polypeptide backbone resonance assignments in the course of the structure determination, an “Assigned NMR-Profile” is generated, which visualizes the variation of the 15N–1H correlation cross peak intensities along the sequence and thus maps the sequence locations of polypeptide segments for which the NMR line shapes are affected by conformational exchange or other processes. The Assigned NMR-Profile provides a guiding reference during later stages of the structure determination, and is of special interest for monitoring the protein during functional studies, where dynamic features may be modulated during physiological functions. PMID:23839514

  8. New generation NMR bioreactor coupled with high-resolution NMR spectroscopy leads to novel discoveries in Moorella thermoaceticum metabolic profiles

    SciTech Connect

    Xue, Junfeng; Isern, Nancy G.; Ewing, R James; Liyu, Andrey V.; Sears, Jesse A.; Knapp, Harlan; Iversen, Jens; Sisk, Daniel R.; Ahring, Birgitte K.; Majors, Paul D.

    2014-06-20

    An in-situ nuclear magnetic resonance (NMR) bioreactor was developed and employed to monitor microbial metabolism under batch-growth conditions in real time. We selected Moorella thermoacetica ATCC 49707 as a test case. M. thermoacetica (formerly Clostridium thermoaceticum) is a strictly anaerobic, thermophilic, acetogenic, gram-positive bacterium with potential for industrial production of chemicals. The metabolic profiles of M. thermoacetica were characterized during growth in batch mode on xylose (a component of lignocellulosic biomass) using the new generation NMR bioreactor in combination with high-resolution, high sensitivity NMR (HR-NMR) spectroscopy. In-situ NMR measurements were performed using water-suppressed H-1 NMR spectroscopy at an NMR frequency of 500 MHz, and aliquots of the bioreactor contents were taken for 600 MHz HR-NMR spectroscopy at specific intervals to confirm metabolite identifications and expand metabolite coverage. M. thermoacetica demonstrated the metabolic potential to produce formate, ethanol and methanol from xylose, in addition to its known capability of producing acetic acid. Real-time monitoring of bioreactor conditions showed a temporary pH decrease, with a concomitant increase in formic acid during exponential growth. Fermentation experiments performed outside of the magnet showed that the strong magnetic field employed for NMR detection did not significantly affect cell metabolism. Use of the in-situ NMR bioreactor facilitated monitoring of the fermentation process in real time, enabling identification of intermediate and end-point metabolites and their correlation with pH and biomass produced during culture growth. Real-time monitoring of culture metabolism using the NMR bioreactor in combination with the HR-NMR spectroscopy will allow optimization of the metabolism of microorganisms producing valuable bioproducts.

  9. Discrimination of wild types and hybrids of Duboisia myoporoides and Duboisia leichhardtii at different growth stages using (1)H NMR-based metabolite profiling and tropane alkaloids-targeted HPLC-MS analysis.

    PubMed

    Ullrich, Sophie Friederike; Averesch, Nils J H; Castellanos, Leonardo; Choi, Young Hae; Rothauer, Andreas; Kayser, Oliver

    2016-11-01

    Duboisia species, which belong to the family of Solanaceae, are commercially cultivated in large scale, as they are main source of the pharmaceutically-used active compound scopolamine. In this study, (1)H NMR-based metabolite profiling linking primary with secondary metabolism and additional quantification via HPCL-MS with special focus on the tropane alkaloids were applied to compare leaf and root extracts of three wild types and two hybrids of Duboisia myoporoides and D. leichhardtii at different developmental stages grown under controlled conditions in climate chambers and under agricultural field plantation. Based on the leaf extracts, a clear distinction between the Duboisia hybrids and the wild types Duboisia myoporoides and D. leichhardtii using principal component analysis of (1)H NMR data was observed. The average content in scopolamine in the hybrids of Duboisia cultivated in climate chambers increased significantly from month 3-6 after potting of the rooted cuttings, however not so for the examined wild types. The Duboisia hybrids grown in climate chambers showed higher growth and contained more sugars and amino acids than Duboisia hybrids grown in the field, which in contrast showed an enhanced flux towards tropane alkaloids as well as flavonoids. For a more detailed analysis of tropane alkaloids, an appropriate HPLC-MS method was developed and validated. The measurements revealed large differences in the alkaloid pattern within the different genotypes under investigation, especially regarding the last enzymatic step, the conversion from hyoscamine to scopolamine by the hyoscyamine 6β-hydroxylase. Scopolamine was found in highest concentrations in Duboisia hybrids (20.04 ± 4.05 and 17.82 ± 3.52 mg/g dry wt) followed by Duboisia myoporoides (12.71 ± 2.55 mg/g dry wt), both showing a high selectivity for scopolamine in contrast to Duboisia leichhardtii (3.38 ± 0.59 and 5.09 ± 1.24 mg/g dry wt) with hyoscyamine being the

  10. The 1H NMR Profile of Healthy Dog Cerebrospinal Fluid

    PubMed Central

    Musteata, Mihai; Nicolescu, Alina; Solcan, Gheorghe; Deleanu, Calin

    2013-01-01

    The availability of data for reference values in cerebrospinal fluid for healthy humans is limited due to obvious practical and ethical issues. The variability of reported values for metabolites in human cerebrospinal fluid is quite large. Dogs present great similarities with humans, including in cases of central nervous system pathologies. The paper presents the first study on healthy dog cerebrospinal fluid metabolomic profile using 1H NMR spectroscopy. A number of 13 metabolites have been identified and quantified from cerebrospinal fluid collected from a group of 10 mix breed healthy dogs. The biological variability as resulting from the relative standard deviation of the physiological concentrations of the identified metabolites had a mean of 18.20% (range between 9.3% and 44.8%). The reported concentrations for metabolites may be used as normal reference values. The homogeneity of the obtained results and the low biologic variability show that the 1H NMR analysis of the dog’s cerebrospinal fluid is reliable in designing and interpreting clinical and therapeutic trials in dogs with central nervous system pathologies. PMID:24376499

  11. Metabolite Profiles During Oral Glucose Challenge

    PubMed Central

    Ho, Jennifer E.; Larson, Martin G.; Vasan, Ramachandran S.; Ghorbani, Anahita; Cheng, Susan; Rhee, Eugene P.; Florez, Jose C.; Clish, Clary B.; Gerszten, Robert E.; Wang, Thomas J.

    2013-01-01

    To identify distinct biological pathways of glucose metabolism, we conducted a systematic evaluation of biochemical changes after an oral glucose tolerance test (OGTT) in a community-based population. Metabolic profiling was performed on 377 nondiabetic Framingham Offspring cohort participants (mean age 57 years, 42% women, BMI 30 kg/m2) before and after OGTT. Changes in metabolite levels were evaluated with paired Student t tests, cluster-based analyses, and multivariable linear regression to examine differences associated with insulin resistance. Of 110 metabolites tested, 91 significantly changed with OGTT (P ≤ 0.0005 for all). Amino acids, β-hydroxybutyrate, and tricarboxylic acid cycle intermediates decreased after OGTT, and glycolysis products increased, consistent with physiological insulin actions. Other pathways affected by OGTT included decreases in serotonin derivatives, urea cycle metabolites, and B vitamins. We also observed an increase in conjugated, and a decrease in unconjugated, bile acids. Changes in β-hydroxybutyrate, isoleucine, lactate, and pyridoxate were blunted in those with insulin resistance. Our findings demonstrate changes in 91 metabolites representing distinct biological pathways that are perturbed in response to an OGTT. We also identify metabolite responses that distinguish individuals with and without insulin resistance. These findings suggest that unique metabolic phenotypes can be unmasked by OGTT in the prediabetic state. PMID:23382451

  12. Metabolite Profiling of Alzheimer's Disease Cerebrospinal Fluid

    PubMed Central

    Czech, Christian; Berndt, Peter; Busch, Kristina; Schmitz, Oliver; Wiemer, Jan; Most, Veronique; Hampel, Harald; Kastler, Jürgen; Senn, Hans

    2012-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive functions. Today the diagnosis of AD relies on clinical evaluations and is only late in the disease. Biomarkers for early detection of the underlying neuropathological changes are still lacking and the biochemical pathways leading to the disease are still not completely understood. The aim of this study was to identify the metabolic changes resulting from the disease phenotype by a thorough and systematic metabolite profiling approach. For this purpose CSF samples from 79 AD patients and 51 healthy controls were analyzed by gas and liquid chromatography-tandem mass spectrometry (GC-MS and LC-MS/MS) in conjunction with univariate and multivariate statistical analyses. In total 343 different analytes have been identified. Significant changes in the metabolite profile of AD patients compared to healthy controls have been identified. Increased cortisol levels seemed to be related to the progression of AD and have been detected in more severe forms of AD. Increased cysteine associated with decreased uridine was the best paired combination to identify light AD (MMSE>22) with specificity and sensitivity above 75%. In this group of patients, sensitivity and specificity above 80% were obtained for several combinations of three to five metabolites, including cortisol and various amino acids, in addition to cysteine and uridine. PMID:22359596

  13. Alterations of urinary metabolite profile in model diabetic nephropathy

    SciTech Connect

    Stec, Donald F.; Wang, Suwan; Stothers, Cody; Avance, Josh; Denson, Deon; Harris, Raymond; Voziyan, Paul

    2015-01-09

    Highlights: • {sup 1}H NMR spectroscopy was employed to study urinary metabolite profile in diabetic mouse models. • Mouse urinary metabolome showed major changes that are also found in human diabetic nephropathy. • These models can be new tools to study urinary biomarkers that are relevant to human disease. - Abstract: Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions characteristic of human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS{sup −/−} C57BLKS and eNOS{sup −/−} C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS{sup −/−} C57BLKS and eNOS{sup −/−} C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be

  14. Identifying Neighborhoods of Coordinated Gene Expression and Metabolite Profiles

    PubMed Central

    Hancock, Timothy; Wicker, Nicolas; Takigawa, Ichigaku; Mamitsuka, Hiroshi

    2012-01-01

    In this paper we investigate how metabolic network structure affects any coordination between transcript and metabolite profiles. To achieve this goal we conduct two complementary analyses focused on the metabolic response to stress. First, we investigate the general size of any relationship between metabolic network gene expression and metabolite profiles. We find that strongly correlated transcript-metabolite profiles are sustained over surprisingly long network distances away from any target metabolite. Secondly, we employ a novel pathway mining method to investigate the structure of this transcript-metabolite relationship. The objective of this method is to identify a minimum set of metabolites which are the target of significantly correlated gene expression pathways. The results reveal that in general, a global regulation signature targeting a small number of metabolites is responsible for a large scale metabolic response. However, our method also reveals pathway specific effects that can degrade this global regulation signature and complicates the observed coordination between transcript-metabolite profiles. PMID:22355360

  15. Metabolite Profiling of Sugarcane Genotypes and Identification of Flavonoid Glycosides and Phenolic Acids.

    PubMed

    Coutinho, Isabel D; Baker, John M; Ward, Jane L; Beale, Michael H; Creste, Silvana; Cavalheiro, Alberto J

    2016-06-01

    Sugarcane is an important agricultural crop in the economy of tropical regions, and Brazil has the largest cultivated acreage in the world. Sugarcane accumulates high levels of sucrose in its stalks. Other compounds produced by sugarcane are currently not of economic importance. To explore potential coproducts, we have studied the chemical diversity of sugarcane genotypes, via metabolite profiling of leaves by NMR and LC-DAD-MS. Metabolites were identified via in-house and public databases. From the analysis of 60 HPLC-fractionated extracts, LC-DAD-MS detected 144 metabolites, of which 56 were identified (MS-MS and (1)H NMR), including 19 phenolics and 25 flavones, with a predominance of isomeric flavone C-glycosides. Multivariate analysis of the profiles from genotypes utilized in Brazilian breeding programs revealed clustering according to sugar, phenolic acid, and flavone contents.

  16. (1)H-NMR, (1)H-NMR T2-edited, and 2D-NMR in bipolar disorder metabolic profiling.

    PubMed

    Sethi, Sumit; Pedrini, Mariana; Rizzo, Lucas B; Zeni-Graiff, Maiara; Mas, Caroline Dal; Cassinelli, Ana Cláudia; Noto, Mariane N; Asevedo, Elson; Cordeiro, Quirino; Pontes, João G M; Brasil, Antonio J M; Lacerda, Acioly; Hayashi, Mirian A F; Poppi, Ronei; Tasic, Ljubica; Brietzke, Elisa

    2017-12-01

    The objective of this study was to identify molecular alterations in the human blood serum related to bipolar disorder, using nuclear magnetic resonance (NMR) spectroscopy and chemometrics. Metabolomic profiling, employing (1)H-NMR, (1)H-NMR T2-edited, and 2D-NMR spectroscopy and chemometrics of human blood serum samples from patients with bipolar disorder (n = 26) compared with healthy volunteers (n = 50) was performed. The investigated groups presented distinct metabolic profiles, in which the main differential metabolites found in the serum sample of bipolar disorder patients compared with those from controls were lipids, lipid metabolism-related molecules (choline, myo-inositol), and some amino acids (N-acetyl-L-phenyl alanine, N-acetyl-L-aspartyl-L-glutamic acid, L-glutamine). In addition, amygdalin, α-ketoglutaric acid, and lipoamide, among other compounds, were also present or were significantly altered in the serum of bipolar disorder patients. The data presented herein suggest that some of these metabolites differentially distributed between the groups studied may be directly related to the bipolar disorder pathophysiology. The strategy employed here showed significant potential for exploring pathophysiological features and molecular pathways involved in bipolar disorder. Thus, our findings may contribute to pave the way for future studies aiming at identifying important potential biomarkers for bipolar disorder diagnosis or progression follow-up.

  17. Structural characterization of plasma metabolites detected via LC-electrochemical coulometric array using LC-UV fractionation, MS, and NMR.

    PubMed

    Bird, Susan S; Sheldon, Diane P; Gathungu, Rose M; Vouros, Paul; Kautz, Roger; Matson, Wayne R; Kristal, Bruce S

    2012-11-20

    Liquid chromatography (LC) separation combined with electrochemical coulometric array detection (EC) is a sensitive, reproducible, and robust technique that can detect hundreds of redox-active metabolites down to the level of femtograms on column, making it ideal for metabolomics profiling. EC detection cannot, however, structurally characterize unknown metabolites that comprise these profiles. Several aspects of LC-EC methods prevent a direct transfer to other structurally informative analytical methods, such as LC-MS and NMR. These include system limits of detection, buffer requirements, and detection mechanisms. To address these limitations, we developed a workflow based on the concentration of plasma, metabolite extraction, and offline LC-UV fractionation. Pooled human plasma was used to provide sufficient material necessary for multiple sample concentrations and platform analyses. Offline parallel LC-EC and LC-MS methods were established that correlated standard metabolites between the LC-EC profiling method and the mass spectrometer. Peak retention times (RT) from the LC-MS and LC-EC system were linearly related (r(2) = 0.99); thus, LC-MS RTs could be directly predicted from the LC-EC signals. Subsequent offline microcoil-NMR analysis of these collected fractions was used to confirm LC-MS characterizations by providing complementary, structural data. This work provides a validated workflow that is transferrable across multiple platforms and provides the unambiguous structural identifications necessary to move primary mathematically driven LC-EC biomarker discovery into biological and clinical utility.

  18. ¹H NMR-based metabolic profiling of human rectal cancer tissue

    PubMed Central

    2013-01-01

    Background Rectal cancer is one of the most prevalent tumor types. Understanding the metabolic profile of rectal cancer is important for developing therapeutic approaches and molecular diagnosis. Methods Here, we report a metabonomics profiling of tissue samples on a large cohort of human rectal cancer subjects (n = 127) and normal controls (n = 43) using 1H nuclear magnetic resonance (1H NMR) based metabonomics assay, which is a highly sensitive and non-destructive method for the biomarker identification in biological systems. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structure with discriminant analysis (OPLS-DA) were applied to analyze the 1H-NMR profiling data to identify the distinguishing metabolites of rectal cancer. Results Excellent separation was obtained and distinguishing metabolites were observed among the different stages of rectal cancer tissues (stage I = 35; stage II = 37; stage III = 37 and stage IV = 18) and normal controls. A total of 38 differential metabolites were identified, 16 of which were closely correlated with the stage of rectal cancer. The up-regulation of 10 metabolites, including lactate, threonine, acetate, glutathione, uracil, succinate, serine, formate, lysine and tyrosine, were detected in the cancer tissues. On the other hand, 6 metabolites, including myo-inositol, taurine, phosphocreatine, creatine, betaine and dimethylglycine were decreased in cancer tissues. These modified metabolites revealed disturbance of energy, amino acids, ketone body and choline metabolism, which may be correlated with the progression of human rectal cancer. Conclusion Our findings firstly identify the distinguishing metabolites in different stages of rectal cancer tissues, indicating possibility of the attribution of metabolites disturbance to the progression of rectal cancer. The altered metabolites may be as potential biomarkers, which would

  19. Species identification of Papaver by metabolite profiling.

    PubMed

    Choe, Sanggil; Kim, Suncheun; Lee, Chul; Yang, Wonkyung; Park, Yuran; Choi, Hwakyung; Chung, Heesun; Lee, Dongho; Hwang, Bang Yeon

    2011-09-10

    Papaver somniferum L. and Papaver setigerum D.C. are controlled as opium poppy in Korea because they contain narcotic substances such as morphine and codeine. It is one of the critical issues whether the plants similar to opium poppy in shape are controlled plants or not. There are more than 110 species in the genus Papaver worldwide and about 10 species in Korea. As the morphological features of some species are very similar and the alkaloid contents and the ratios among the major alkaloids vary even within the same species, it is often difficult to identify the exact species by the morphological features and/or major alkaloids analysis. To develop a new method that uses metabolite profiling for species discrimination between P. somniferum, Papaver rhoeas and P. setigerum, the gas chromatography/mass spectrometry (GC-MS) data of the alkaline extract were processed with in-house Microsoft Visual Basic(®) modules and the chemical information was analyzed through multivariate statistical analyses such as Hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (DA). The GC-MS results combined with multivariate analysis demonstrated that the metabolite profiling was an efficient technique for the classification and this method will provide a powerful tool for the identification of Korean Papaver species.

  20. Alterations of Urinary Metabolite Profile in Model Diabetic Nephropathy

    PubMed Central

    Stec, Donald F.; Wang, Suwan; Stothers, Cody; Avance, Josh; Denson, Deon; Harris, Raymond; Voziyan, Paul

    2014-01-01

    Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions found in human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS−/− C57BLKS and eNOS−/− C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS−/− C57BLKS and eNOS−/− C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be useful new tools in metabolomic studies relevant to human pathology. PMID:25499815

  1. Alterations of urinary metabolite profile in model diabetic nephropathy.

    PubMed

    Stec, Donald F; Wang, Suwan; Stothers, Cody; Avance, Josh; Denson, Deon; Harris, Raymond; Voziyan, Paul

    2015-01-09

    Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions characteristic of human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be useful new tools in metabolomic studies relevant to human pathology. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Signal intensities derived from different NMR probes and parameters contribute to variations in quantification of metabolites.

    PubMed

    Lacy, Paige; McKay, Ryan T; Finkel, Michael; Karnovsky, Alla; Woehler, Scott; Lewis, Michael J; Chang, David; Stringer, Kathleen A

    2014-01-01

    We discovered that serious issues could arise that may complicate interpretation of metabolomic data when identical samples are analyzed at more than one NMR facility, or using slightly different NMR parameters on the same instrument. This is important because cross-center validation metabolomics studies are essential for the reliable application of metabolomics to clinical biomarker discovery. To test the reproducibility of quantified metabolite data at multiple sites, technical replicates of urine samples were assayed by 1D-(1)H-NMR at the University of Alberta and the University of Michigan. Urine samples were obtained from healthy controls under a standard operating procedure for collection and processing. Subsequent analysis using standard statistical techniques revealed that quantitative data across sites can be achieved, but also that previously unrecognized NMR parameter differences can dramatically and widely perturb results. We present here a confirmed validation of NMR analysis at two sites, and report the range and magnitude that common NMR parameters involved in solvent suppression can have on quantitated metabolomics data. Specifically, saturation power levels greatly influenced peak height intensities in a frequency-dependent manner for a number of metabolites, which markedly impacted the quantification of metabolites. We also investigated other NMR parameters to determine their effects on further quantitative accuracy and precision. Collectively, these findings highlight the importance of and need for consistent use of NMR parameter settings within and across centers in order to generate reliable, reproducible quantified NMR metabolomics data.

  3. Expanding the Limits of Human Blood Metabolite Quantitation Using NMR Spectroscopy

    PubMed Central

    2015-01-01

    A current challenge in metabolomics is the reliable quantitation of many metabolites. Limited resolution and sensitivity combined with the challenges associated with unknown metabolite identification have restricted both the number and the quantitative accuracy of blood metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics, investigations of pooled human serum combining an array of 1D/2D NMR experiments at 800 MHz, database searches, and spiking with authentic compounds enabled the identification of 67 blood metabolites. Many of these (∼1/3) are new compared with those reported previously as a part of the Human Serum Metabolome Database. In addition, considering both the high reproducibility and quantitative nature of NMR as well as the sensitivity of NMR chemical shifts to altered sample conditions, experimental protocols and comprehensive peak annotations are provided here as a guide for identification and quantitation of the new pool of blood metabolites for routine applications. Further, investigations focused on the evaluation of quantitation using organic solvents revealed a surprisingly poor performance for protein precipitation using acetonitrile. One-third of the detected metabolites were attenuated by 10–67% compared with methanol precipitation at the same solvent-to-serum ratio of 2:1 (v/v). Nearly 2/3 of the metabolites were further attenuated by up to 65% upon increasing the acetonitrile-to-serum ratio to 4:1 (v/v). These results, combined with the newly established identity for many unknown metabolites in the NMR spectrum, offer new avenues for human serum/plasma-based metabolomics. Further, the ability to quantitatively evaluate nearly 70 blood metabolites that represent numerous classes, including amino acids, organic acids, carbohydrates, and heterocyclic compounds, using a simple and highly reproducible analytical method such as NMR may potentially guide the evaluation of samples for analysis using mass spectrometry

  4. Application of 1H-NMR Metabolomic Profiling for Reef-Building Corals

    PubMed Central

    Sogin, Emilia M.; Anderson, Paul; Williams, Philip; Chen, Chii-Shiarng; Gates, Ruth D.

    2014-01-01

    In light of global reef decline new methods to accurately, cheaply, and quickly evaluate coral metabolic states are needed to assess reef health. Metabolomic profiling can describe the response of individuals to disturbance (i.e., shifts in environmental conditions) across biological models and is a powerful approach for characterizing and comparing coral metabolism. For the first time, we assess the utility of a proton-nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomics approach in characterizing coral metabolite profiles by 1) investigating technical, intra-, and inter-sample variation, 2) evaluating the ability to recover targeted metabolite spikes, and 3) assessing the potential for this method to differentiate among coral species. Our results indicate 1H-NMR profiling of Porites compressa corals is highly reproducible and exhibits low levels of variability within and among colonies. The spiking experiments validate the sensitivity of our methods and showcase the capacity of orthogonal partial least squares discriminate analysis (OPLS-DA) to distinguish between profiles spiked with varying metabolite concentrations (0 mM, 0.1 mM, and 10 mM). Finally, 1H-NMR metabolomics coupled with OPLS-DA, revealed species-specific patterns in metabolite profiles among four reef-building corals (Pocillopora damicornis, Porites lobata, Montipora aequituberculata, and Seriatopora hystrix). Collectively, these data indicate that 1H-NMR metabolomic techniques can profile reef-building coral metabolomes and have the potential to provide an integrated picture of the coral phenotype in response to environmental change. PMID:25354140

  5. Metabolite Profiling and Classification of DNA-Authenticated Licorice Botanicals

    PubMed Central

    Simmler, Charlotte; Anderson, Jeffrey R.; Gauthier, Laura; Lankin, David C.; McAlpine, James B.; Chen, Shao-Nong; Pauli, Guido F.

    2015-01-01

    Raw licorice roots represent heterogeneous materials obtained from mainly three Glycyrrhiza species. G. glabra, G. uralensis, and G. inflata exhibit marked metabolite differences in terms of flavanones (Fs), chalcones (Cs), and other phenolic constituents. The principal objective of this work was to develop complementary chemometric models for the metabolite profiling, classification, and quality control of authenticated licorice. A total of 51 commercial and macroscopically verified samples were DNA authenticated. Principal component analysis and canonical discriminant analysis were performed on 1H NMR spectra and area under the curve values obtained from UHPLC-UV chromatograms, respectively. The developed chemometric models enable the identification and classification of Glycyrrhiza species according to their composition in major Fs, Cs, and species specific phenolic compounds. Further key outcomes demonstrated that DNA authentication combined with chemometric analyses enabled the characterization of mixtures, hybrids, and species outliers. This study provides a new foundation for the botanical and chemical authentication, classification, and metabolomic characterization of crude licorice botanicals and derived materials. Collectively, the proposed methods offer a comprehensive approach for the quality control of licorice as one of the most widely used botanical dietary supplements. PMID:26244884

  6. Metabolite Profiling and Classification of DNA-Authenticated Licorice Botanicals.

    PubMed

    Simmler, Charlotte; Anderson, Jeffrey R; Gauthier, Laura; Lankin, David C; McAlpine, James B; Chen, Shao-Nong; Pauli, Guido F

    2015-08-28

    Raw licorice roots represent heterogeneous materials obtained from mainly three Glycyrrhiza species. G. glabra, G. uralensis, and G. inflata exhibit marked metabolite differences in terms of flavanones (Fs), chalcones (Cs), and other phenolic constituents. The principal objective of this work was to develop complementary chemometric models for the metabolite profiling, classification, and quality control of authenticated licorice. A total of 51 commercial and macroscopically verified samples were DNA authenticated. Principal component analysis and canonical discriminant analysis were performed on (1)H NMR spectra and area under the curve values obtained from UHPLC-UV chromatograms, respectively. The developed chemometric models enable the identification and classification of Glycyrrhiza species according to their composition in major Fs, Cs, and species specific phenolic compounds. Further key outcomes demonstrated that DNA authentication combined with chemometric analyses enabled the characterization of mixtures, hybrids, and species outliers. This study provides a new foundation for the botanical and chemical authentication, classification, and metabolomic characterization of crude licorice botanicals and derived materials. Collectively, the proposed methods offer a comprehensive approach for the quality control of licorice as one of the most widely used botanical dietary supplements.

  7. Functional Analysis of the Nitrogen Metabolite Repression Regulator Gene nmrA in Aspergillus flavus.

    PubMed

    Han, Xiaoyun; Qiu, Mengguang; Wang, Bin; Yin, Wen-Bing; Nie, Xinyi; Qin, Qiuping; Ren, Silin; Yang, Kunlong; Zhang, Feng; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    In Aspergillus nidulans, the nitrogen metabolite repression (NMR) regulator NmrA plays a major role in regulating the activity of the GATA transcription factor AreA during nitrogen metabolism. However, the function of nmrA in A. flavus has not been previously studied. Here, we report the identification and functional analysis of nmrA in A. flavus. Our work showed that the amino acid sequences of NmrA are highly conserved among Aspergillus species and that A. flavus NmrA protein contains a canonical Rossmann fold motif. Deletion of nmrA slowed the growth of A. flavus but significantly increased conidiation and sclerotia production. Moreover, seed infection experiments indicated that nmrA is required for the invasive virulence of A. flavus. In addition, the ΔnmrA mutant showed increased sensitivity to rapamycin and methyl methanesulfonate, suggesting that nmrA could be responsive to target of rapamycin signaling and DNA damage. Furthermore, quantitative real-time reverse transcription polymerase chain reaction analysis suggested that nmrA might interact with other nitrogen regulatory and catabolic genes. Our study provides a better understanding of NMR and the nitrogen metabolism network in fungi.

  8. Functional Analysis of the Nitrogen Metabolite Repression Regulator Gene nmrA in Aspergillus flavus

    PubMed Central

    Han, Xiaoyun; Qiu, Mengguang; Wang, Bin; Yin, Wen-Bing; Nie, Xinyi; Qin, Qiuping; Ren, Silin; Yang, Kunlong; Zhang, Feng; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    In Aspergillus nidulans, the nitrogen metabolite repression (NMR) regulator NmrA plays a major role in regulating the activity of the GATA transcription factor AreA during nitrogen metabolism. However, the function of nmrA in A. flavus has not been previously studied. Here, we report the identification and functional analysis of nmrA in A. flavus. Our work showed that the amino acid sequences of NmrA are highly conserved among Aspergillus species and that A. flavus NmrA protein contains a canonical Rossmann fold motif. Deletion of nmrA slowed the growth of A. flavus but significantly increased conidiation and sclerotia production. Moreover, seed infection experiments indicated that nmrA is required for the invasive virulence of A. flavus. In addition, the ΔnmrA mutant showed increased sensitivity to rapamycin and methyl methanesulfonate, suggesting that nmrA could be responsive to target of rapamycin signaling and DNA damage. Furthermore, quantitative real-time reverse transcription polymerase chain reaction analysis suggested that nmrA might interact with other nitrogen regulatory and catabolic genes. Our study provides a better understanding of NMR and the nitrogen metabolism network in fungi. PMID:27933036

  9. Current approaches and challenges for the metabolite profiling of complex natural extracts.

    PubMed

    Wolfender, Jean-Luc; Marti, Guillaume; Thomas, Aurélien; Bertrand, Samuel

    2015-02-20

    Metabolite profiling is critical in many aspects of the life sciences, particularly natural product research. Obtaining precise information on the chemical composition of complex natural extracts (metabolomes) that are primarily obtained from plants or microorganisms is a challenging task that requires sophisticated, advanced analytical methods. In this respect, significant advances in hyphenated chromatographic techniques (LC-MS, GC-MS and LC-NMR in particular), as well as data mining and processing methods, have occurred over the last decade. Together, these tools, in combination with bioassay profiling methods, serve an important role in metabolomics for the purposes of both peak annotation and dereplication in natural product research. In this review, a survey of the techniques that are used for generic and comprehensive profiling of secondary metabolites in natural extracts is provided. The various approaches (chromatographic methods: LC-MS, GC-MS, and LC-NMR and direct spectroscopic methods: NMR and DIMS) are discussed with respect to their resolution and sensitivity for extract profiling. In addition the structural information that can be generated through these techniques or in combination, is compared in relation to the identification of metabolites in complex mixtures. Analytical strategies with applications to natural extracts and novel methods that have strong potential, regardless of how often they are used, are discussed with respect to their potential applications and future trends.

  10. Microscale LC-MS-NMR Platform Applied to the Identification of Active Cyanobacterial Metabolites

    PubMed Central

    Lin, Yiqing; Schiavo, Susan; Orjala, Jimmy; Vouros, Paul; Kautz, Roger

    2009-01-01

    An LC-MS-NMR platform is demonstrated, which combines two innovations in microscale analysis, nanoSplitter LC–MS and microdroplet NMR, for the identification of unknown compounds found at low concentrations in complex sample matrixes as frequently encountered in metabolomics or natural products discovery. The nano-Splitter provides the high sensitivity of nanoelectrospray MS while allowing 98% of the HPLC effluent from a large-bore LC column to be collected and concentrated for NMR. Microdroplet NMR is a droplet microfluidic NMR loading method providing severalfold higher sample efficiency than conventional flow injection methods. Performing NMR offline from LC-UV-MS accommodates the disparity between MS and NMR in their sample mass and time requirements, as well as allowing NMR spectra to be requested retrospectively, after review of the LC–MS data. Interpretable 1D NMR spectra were obtained from analytes at the 200-ng level, in 1 h/well automated NMR data acquisitions. The system also showed excellent intra- and interdetector reproducibility with retention time RSD values less than 2% and sample recovery on the order of 93%. When applied to a cyanobacterial extract showing antibacterial activity, the platform recognized several previously known metabolites, down to the 1% level, in a single 30-μg injection, and prioritized one unknown for further study. PMID:18834150

  11. Bayesian deconvolution and quantification of metabolites in complex 1D NMR spectra using BATMAN.

    PubMed

    Hao, Jie; Liebeke, Manuel; Astle, William; De Iorio, Maria; Bundy, Jacob G; Ebbels, Timothy M D

    2014-01-01

    Data processing for 1D NMR spectra is a key bottleneck for metabolomic and other complex-mixture studies, particularly where quantitative data on individual metabolites are required. We present a protocol for automated metabolite deconvolution and quantification from complex NMR spectra by using the Bayesian automated metabolite analyzer for NMR (BATMAN) R package. BATMAN models resonances on the basis of a user-controllable set of templates, each of which specifies the chemical shifts, J-couplings and relative peak intensities for a single metabolite. Peaks are allowed to shift position slightly between spectra, and peak widths are allowed to vary by user-specified amounts. NMR signals not captured by the templates are modeled non-parametrically by using wavelets. The protocol covers setting up user template libraries, optimizing algorithmic input parameters, improving prior information on peak positions, quality control and evaluation of outputs. The outputs include relative concentration estimates for named metabolites together with associated Bayesian uncertainty estimates, as well as the fit of the remainder of the spectrum using wavelets. Graphical diagnostics allow the user to examine the quality of the fit for multiple spectra simultaneously. This approach offers a workflow to analyze large numbers of spectra and is expected to be useful in a wide range of metabolomics studies.

  12. Nicotine metabolite ratio (NMR) prospectively predicts smoking relapse: Longitudinal findings from ITC Surveys in five countries.

    PubMed

    Fix, Brian V; O'Connor, Richard J; Benowitz, Neal; Heckman, Bryan W; Cummings, K Michael; Fong, Geoffrey T; Thrasher, James F

    2017-04-06

    The ratio of trans 3'-hydroxycotinine (3HC) to cotinine (nicotine metabolite ratio, NMR) is a biomarker of the rate of nicotine metabolism, with higher NMR indicating faster metabolism. Higher NMR has been found to be associated with higher daily cigarette consumption and less success stopping smoking in cessation trials. This study examines differences in NMR among population-based samples of smokers in the 5 countries and explores the relationship between NMR and smoking abstinence. Participants (N=874) provided saliva samples during International Tobacco Control (ITC) surveys in the US, UK, Mauritius, Mexico, and Thailand conducted in 2010/2011 with follow-up surveys in 2012/2013. When all samples were received, they were sent to a common laboratory for analysis using liquid chromatography and tandem mass spectroscopy. There was significant variation in NMR across countries (F=15.49, p<.001). Those who reported smoking at follow-up had a mean NMR of 0.32, compared to a mean NMR of 0.42 in participants who reported that they had stopped (F=8.93; p=0.003). Higher mean NMR values were also associated with longer quit duration (p=0.007). There was no substantial difference in NMR between current smokers who made a failed quit attempt and those who made no attempt - both had significantly lower NMR compared to those who quit and remained abstinent. Smokers with a higher NMR were more likely to report that they stopped smoking compared to those with a lower NMR (OR=2.67; 95%CI: 1.25-5.68). These results suggest faster nicotine metabolizers may be less likely to relapse following a quit attempt. This finding differs from results of clinical trials testing stop smoking medications, where slower metabolizers have been found to be more likely to maintain abstinence from smoking.

  13. 1H NMR-Based Analysis of Serum Metabolites in Monocrotaline-Induced Pulmonary Arterial Hypertensive Rats

    PubMed Central

    Lin, Taijie; Gu, Jinping; Huang, Caihua; Zheng, Suli; Lin, Xu; Xie, Liangdi; Lin, Donghai

    2016-01-01

    Aims. To study the changes of the metabolic profile during the pathogenesis in monocrotaline (MCT) induced pulmonary arterial hypertension (PAH). Methods. Forty male Sprague-Dawley (SD) rats were randomly divided into 5 groups (n = 8, each). PAH rats were induced by a single dose intraperitoneal injection of 60 mg/kg MCT, while 8 rats given intraperitoneal injection of 1 ml normal saline and scarified in the same day (W0) served as control. Mean pulmonary arterial pressure (mPAP) was measured through catherization. The degree of right ventricular hypertrophy and pulmonary hyperplasia were determined at the end of first to fourth weeks; nuclear magnetic resonance (NMR) spectra of sera were then acquired for the analysis of metabolites. Principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were used to discriminate different metabolic profiles. Results. The prominent changes of metabolic profiles were seen during these four weeks. Twenty specific metabolites were identified, which were mainly involved in lipid metabolism, glycolysis, energy metabolism, ketogenesis, and methionine metabolism. Profiles of correlation between these metabolites in each stage changed markedly, especially in the fourth week. Highly activated methionine and betaine metabolism pathways were selected by the pathway enrichment analysis. Conclusions. Metabolic dysfunction is involved in the development and progression of PAH. PMID:27057080

  14. Profile of urinary arsenic metabolites during pregnancy.

    PubMed

    Hopenhayn, Claudia; Huang, Bin; Christian, Jay; Peralta, Cecilia; Ferreccio, Catterina; Atallah, Raja; Kalman, David

    2003-12-01

    Chronic exposure to inorganic arsenic (In-As) from drinking water is associated with different health effects, including skin, lung, bladder, and kidney cancer as well as vascular and possibly reproductive effects. In-As is metabolized through the process of methylation, resulting in the production and excretion of methylated species, mainly monomethylarsenate (MMA) and dimethylarsenate (DMA). Because a large percentage of the dose is excreted in urine, the distribution of urinary In-As, MMA, and DMA is considered a useful indicator of methylation patterns in human populations. Several factors affect these patterns, including sex and exposure level. In this study, we investigated the profile of urinary In-As, MMA, and DMA of pregnant women. Periodic urine samples were collected from early to late pregnancy among 29 pregnant women living in Antofagasta, Chile, who drank tap water containing 40 micro g/L In-As. The total urinary arsenic across four sampling periods increased with increasing weeks of gestation, from an initial mean value of 36.1 to a final value of 54.3 micro g/L. This increase was mainly due to an increase in DMA, resulting in lower percentages of In-As and MMA and a higher percentage of DMA. Our findings indicate that among women exposed to moderate arsenic from drinking water during pregnancy, changes occur in the pattern of urinary arsenic excretion and metabolite distribution. The toxicologic significance of this is not clear, given recent evidence suggesting that intermediate methylated species may be highly toxic. Nevertheless, this study suggests that arsenic metabolism changes throughout the course of pregnancy, which in turn may have toxicologic effects on the developing fetus. Key words: arsenic, arsenic metabolism, arsenic methylation, Chile, pregnancy, urinary arsenic.

  15. Profile of urinary arsenic metabolites during pregnancy.

    PubMed Central

    Hopenhayn, Claudia; Huang, Bin; Christian, Jay; Peralta, Cecilia; Ferreccio, Catterina; Atallah, Raja; Kalman, David

    2003-01-01

    Chronic exposure to inorganic arsenic (In-As) from drinking water is associated with different health effects, including skin, lung, bladder, and kidney cancer as well as vascular and possibly reproductive effects. In-As is metabolized through the process of methylation, resulting in the production and excretion of methylated species, mainly monomethylarsenate (MMA) and dimethylarsenate (DMA). Because a large percentage of the dose is excreted in urine, the distribution of urinary In-As, MMA, and DMA is considered a useful indicator of methylation patterns in human populations. Several factors affect these patterns, including sex and exposure level. In this study, we investigated the profile of urinary In-As, MMA, and DMA of pregnant women. Periodic urine samples were collected from early to late pregnancy among 29 pregnant women living in Antofagasta, Chile, who drank tap water containing 40 micro g/L In-As. The total urinary arsenic across four sampling periods increased with increasing weeks of gestation, from an initial mean value of 36.1 to a final value of 54.3 micro g/L. This increase was mainly due to an increase in DMA, resulting in lower percentages of In-As and MMA and a higher percentage of DMA. Our findings indicate that among women exposed to moderate arsenic from drinking water during pregnancy, changes occur in the pattern of urinary arsenic excretion and metabolite distribution. The toxicologic significance of this is not clear, given recent evidence suggesting that intermediate methylated species may be highly toxic. Nevertheless, this study suggests that arsenic metabolism changes throughout the course of pregnancy, which in turn may have toxicologic effects on the developing fetus. Key words: arsenic, arsenic metabolism, arsenic methylation, Chile, pregnancy, urinary arsenic. PMID:14644662

  16. Investigating Compensation and Recovery of Fathead Minnow (Pimephales promelas) Exposed to 17α-Ethynylestradiol with Metabolite Profiling

    EPA Science Inventory

    1H-NMR spectroscopy was used to profile metabolite changes in the livers of fathead minnows (Pimephales promelas) exposed to the synthetic estrogen 17α ethynylestradiol (EE2) via a continuous flow water exposure. Fish were exposed to either 10 or 100 ng EE2/L for 8 days, followed...

  17. Investigating Compensation and Recovery of Fathead Minnow (Pimephales promelas) Exposed to 17α-Ethynylestradiol with Metabolite Profiling

    EPA Science Inventory

    1H-NMR spectroscopy was used to profile metabolite changes in the livers of fathead minnows (Pimephales promelas) exposed to the synthetic estrogen 17α ethynylestradiol (EE2) via a continuous flow water exposure. Fish were exposed to either 10 or 100 ng EE2/L for 8 days, followed...

  18. Application of rotated PCA models to facilitate interpretation of metabolite profiles: commercial preparations of St. John's Wort.

    PubMed

    Lawaetz, Anders Juul; Schmidt, Bonnie; Staerk, Dan; Jaroszewski, Jerzy W; Bro, Rasmus

    2009-02-01

    This paper describes the application of orthogonal rotation of models based on principal component analysis (PCA) of (1)H nuclear magnetic resonance (NMR) spectra and high-performance liquid chromatography-photo diode array detection (HPLC-PDA) profiles of natural product mixtures using extracts of antidepressive pharmaceutical preparations of St. John's wort as an example. (1)H-NMR spectroscopy of complex mixtures is often used in metabolomic, metabonomic and metabolite profiling studies for assessment of sample composition. Interpretation of the derived chemometric models may be complicated because several sample properties often contribute to each principal component and because the influence of individual metabolites may be shared by several principal components. Furthermore, extensive signal overlap in (1)H-NMR spectra poses additional challenges to the interpretation of PCA models derived from such data. Orthogonal rotation of PCA models derived from (1)H-NMR spectra and HPLC-PDA profiles of the extracts of St. John's wort preparations facilitate interpretation of the model. Using the varimax criterion, rotation of loadings provides simpler conditions for understanding the influence of individual metabolites on the observed clustering. Alternatively, rotation of scores simplifies the understanding of the influence of whole metabolite profiles on the clustering of individual samples.

  19. NMR Metabolic profiling of green tea (Camellia sinensis L.) leaves grown at Kemuning, Indonesia

    NASA Astrophysics Data System (ADS)

    Wahyuni, D. S. C.; Kristanti, M. W.; Putri, R. K.; Rinanto, Y.

    2017-01-01

    Green tea (Camellia sinensis L.) has been famous as a beverage and natural medicine. It contains a broad range of primary and secondary metabolites i.e. polyphenols. Nuclear Magnetic Resonance (NMR) has been widely used for metabolic profiling in medicinal plants. It provides a very fast and detailed analysis of the biomolecular composition of crude extracts. Moreover, an NMR spectrum is a physical characteristic of a compound and thus highly reproducible. Therefore, this study aims to profile metabolites of three different varieties of green tea C. Sinensis grown in Kemuning, Middle Java. Three varieties of green tea collected on Kemuning (TR1 2025, Gambung 4/5, and Chiaruan 143) were used in this study. 1H-NMR spectra were recorded at 230C on a 400 MHz Agilent WB (Widebore). The analysis was performed on dried green tea leaves and analyzed by 1H-NMR, 2D-J-resolved and 1H-1H correlated spectroscopy (COSY). MestRenova version 11.0.0 applied to identify metabolites in samples. A 1H-NMR spectrum of tea showed amino acids and organic acids signal at the area δ 0.8-4.0. These were theanine, alanine, threonine, succinic acid, aspartic acid, lactic acid. Anomeric protons of carbohydrate were shown by the region of β-glucose, α-glucose, fructose and sucrose. The phenolic region was depicted at area δ 5.5-8.5. Epigallocatechin derivates and caffeine were detected in the tea leaves. The detail compound identification was observed and discussed in the text.

  20. Strategies for metabolite profiling based on liquid chromatography.

    PubMed

    Saurina, Javier; Sentellas, Sonia

    2017-02-15

    This paper aims at covering the principal strategies based on liquid chromatography (LC) for metabolite profiling in the field of drug discovery and development. The identification of metabolites generated in the organism is an important task during the early stages of preclinical research to define the most proper strategy for optimizing, adjusting metabolic clearance and minimizing bioactivation. An early assessment of the metabolite profile may be critical since metabolites can contribute to pharmacological and/or toxicological effects. The study of metabolites first involves their synthesis/generation and their further characterization and structural elucidation. For such a purpose, both in vitro and in vivo methods are commonly used for the generation of the corresponding metabolites. Next, analytical methods are used to tackle identification and characterization studies. Among the arsenal of techniques available in our labs, we will focus on LC, especially coupled to mass spectrometry (LC-MS), as one of the most powerful approaches for metabolite identification, characterization and quantification. Here, the topic of metabolite profiling based on LC will be addressed and representative examples of different possibilities will be discussed.

  1. Building blocks for automated elucidation of metabolites: Machine learning methods for NMR prediction

    PubMed Central

    Kuhn, Stefan; Egert, Björn; Neumann, Steffen; Steinbeck, Christoph

    2008-01-01

    Background Current efforts in Metabolomics, such as the Human Metabolome Project, collect structures of biological metabolites as well as data for their characterisation, such as spectra for identification of substances and measurements of their concentration. Still, only a fraction of existing metabolites and their spectral fingerprints are known. Computer-Assisted Structure Elucidation (CASE) of biological metabolites will be an important tool to leverage this lack of knowledge. Indispensable for CASE are modules to predict spectra for hypothetical structures. This paper evaluates different statistical and machine learning methods to perform predictions of proton NMR spectra based on data from our open database NMRShiftDB. Results A mean absolute error of 0.18 ppm was achieved for the prediction of proton NMR shifts ranging from 0 to 11 ppm. Random forest, J48 decision tree and support vector machines achieved similar overall errors. HOSE codes being a notably simple method achieved a comparatively good result of 0.17 ppm mean absolute error. Conclusion NMR prediction methods applied in the course of this work delivered precise predictions which can serve as a building block for Computer-Assisted Structure Elucidation for biological metabolites. PMID:18817546

  2. The metabolite profiling of coastal coccolithophorid species Pleurochrysis carterae (Haptophyta)

    NASA Astrophysics Data System (ADS)

    Zhou, Chengxu; Luo, Jie; Ye, Yangfang; Yan, Xiaojun; Liu, Baoning; Wen, Xin

    2016-07-01

    Pleurochrysis carterae is a calcified coccolithophorid species that usually blooms in the coastal area and causes aquaculture losses. The cellular calcification, blooming and many other critical species specific eco-physiological processes are closely related to various metabolic pathways. The purpose of this study is to apply the unbiased and non-destructive method of nuclear magnetic resonance (NMR) to detect the unknown holistic metabolite of P. carterae. The results show that NMR spectroscopic method is practical in the analysis of metabolites of phytoplankton. The metabolome of P. carterae was dominated by 26 metabolites involved in a number of different primary and secondary metabolic pathways. Organic acids and their derivatives, amino acids, sugars, nucleic aides were mainly detected. The abundant metabolites are that closely related to the process of cellular osmotic adjustment, which possibly reflect the active ability of P. carterae to adapt to the versatile coastal niche. DMSP (dimethylsulphoniopropionate) was the most dominant metabolite in P. carterae, up to 2.065±0.278 mg/g lyophilized cells, followed by glutamate and lactose, the contents were 0.349±0.035 and 0.301±0.073 mg/g lyophilized cells respectively. Other metabolites that had the content ranged between 0.1-0.2 mg/g lyophilized cells were alanine, isethionate and arabinose. Amino acid (valine, phenylalanine, isoleucine, tyrosine), organic acid salts (lactate, succinate), scyllitol and uracil had content ranged from 0.01 to below 0.1 mg/g lyophilized cells. Trigonelline, fumarate and formate were detected in very low content (only thousandths of 1 mg per gram of lyophilized cells or below). Our results of the holistic metabolites of P. carterae are the basic references for the further studies when multiple problems will be addressed to this notorious blooming calcifying species.

  3. Computer-Aided (13)C NMR Chemical Profiling of Crude Natural Extracts without Fractionation.

    PubMed

    Bakiri, Ali; Hubert, Jane; Reynaud, Romain; Lanthony, Sylvie; Harakat, Dominique; Renault, Jean-Hugues; Nuzillard, Jean-Marc

    2017-05-26

    A computer-aided, (13)C NMR-based dereplication method is presented for the chemical profiling of natural extracts without any fractionation. An algorithm was developed in order to compare the (13)C NMR chemical shifts obtained from a single routine spectrum with a set of predicted NMR data stored in a natural metabolite database. The algorithm evaluates the quality of the matching between experimental and predicted data by calculating a score function and returns the list of metabolites that are most likely to be present in the studied extract. The proof of principle of the method is demonstrated on a crude alkaloid extract obtained from the leaves of Peumus boldus, resulting in the identification of eight alkaloids, including isocorydine, rogersine, boldine, reticuline, coclaurine, laurotetanine, N-methylcoclaurine, and norisocorydine, as well as three monoterpenes, namely, p-cymene, eucalyptol, and α-terpinene. The results were compared to those obtained with other methods, either involving a fractionation step before the chemical profiling process or using mass spectrometry detection in the infusion mode or coupled to gas chromatography.

  4. Application of (1)h NMR profiling to assess seed metabolomic diversity. A case study on a soybean era population.

    PubMed

    Harrigan, George G; Skogerson, Kirsten; MacIsaac, Susan; Bickel, Anna; Perez, Tim; Li, Xin

    2015-05-13

    (1)H NMR spectroscopy offers advantages in metabolite quantitation and platform robustness when applied in food metabolomics studies. This paper provides a (1)H NMR-based assessment of seed metabolomic diversity in conventional and glyphosate-resistant genetically modified (GM) soybean from a genetic lineage representing ∼35 years of breeding and differing yield potential. (1)H NMR profiling of harvested seed allowed quantitation of 27 metabolites, including free amino acids, sugars, and organic acids, as well as choline, O-acetylcholine, dimethylamine, trigonelline, and p-cresol. Data were analyzed by canonical discriminant analysis (CDA) and principal variance component analysis (PVCA). Results demonstrated that (1)H NMR spectroscopy was effective in highlighting variation in metabolite levels in the genetically diverse sample set presented. The results also confirmed that metabolite variability is influenced by selective breeding and environment, but not genetic modification. Therefore, metabolite variability is an integral part of crop improvement that has occurred for decades and is associated with a history of safe use.

  5. Diurnal variation of NMR based blood metabolites in calves fed a high plane of milk replacer: a pilot study.

    PubMed

    Ghaffari, Morteza H; MacPherson, Jayden A R; Berends, Harma; Steele, Michael A

    2017-08-23

    Blood profiles have been used to monitor herd health status, diagnose disorders, and predict the risk of diseases in cattle and calves. Characterizing plasma metabolites in dairy calves could provide further insight into daily metabolic variations and the mechanisms that lead to metabolic diseases. In addition, by understanding physiological ranges of plasma metabolites relative to meal and the time of feeding in healthy animals, veterinarians can accurately diagnose abnormalities with a blood test. For diagnostic purposes, nuclear magnetic resonance (NMR) spectroscopy shows promise as a new and reliable method to determine a large number of blood metabolites simultaneously. Results demonstrated that the concentration of specific metabolites in plasma (i.e., lysine, isoleucine, leucine, tyrosine, glutamine, creatine, and 1-methylhistidine) fluctuated around meal times, while others (i.e., glutamic acid, methanol, formic acid, and acetic acid) maintained a stable temporal concentration. In addition to temporal changes in concentration, results also characterized differences for overall plasma metabolite concentrations; for example, methionine had the lowest (38 μM) while glutamine had the highest concentration (239 μM) amongst plasma AA. This is the first report describing how the plasma metabolome changes during 24-h period in young calves fed an elevated plane of milk replacer twice daily. Data from this pilot study will help to establish reference standards for future metabolic diagnostics in dairy calves. In addition, this pilot study illustrated that feeding milk replacer may influence plasma metabolite concentrations. With the rapid implementation of blood metabolomics in monitoring animal health, it is then important to consider the time of feeding during the day when interpreting metabolomics analysis results.

  6. Quantification of Water-Soluble Metabolites in Medicinal Mushrooms Using Proton NMR Spectroscopy.

    PubMed

    Lo, Yu-Chang; Chien, Shih-Chang; Mishchuk, Darya O; Slupsky, Carolyn M; Mau, Jeng-Leun

    2016-01-01

    The water-soluble metabolites in 5 mushrooms were identified and quantified using proton nuclear magnetic resonance (NMR) spectroscopy and software for targeted metabolite detection and quantification. In total, 35 compounds were found in Agaricus brasiliensis, 25 in Taiwanofungus camphoratus, 23 in Ganoderma lucidum (Taiwan) and Lentinus edodes, and 16 in G. lucidum (China). Total amounts of all identified metabolites in A. brasiliensis, T. camphoratus, G. lucidum, G. lucidum (China), and L. edodes were 149,950.51, 12,834.18, 9,549.09, 2,788.41, and 111,726.51 mg/kg dry weight, respectively. These metabolites were categorized into 4 groups: free amino acids and derivatives, carbohydrates, carboxylic acids, and nucleosides. Carbohydrates were the most abundant metabolites among all 4 groups, with mannitol having the highest concentration among all analyzed metabolites (848-94,104 mg/kg dry weight). Principal components analysis (PCA) showed obvious distinction among the metabolites of the 5 different kinds of mushrooms analyzed in this study. Thus PCA could provide an optional analytical way of identifying and recognizing the compositions of flavor products. Furthermore, the results of this study demonstrate that NMRbased metabolomics is a powerful tool for differentiating between various medicinal mushrooms.

  7. Characterization of a new rat urinary metabolite of piperine by LC/NMR/MS studies.

    PubMed

    Bajad, Sunil; Coumar, Mohane; Khajuria, Ravi; Suri, Om P; Bedi, Kasturi L

    2003-08-01

    Potential of piperine, an active alkaloid of black and long peppers, to increase the bioavailability of drugs in humans is of great clinical significance owing to its omnipresence in food. In an attempt to further study the reported differences in its metabolism in rats and humans, a new major urinary metabolite was detected in rat urine and plasma using HPLC. The metabolite was partially purified using reverse phase column chromatography on Sephadex((R))-LH 20 and characterized as 5-(3, 4-methylenedioxy phenyl)-2E,4E-pentadienoic acid-N-(3-yl propionic acid)-amide with the help of LC/NMR/positive ESI-MS studies. Complete mass fragmentation pattern could be assigned with MS/MS studies. The metabolite has a unique structure compared to the previously reported metabolites in that it retains methylenedioxy ring and conjugated double bonds while the piperidine ring is modified to form propionic acid group. Mechanism of formation of the metabolite by oxidation and cleavage of piperidine ring is proposed. Kidney appears to be the major excretion route for piperine metabolites in rats as no metabolite could be detected in feces.

  8. In Vivo NMR Metabolic Profiling of Fabrea salina Reveals Sequential Defense Mechanisms against Ultraviolet Radiation

    PubMed Central

    Marangoni, Roberto; Paris, Debora; Melck, Dominique; Fulgentini, Lorenzo; Colombetti, Giuliano; Motta, Andrea

    2011-01-01

    Fabrea salina is a hypersaline ciliate that is known to be among the strongest ultraviolet (UV)-resistant microorganisms; however, the molecular mechanisms of this resistance are almost unknown. By means of in vivo NMR spectroscopy, we determined the metabolic profile of living F. salina cells exposed to visible light and to polychromatic UV-B + UV-A + Vis radiation for several different exposure times. We used unsupervised pattern-recognition analysis to compare these profiles and discovered some metabolites whose concentration changed specifically upon UV exposure and in a dose-dependent manner. This variation was interpreted in terms of a two-phase cell reaction involving at least two different pathways: an early response consisting of degradation processes, followed by a late response activating osmoprotection mechanisms. The first step alters the concentration of formate, acetate, and saturated fatty-acid metabolites, whereas the osmoprotection modifies the activity of betaine moieties and other functionally related metabolites. In the latter pathway, alanine, proline, and sugars suggest a possible incipient protein synthesis as defense and/or degeneration mechanisms. We conclude that NMR spectroscopy on in vivo cells is an optimal approach for investigating the effect of UV-induced stress on the whole metabolome of F. salina because it minimizes the invasiveness of the measurement. PMID:21190674

  9. In vivo NMR metabolic profiling of Fabrea salina reveals sequential defense mechanisms against ultraviolet radiation.

    PubMed

    Marangoni, Roberto; Paris, Debora; Melck, Dominique; Fulgentini, Lorenzo; Colombetti, Giuliano; Motta, Andrea

    2011-01-05

    Fabrea salina is a hypersaline ciliate that is known to be among the strongest ultraviolet (UV)-resistant microorganisms; however, the molecular mechanisms of this resistance are almost unknown. By means of in vivo NMR spectroscopy, we determined the metabolic profile of living F. salina cells exposed to visible light and to polychromatic UV-B + UV-A + Vis radiation for several different exposure times. We used unsupervised pattern-recognition analysis to compare these profiles and discovered some metabolites whose concentration changed specifically upon UV exposure and in a dose-dependent manner. This variation was interpreted in terms of a two-phase cell reaction involving at least two different pathways: an early response consisting of degradation processes, followed by a late response activating osmoprotection mechanisms. The first step alters the concentration of formate, acetate, and saturated fatty-acid metabolites, whereas the osmoprotection modifies the activity of betaine moieties and other functionally related metabolites. In the latter pathway, alanine, proline, and sugars suggest a possible incipient protein synthesis as defense and/or degeneration mechanisms. We conclude that NMR spectroscopy on in vivo cells is an optimal approach for investigating the effect of UV-induced stress on the whole metabolome of F. salina because it minimizes the invasiveness of the measurement. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Evaluation of ensemble Monte Carlo variable selection for identification of metabolite markers on NMR data.

    PubMed

    Esquerre, C A; Gowen, A A; O'Gorman, A; Downey, G; O'Donnell, C P

    2017-04-29

    The aim of this study was to investigate the potential of the recently developed ensemble Monte Carlo Variable Selection (EMCVS) method to identify the relevant portions of high resolution (1)H NMR spectra as a metabolite fingerprinting tool and compare to a widely used method (Variable importance on projection (VIP)) and recently proposed variable selected methods i.e. selectivity ratio (SR) and significance multivariate correlation (sMC). As case studies two quantitative publicly available datasets: wine samples, urine samples of rats, and an experiment on mushroom (Agaricus bisporus) were examined. EMCVS outperformed the three other variable selection methods in most cases, selecting fewer chemical shifts and leading to improved classification of mushrooms and prediction of onion by-products intake and wine components. These fewer chemical shift regions facilitate the interpretation of the NMR spectra, fingerprinting and identification of metabolite markers. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Metabolite profiling in plant biology: platforms and destinations

    PubMed Central

    Kopka, Joachim; Fernie, Alisdair; Weckwerth, Wolfram; Gibon, Yves; Stitt, Mark

    2004-01-01

    Optimal use of genome sequences and gene-expression resources requires powerful phenotyping platforms, including those for systematic analysis of metabolite composition. The most used technologies for metabolite profiling, including mass spectral, nuclear magnetic resonance and enzyme-based approaches, have various advantages and disadvantages, and problems can arise with reliability and the interpretation of the huge datasets produced. These techniques will be useful for answering important biological questions in the future. PMID:15186482

  12. Unambiguous Metabolite Identification in High-Throughput Metabolomics by Hybrid 1H-NMR/ESI-MS1 Approach

    SciTech Connect

    2016-10-18

    The invention improves accuracy of metabolite identification by combining direct infusion ESI-MS with one-dimensional 1H-NMR spectroscopy. First, we apply a standard 1H-NMR metabolite identification protocol by matching the chemical shift, J-coupling and intensity information of experimental NMR signals against the NMR signals of standard metabolites in a metabolomics reference libraries. This generates a list of candidate metabolites. The list contains both false positive and ambiguous identifications. The software tool (the invention) takes the list of candidate metabolites, generated from NMRbased metabolite identification, and then calculates, for each of the candidate metabolites, the monoisotopic mass-tocharge (m/z) ratios for each commonly observed ion, fragment and adduct feature. These are then used to assign m/z ratios in experimental ESI-MS spectra of the same sample. Detection of the signals of a given metabolite in both NMR and MS spectra resolves the ambiguities, and therefore, significantly improves the confidence of the identification.

  13. Suppression of CCR impacts metabolite profile and cell wall composition in Pinus radiata tracheary elements.

    PubMed

    Wagner, Armin; Tobimatsu, Yuki; Goeminne, Geert; Phillips, Lorelle; Flint, Heather; Steward, Diane; Torr, Kirk; Donaldson, Lloyd; Boerjan, Wout; Ralph, John

    2013-01-01

    Suppression of the lignin-related gene cinnamoyl-CoA reductase (CCR) in the Pinus radiata tracheary element (TE) system impacted both the metabolite profile and the cell wall matrix in CCR-RNAi lines. UPLC-MS/MS-based metabolite profiling identified elevated levels of p-coumaroyl hexose, caffeic acid hexoside and ferulic acid hexoside in CCR-RNAi lines, indicating a redirection of metabolite flow within phenylpropanoid metabolism. Dilignols derived from coniferyl alcohol such as G(8-5)G, G(8-O-4)G and isodihydrodehydrodiconiferyl alcohol (IDDDC) were substantially depleted, providing evidence for CCR's involvement in coniferyl alcohol biosynthesis. Severe CCR suppression almost halved lignin content in TEs based on a depletion of both H-type and G-type lignin, providing evidence for CCR's involvement in the biosynthesis of both lignin types. 2D-NMR studies revealed minor changes in the H:G-ratio and consequently a largely unchanged interunit linkage distribution in the lignin polymer. However, unusual cell wall components including ferulate and unsaturated fatty acids were identified in TEs by thioacidolysis, pyrolysis-GC/MS and/or 2D-NMR in CCR-RNAi lines, providing new insights into the consequences of CCR suppression in pine. Interestingly, CCR suppression substantially promoted pyrolytic breakdown of cell wall polysaccharides, a phenotype most likely caused by the incorporation of acidic compounds into the cell wall matrix in CCR-RNAi lines.

  14. Autonomous Metabolomics for Rapid Metabolite Identification in Global Profiling

    PubMed Central

    2015-01-01

    An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. As a result of this unique integration, we can analyze large profiling datasets and simultaneously obtain structural identifications. Validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level. PMID:25496351

  15. Autonomous Metabolomics for Rapid Metabolite Identification in Global Profiling

    SciTech Connect

    Benton, H. Paul; Ivanisevic, Julijana; Mahieu, Nathaniel G.; Kurczy, Michael E.; Johnson, Caroline H.; Franco, Lauren; Rinehart, Duane; Valentine, Elizabeth; Gowda, Harsha; Ubhi, Baljit K.; Tautenhahn, Ralf; Gieschen, Andrew; Fields, Matthew W.; Patti, Gary J.; Siuzdak, Gary

    2014-12-12

    An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. We can analyze large profiling datasets and simultaneously obtain structural identifications, as a result of this unique integration. Furthermore, validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometry data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level.

  16. NMR method for measuring carbon-13 isotopic enrichment of metabolites in complex solutions.

    PubMed

    Lewis, Ian A; Karsten, Ryan H; Norton, Mark E; Tonelli, Marco; Westler, William M; Markley, John L

    2010-06-01

    Isotope-based methods are commonly used for metabolic flux analysis and metabolite quantification in biological extracts. Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for these studies because NMR can unambiguously identify compounds and accurately measure (13)C enrichment. We have developed a new pulse sequence, isotope-edited total correlation spectroscopy (ITOCSY), that filters two-dimensional (1)H-(1)H NMR spectra from (12)C- and (13)C-containing molecules into separate, quantitatively equivalent spectra. The ITOCSY spectra of labeled and unlabeled molecules are directly comparable and can be assigned using existing bioinformatics tools. In this study, we evaluate ITOCSY using synthetic mixtures of standards and extracts from Escherichia coli . We show that ITOCSY has low technical error (6.6% for metabolites ranging from 0.34 to 6.2 mM) and can detect molecules at concentrations less than 10 muM. We propose ITOCSY as a practical NMR strategy for metabolic flux analysis, isotope dilution experiments, and other methods that rely on carbon-13 labeling.

  17. NMR Method for Measuring Carbon-13 Isotopic Enrichment of Metabolites in Complex Solutions

    PubMed Central

    2010-01-01

    Isotope-based methods are commonly used for metabolic flux analysis and metabolite quantification in biological extracts. Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for these studies because NMR can unambiguously identify compounds and accurately measure 13C enrichment. We have developed a new pulse sequence, isotope-edited total correlation spectroscopy (ITOCSY), that filters two-dimensional 1H−1H NMR spectra from 12C- and 13C-containing molecules into separate, quantitatively equivalent spectra. The ITOCSY spectra of labeled and unlabeled molecules are directly comparable and can be assigned using existing bioinformatics tools. In this study, we evaluate ITOCSY using synthetic mixtures of standards and extracts from Escherichia coli. We show that ITOCSY has low technical error (6.6% for metabolites ranging from 0.34 to 6.2 mM) and can detect molecules at concentrations less than 10 μM. We propose ITOCSY as a practical NMR strategy for metabolic flux analysis, isotope dilution experiments, and other methods that rely on carbon-13 labeling. PMID:20459129

  18. NMR metabolic profiling of organic and aqueous sea bass extracts: implications in the discrimination of wild and cultured sea bass.

    PubMed

    Mannina, L; Sobolev, A P; Capitani, D; Iaffaldano, N; Rosato, M P; Ragni, P; Reale, A; Sorrentino, E; D'Amico, I; Coppola, R

    2008-10-19

    The nuclear magnetic resonance (NMR) technique was used as analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples. Preliminary results show that discrimination between wild and cultured sea bass was obtained not only using fatty acid composition but also cholesterol and phosphatidylethanolamine and some water-soluble metabolites such as choline, trimethylamine oxide, glutamine, fumaric and malic acids.

  19. Association between Metabolite Profiles, Metabolic Syndrome and Obesity Status

    PubMed Central

    Allam-Ndoul, Bénédicte; Guénard, Frédéric; Garneau, Véronique; Cormier, Hubert; Barbier, Olivier; Pérusse, Louis; Vohl, Marie-Claude

    2016-01-01

    Underlying mechanisms associated with the development of abnormal metabolic phenotypes among obese individuals are not yet clear. Our aim is to investigate differences in plasma metabolomics profiles between normal weight (NW) and overweight/obese (Ov/Ob) individuals, with or without metabolic syndrome (MetS). Mass spectrometry-based metabolite profiling was used to compare metabolite levels between each group. Three main principal components factors explaining a maximum of variance were retained. Factor 1’s (long chain glycerophospholipids) metabolite profile score was higher among Ov/Ob with MetS than among Ov/Ob and NW participants without MetS. This factor was positively correlated to plasma total cholesterol (total-C) and triglyceride levels in the three groups, to high density lipoprotein -cholesterol (HDL-C) among participants without MetS. Factor 2 (amino acids and short to long chain acylcarnitine) was positively correlated to HDL-C and negatively correlated with insulin levels among NW participants. Factor 3’s (medium chain acylcarnitines) metabolite profile scores were higher among NW participants than among Ov/Ob with or without MetS. Factor 3 was negatively associated with glucose levels among the Ov/Ob with MetS. Factor 1 seems to be associated with a deteriorated metabolic profile that corresponds to obesity, whereas Factors 2 and 3 seem to be rather associated with a healthy metabolic profile. PMID:27240400

  20. 1H HR-MAS NMR Spectroscopy and the Metabolite Determination of Typical Foods in Mediterranean Diet

    PubMed Central

    Corsaro, Carmelo; Mallamace, Domenico; Vasi, Sebastiano; Ferrantelli, Vincenzo; Dugo, Giacomo; Cicero, Nicola

    2015-01-01

    NMR spectroscopy has become an experimental technique widely used in food science. The experimental procedures that allow precise and quantitative analysis on different foods are relatively simple. For a better sensitivity and resolution, NMR spectroscopy is usually applied to liquid sample by means of extraction procedures that can be addressed to the observation of particular compounds. For the study of semisolid systems such as intact tissues, High-Resolution Magic Angle Spinning (HR-MAS) has received great attention within the biomedical area and beyond. Metabolic profiling and metabolism changes can be investigated both in animal organs and in foods. In this work we present a proton HR-MAS NMR study on the typical vegetable foods of Mediterranean diet such as the Protected Geographical Indication (PGI) cherry tomato of Pachino, the PGI Interdonato lemon of Messina, several Protected Designation of Origin (PDO) extra virgin olive oils from Sicily, and the Traditional Italian Food Product (PAT) red garlic of Nubia. We were able to identify and quantify the main metabolites within the studied systems that can be used for their characterization and authentication. PMID:26495154

  1. (1)H HR-MAS NMR Spectroscopy and the Metabolite Determination of Typical Foods in Mediterranean Diet.

    PubMed

    Corsaro, Carmelo; Mallamace, Domenico; Vasi, Sebastiano; Ferrantelli, Vincenzo; Dugo, Giacomo; Cicero, Nicola

    2015-01-01

    NMR spectroscopy has become an experimental technique widely used in food science. The experimental procedures that allow precise and quantitative analysis on different foods are relatively simple. For a better sensitivity and resolution, NMR spectroscopy is usually applied to liquid sample by means of extraction procedures that can be addressed to the observation of particular compounds. For the study of semisolid systems such as intact tissues, High-Resolution Magic Angle Spinning (HR-MAS) has received great attention within the biomedical area and beyond. Metabolic profiling and metabolism changes can be investigated both in animal organs and in foods. In this work we present a proton HR-MAS NMR study on the typical vegetable foods of Mediterranean diet such as the Protected Geographical Indication (PGI) cherry tomato of Pachino, the PGI Interdonato lemon of Messina, several Protected Designation of Origin (PDO) extra virgin olive oils from Sicily, and the Traditional Italian Food Product (PAT) red garlic of Nubia. We were able to identify and quantify the main metabolites within the studied systems that can be used for their characterization and authentication.

  2. Profiling Lipid Metabolites Yields Unique Information on Sex- and Time-dependent Responses of Fathead Minnows (Pimephales promelas) Exposed to 17α-Ethynylestradiol

    EPA Science Inventory

    Alterations in hepatic lipid profiles of fathead minnows (FHM) exposed to the synthetic estrogen 17α-ethynylestradiol (EE2) were determined using 1H-NMR spectroscopy-based metabolite profiling. The exposures were conducted using either 10 ng/l or 100 ng/l EE2 via a continuous flo...

  3. Profiling Lipid Metabolites Yields Unique Information on Sex- and Time-dependent Responses of Fathead Minnows (Pimephales promelas) Exposed to 17α-Ethynylestradiol

    EPA Science Inventory

    Alterations in hepatic lipid profiles of fathead minnows (FHM) exposed to the synthetic estrogen 17α-ethynylestradiol (EE2) were determined using 1H-NMR spectroscopy-based metabolite profiling. The exposures were conducted using either 10 ng/l or 100 ng/l EE2 via a continuous flo...

  4. Probing Metabolite Space of Escherichia coli via Growth Medium Composition as Monitored by Two-Dimensional NMR Spectroscopy.

    PubMed

    Kee Chae, Young; Hyun Kim, Seol; Seong Hyun, Jin

    2015-06-01

    As for recombinant protein production, Escherichia coli is one of the most frequently employed hosts because it offers a simple and inexpensive, but rapid and high-yield system in addition to the vast information on its molecular genetics and biology. However, due to its prokaryotic nature, it often fails to produce eukaryotic proteins in a desired form. To devise a systematic way leading to a condition that produces a large amount of usable proteins, we attempted to monitor intracellular metabolites under various conditions, and to link them to recombinant protein production. With such an intention, we identified 31 metabolites from cells grown in different media by using two-dimensional (2D) NMR spectroscopy. Our results revealed that 1) the level of betaine was low, while that of glutamic acid was high when grown in minimal media; 2) the level of glycerol was constantly high in all cases; 3) the level of oxidized glutathione was lower in Luria broth (LB); and 4) the level of leucine was low in minimal media. We hope this work might shed light onto how to improve production of the target proteins by metabolite profiling. Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

  5. Comparing metabolite profiles of habitual diet in serum and urine.

    PubMed

    Playdon, Mary C; Sampson, Joshua N; Cross, Amanda J; Sinha, Rashmi; Guertin, Kristin A; Moy, Kristin A; Rothman, Nathaniel; Irwin, Melinda L; Mayne, Susan T; Stolzenberg-Solomon, Rachael; Moore, Steven C

    2016-09-01

    Diet plays an important role in chronic disease etiology, but some diet-disease associations remain inconclusive because of methodologic limitations in dietary assessment. Metabolomics is a novel method for identifying objective dietary biomarkers, although it is unclear what dietary information is captured from metabolites found in serum compared with urine. We compared metabolite profiles of habitual diet measured from serum with those measured from urine. We first estimated correlations between consumption of 56 foods, beverages, and supplements assessed by a food-frequency questionnaire, with 676 serum and 848 urine metabolites identified by untargeted liquid chromatography mass spectrometry, ultra-high performance liquid chromatography tandem mass spectrometry, and gas chromatography mass spectrometry in a colon adenoma case-control study (n = 125 cases and 128 controls) while adjusting for age, sex, smoking, fasting, case-control status, body mass index, physical activity, education, and caloric intake. We controlled for multiple comparisons with the use of a false discovery rate of <0.1. Next, we created serum and urine multiple-metabolite models to predict food intake with the use of 10-fold crossvalidation least absolute shrinkage and selection operator regression for 80% of the data; predicted values were created in the remaining 20%. Finally, we compared predicted values with estimates obtained from self-reported intake for metabolites measured in serum and urine. We identified metabolites associated with 46 of 56 dietary items; 417 urine and 105 serum metabolites were correlated with ≥1 food, beverage, or supplement. More metabolites in urine (n = 154) than in serum (n = 39) were associated uniquely with one food. We found previously unreported metabolite associations with leafy green vegetables, sugar-sweetened beverages, citrus, added sugar, red meat, shellfish, desserts, and wine. Prediction of dietary intake from multiple-metabolite profiles was

  6. Metabolite Profiles and the Risk of Developing Diabetes

    PubMed Central

    Wang, Thomas J.; Larson, Martin G.; Vasan, Ramachandran S.; Cheng, Susan; Rhee, Eugene P.; McCabe, Elizabeth; Lewis, Gregory D.; Fox, Caroline S.; Jacques, Paul F.; Fernandez, Céline; O’Donnell, Christopher J.; Carr, Stephen A.; Mootha, Vamsi K.; Florez, Jose C.; Souza, Amanda; Melander, Olle; Clish, Clary B.; Gerszten, Robert E.

    2011-01-01

    Emerging technologies allow the high-throughput profiling of metabolic status from a blood specimen (metabolomics). We investigated whether metabolite profiles could predict the development of diabetes. Among 2,422 normoglycemic individuals followed for 12 years, 201 developed diabetes. Amino acids, amines, and other polar metabolites were profiled in baseline specimens using liquid chromatography-tandem mass spectrometry. Cases and controls were matched for age, body mass index and fasting glucose. Five branched-chain and aromatic amino acids had highly-significant associations with future diabetes: isoleucine, leucine, valine, tyrosine, and phenylalanine. A combination of three amino acids predicted future diabetes (>5-fold higher risk for individuals in top quartile). The results were replicated in an independent, prospective cohort. These findings underscore the potential importance of amino acid metabolism early in the pathogenesis of diabetes, and suggest that amino acid profiles could aid in diabetes risk assessment. PMID:21423183

  7. Global metabolite profiling of human colorectal cancer xenografts in mice using HPLC-MS/MS.

    PubMed

    Loftus, Neil J; Lai, Lindsay; Wilkinson, Robert W; Odedra, Rajesh; Wilson, Ian D; Barnes, Alan J

    2013-06-07

    Reversed-phase gradient LC-MS was used to perform untargeted metabonomic analysis on extracts of human colorectal cancer (CRC) cell lines (COLO 205, HT-29, HCT 116 and SW620) subcutaneously implanted into age-matched athymic nude male mice to study small molecule metabolic profiles and examine possible correlations with human cancer biopsies. Following high mass accuracy data analysis using MS and MS/MS, metabolites were identified by searching against major metabolite databases including METLIN, MASSBANK, The Human Metabolome Database, PubChem, Biospider, LipidMaps and KEGG. HT-29 and COLO 205 tumor xenografts showed a distribution of metabolites that differed from SW620 and HCT 116 xenografts (predominantly on the basis of relative differences in the amounts of amino acids and lipids detected). This finding is consistent with NMR-based analysis of human colorectal tissue, where the metabolite profiles of HT-29 tumors exhibit the greatest similarity to human rectal cancer tissue with respect to changes in the relative amounts of lipids and choline-containing compounds. As the metabolic signatures of cancer cells result from oncogene-directed metabolic reprogramming, the HT-29 xenografts in mice may prove to be a useful model to further study the tumor microenvironment and cancer biology.

  8. Plant metabolite profiles and the buffering capacities of ecosystems.

    PubMed

    Fester, Thomas

    2015-02-01

    In spite of some inherent challenges, metabolite profiling is becoming increasingly popular under field conditions. It has been used successfully to address topics like species interactions, connections between growth and chemical stoichiometry or the plant's stress response. Stress exerts a particularly clear impact on plant metabolomes and has become a central topic in many metabolite profiling experiments in the fields. In contrast to phytochambers, however, external stress is often at least partially absorbed by the environment when measuring under field conditions. Such stress-buffering capacities of (agro)-ecosystems are of crucial interest given the ever-increasing anthropogenic impact on ecosystems and this review promotes the idea of using plant metabolite profiles for respective measurements. More specifically I propose to use parameters of the response of key plant species to a given stress treatment as proxies for measuring and comparing stress-buffering capacities of ecosystems. Stress response parameters accessible by metabolite profiling comprise for example the intensity or duration of the impact of stress or the ability of the plant organism to recover from this impact after a given time. Analyses of ecosystem stress-buffering capacities may improve our understanding of how ecosystems cope with stress and may improve our abilities to predict ecosystem changes.

  9. mQTL.NMR: an integrated suite for genetic mapping of quantitative variations of (1)H NMR-based metabolic profiles.

    PubMed

    Hedjazi, Lyamine; Gauguier, Dominique; Zalloua, Pierre A; Nicholson, Jeremy K; Dumas, Marc-Emmanuel; Cazier, Jean-Baptiste

    2015-04-21

    High-throughput (1)H nuclear magnetic resonance (NMR) is an increasingly popular robust approach for qualitative and quantitative metabolic profiling, which can be used in conjunction with genomic techniques to discover novel genetic associations through metabotype quantitative trait locus (mQTL) mapping. There is therefore a crucial necessity to develop specialized tools for an accurate detection and unbiased interpretability of the genetically determined metabolic signals. Here we introduce and implement a combined chemoinformatic approach for objective and systematic analysis of untargeted (1)H NMR-based metabolic profiles in quantitative genetic contexts. The R/Bioconductor mQTL.NMR package was designed to (i) perform a series of preprocessing steps restoring spectral dependency in collinear NMR data sets to reduce the multiple testing burden, (ii) carry out robust and accurate mQTL mapping in human cohorts as well as in rodent models, (iii) statistically enhance structural assignment of genetically determined metabolites, and (iv) illustrate results with a series of visualization tools. Built-in flexibility and implementation in the powerful R/Bioconductor framework allow key preprocessing steps such as peak alignment, normalization, or dimensionality reduction to be tailored to specific problems. The mQTL.NMR package is freely available with its source code through the Comprehensive R/Bioconductor repository and its own website ( http://www.ican-institute.org/tools/ ). It represents a significant advance to facilitate untargeted metabolomic data processing and quantitative analysis and their genetic mapping.

  10. (1)H NMR studies distinguish the water soluble metabolomic profiles of untransformed and RAS-transformed cells.

    PubMed

    Marks, Vered; Munoz, Anisleidys; Rai, Priyamvada; Walls, Jamie D

    2016-01-01

    Metabolomic profiling is an increasingly important method for identifying potential biomarkers in cancer cells with a view towards improved diagnosis and treatment. Nuclear magnetic resonance (NMR) provides a potentially noninvasive means to accurately characterize differences in the metabolomic profiles of cells. In this work, we use (1)H NMR to measure the metabolomic profiles of water soluble metabolites extracted from isogenic control and oncogenic HRAS-, KRAS-, and NRAS-transduced BEAS2B lung epithelial cells to determine the robustness of NMR metabolomic profiling in detecting differences between the transformed cells and their untransformed counterparts as well as differences among the RAS-transformed cells. Unique metabolomic signatures between control and RAS-transformed cell lines as well as among the three RAS isoform-transformed lines were found by applying principal component analysis to the NMR data. This study provides a proof of principle demonstration that NMR-based metabolomic profiling can robustly distinguish untransformed and RAS-transformed cells as well as cells transformed with different RAS oncogenic isoforms. Thus, our data may potentially provide new diagnostic signatures for RAS-transformed cells.

  11. 1H NMR studies distinguish the water soluble metabolomic profiles of untransformed and RAS-transformed cells

    PubMed Central

    Marks, Vered; Munoz, Anisleidys; Rai, Priyamvada

    2016-01-01

    Metabolomic profiling is an increasingly important method for identifying potential biomarkers in cancer cells with a view towards improved diagnosis and treatment. Nuclear magnetic resonance (NMR) provides a potentially noninvasive means to accurately characterize differences in the metabolomic profiles of cells. In this work, we use 1H NMR to measure the metabolomic profiles of water soluble metabolites extracted from isogenic control and oncogenic HRAS-, KRAS-, and NRAS-transduced BEAS2B lung epithelial cells to determine the robustness of NMR metabolomic profiling in detecting differences between the transformed cells and their untransformed counterparts as well as differences among the RAS-transformed cells. Unique metabolomic signatures between control and RAS-transformed cell lines as well as among the three RAS isoform-transformed lines were found by applying principal component analysis to the NMR data. This study provides a proof of principle demonstration that NMR-based metabolomic profiling can robustly distinguish untransformed and RAS-transformed cells as well as cells transformed with different RAS oncogenic isoforms. Thus, our data may potentially provide new diagnostic signatures for RAS-transformed cells. PMID:27330862

  12. 1H-NMR-based metabolic profiling of a colorectal cancer CT-26 lung metastasis model in mice.

    PubMed

    Li, Yan; Wang, Chunting; Li, Dandan; Deng, Pengchi; Shao, Xiaoni; Hu, Jing; Liu, Chunqi; Jie, Hui; Lin, Yiyun; Li, Zhuoling; Qian, Xinying; Zhang, Huaqin; Zhao, Yinglan

    2017-09-13

    Lung metastasis is an important cause for the low 5-year survival rate of colorectal cancer patients. Understanding the metabolic profile of lung metastasis of colorectal cancer is important for developing molecular diagnostic and therapeutic approaches. We carried out the metabonomic profiling of lung tissue samples on a mouse lung metastasis model of colorectal cancer using 1H-nuclear magnetic resonance (1H-NMR). The lung tissues of mice were collected at different intervals after marine colon cancer cell line CT-26 was intravenously injected into BALB/c mice. The distinguishing metabolites of lung tissue were investigated using 1H-NMR-based metabonomic assay, which is a highly sensitive and non-destructive method for biomarker identification. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were applied to analyze 1H-NMR profiling data to seek potential biomarkers. All of the 3 analyses achieved excellent separations between the normal and metastasis groups. A total of 42 metabolites were identified, ~12 of which were closely correlated with the process of metastasis from colon to lung. These altered metabolites indicated the disturbance of metabolism in metastatic tumors including glycolysis, TCA cycle, glutaminolysis, choline metabolism and serine biosynthesis. Our findings firstly identified the distinguishing metabolites in mouse colorectal cancer lung metastasis models, and indicated that the metabolite disturbance may be associated with the progression of lung metastasis from colon cancer. The altered metabolites may be potential biomarkers that provide a promising molecular approach for clinical diagnosis and mechanistic study of colorectal cancer with lung metastasis.

  13. Profiling human gut bacterial metabolism and its kinetics using [U-13C]glucose and NMR.

    PubMed

    de Graaf, Albert A; Maathuis, Annet; de Waard, Pieter; Deutz, Nicolaas E P; Dijkema, Cor; de Vos, Willem M; Venema, Koen

    2010-01-01

    This study introduces a stable-isotope metabolic approach employing [U-(13)C]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well-established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U-(13)C]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a (13)C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed (13)C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the (12)C contents and (13)C labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short-chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner.

  14. Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra.

    PubMed

    Lewis, Ian A; Schommer, Seth C; Hodis, Brendan; Robb, Kate A; Tonelli, Marco; Westler, William M; Sussman, Michael R; Markley, John L

    2007-12-15

    One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) spectroscopy is used extensively for high-throughput analysis of metabolites in biological fluids and tissue extracts. Typically, such spectra are treated as multivariate statistical objects rather than as collections of quantifiable metabolites. We report here a two-dimensional (2D) (1)H-(13)C NMR strategy (fast metabolite quantification, FMQ, by NMR) for identifying and quantifying the approximately 40 most abundant metabolites in biological samples. To validate this technique, we prepared mixtures of synthetic compounds and extracts from Arabidopsis thaliana, Saccharomyces cerevisiae, and Medicago sativa. We show that accurate (technical error 2.7%) molar concentrations can be determined in 12 min using our quantitative 2D (1)H-(13)C NMR strategy. In contrast, traditional 1D (1)H NMR analysis resulted in 16.2% technical error under nearly ideal conditions. We propose FMQ by NMR as a practical alternative to 1D (1)H NMR for metabolomics studies in which 50-mg (extract dry weight) samples can be obtained.

  15. Biomarker Research in Parkinson's Disease Using Metabolite Profiling.

    PubMed

    Havelund, Jesper F; Heegaard, Niels H H; Færgeman, Nils J K; Gramsbergen, Jan Bert

    2017-08-11

    Biomarker research in Parkinson's disease (PD) has long been dominated by measuring dopamine metabolites or alpha-synuclein in cerebrospinal fluid. However, these markers do not allow early detection, precise prognosis or monitoring of disease progression. Moreover, PD is now considered a multifactorial disease, which requires a more precise diagnosis and personalized medication to obtain optimal outcome. In recent years, advanced metabolite profiling of body fluids like serum/plasma, CSF or urine, known as "metabolomics", has become a powerful and promising tool to identify novel biomarkers or "metabolic fingerprints" characteristic for PD at various stages of disease. In this review, we discuss metabolite profiling in clinical and experimental PD. We briefly review the use of different analytical platforms and methodologies and discuss the obtained results, the involved metabolic pathways, the potential as a biomarker and the significance of understanding the pathophysiology of PD. Many of the studies report alterations in alanine, branched-chain amino acids and fatty acid metabolism, all pointing to mitochondrial dysfunction in PD. Aromatic amino acids (phenylalanine, tyrosine, tryptophan) and purine metabolism (uric acid) are also altered in most metabolite profiling studies in PD.

  16. Characterization of metabolites in different kiwifruit varieties by NMR and fluorescence spectroscopy.

    PubMed

    Abdul Hamid, Nur Ashikin; Mediani, Ahmed; Maulidiani, M; Abas, Faridah; Park, Yong Seo; Leontowicz, Hanna; Leontowicz, Maria; Namiesnik, Jacek; Gorinstein, S

    2017-05-10

    It is known from our previous studies that kiwifruits, which are used in common human diet, have preventive properties of coronary artery disease. This study describes a combination of (1)H NMR spectroscopy, multivariate data analyses and fluorescence measurements in differentiating of some kiwifruit varieties, their quenching and antioxidant properties. A total of 41 metabolites were identified by comparing with literature data Chenomx database and 2D NMR. The binding properties of the extracted polyphenols against HSA showed higher reactivity of studied two cultivars in comparison with the common Hayward. The results showed that the fluorescence of HSA was quenched by Bidan as much as twice than by other fruits. The correlation between the binding properties of polyphenols in the investigated fruits, their relative quantification and suggested metabolic pathway was established. These results can provide possible application of fruit extracts in pharmaceutical industry.

  17. NMR Chemical Shift Ranges of Urine Metabolites in Various Organic Solvents

    PubMed Central

    Görling, Benjamin; Bräse, Stefan; Luy, Burkhard

    2016-01-01

    Signal stability is essential for reliable multivariate data analysis. Urine samples show strong variance in signal positions due to inter patient differences. Here we study the exchange of the solvent of a defined urine matrix and how it affects signal and integral stability of the urinary metabolites by NMR spectroscopy. The exchange solvents were methanol, acetonitrile, dimethyl sulfoxide, chloroform, acetone, dichloromethane, and dimethyl formamide. Some of these solvents showed promising results with a single batch of urine. To evaluate further differences between urine samples, various acid, base, and salt solutions were added in a defined way mimicking to some extent inter human differences. Corresponding chemical shift changes were monitored. PMID:27598217

  18. Synthesis and NMR studies of (13)C-labeled vitamin D metabolites.

    PubMed

    Okamura, William H; Zhu, Gui-Dong; Hill, David K; Thomas, Richard J; Ringe, Kerstin; Borchardt, Daniel B; Norman, Anthony W; Mueller, Leonard J

    2002-03-08

    Isotope-labeled drug molecules may be useful for probing by NMR spectroscopy the conformation of ligand associated with biological hosts such as membranes and proteins. Triple-labeled [7,9,19-(13)C(3)]-vitamin D(3) (56), its 25-hydroxylated and 1 alpha,25-dihydroxylated metabolites (58 and 68, respectively), and other labeled materials have been synthesized via coupling of [9-(13)C]-Grundmann's ketone 39 or its protected 25-hydroxy derivative 43 with labeled A ring enyne fragments 25 or 26. The labeled CD-ring fragment 39 was prepared by a sequence involving Grignard addition of [(13)C]-methylmagnesium iodide to Grundmann's enone 28, oxidative cleavage, functional group modifications leading to seco-iodide 38, and finally a kinetic enolate S(N)2 cycloalkylation. The C-7,19 double labeling of the A-ring enyne was achieved by the Corey-Fuchs/Wittig processes on keto aldehyde 11. By employing these labeled fragments in the Wilson-Mazur route, the C-7,9,19 triple-(13)C-labeled metabolites 56, 58, and 68 as well as other (13)C-labeled metabolites have been prepared. In an initial NMR investigation of one of the labeled metabolites prepared in this study, namely [7,9,19-(13)C(3)]-25-hydroxyvitamin D(3) (58), the three (13)C-labeled carbons of the otherwise water insoluble steroid could be clearly detected by (13)C NMR analysis at 0.1 mM in a mixture of CD(3)OD/D(2)O (60/40) or in aqueous dimethylcyclodextrin solution and at 2 mM in 20 mM sodium dodecyl sulfate (SDS) aqueous micellar solution. In the SDS micellar solution, a double half-filter NOESY experiment revealed that the distance between the H(19Z) and H(7) protons is significantly shorter than that of the corresponding distance calculated from the solid state (X-ray) structure of the free ligand. The NMR data in micelles reveals that 58 exists essentially completely in the alpha-conformer with the 3 beta-hydroxyl equatorially oriented, just as in the solid state. The shortened distance (H(19Z))-H(7)) in micellar

  19. NMR Chemical Shift Ranges of Urine Metabolites in Various Organic Solvents.

    PubMed

    Görling, Benjamin; Bräse, Stefan; Luy, Burkhard

    2016-09-02

    Signal stability is essential for reliable multivariate data analysis. Urine samples show strong variance in signal positions due to inter patient differences. Here we study the exchange of the solvent of a defined urine matrix and how it affects signal and integral stability of the urinary metabolites by NMR spectroscopy. The exchange solvents were methanol, acetonitrile, dimethyl sulfoxide, chloroform, acetone, dichloromethane, and dimethyl formamide. Some of these solvents showed promising results with a single batch of urine. To evaluate further differences between urine samples, various acid, base, and salt solutions were added in a defined way mimicking to some extent inter human differences. Corresponding chemical shift changes were monitored.

  20. Tissue metabolic profiling of lymph node metastasis of colorectal cancer assessed by 1H NMR.

    PubMed

    Zhang, Hailong; Qiao, Liang; Li, Xiaopeng; Wan, Yang; Yang, Li; Wang, Huijuan

    2016-12-01

    Lymph node metastasis is a decisive prognostic and therapeutic staging factor for colorectal cancer (CRC), which is one of the most prevalent types of cancer and a malignant tumor. The metabolic profiling of tissue samples from a large cohort of lymph node non‑metastatic CRC patients (n=73), lymph node metastatic CRC patients (n=52) and normal controls (n=41) was performed using 1H nuclear magnetic resonance (NMR) together with multivariate statistical analyses. Excellent separation was obtained between CRC patients and normal controls, and CRC patients were also perfectly classified according to lymph node metastasis. Forty‑two distinguishing metabolites were identified, which revealed disturbance of glycolysis, glutaminolysis, fatty acid metabolism, choline metabolism and amino acids, suggesting that cellular functions in energy production, macromolecular synthesis, oxidative stress and immune escape of cancer cells are affected in CRC. In total, 10 tissue metabolites were significantly disturbed between non‑metastatic and metastatic CRC patients. The present study firstly staged CRC patients by lymph node metastasis by metabolomic approach. The identified metabolites may be associated with the neoplasia, invasion and metastasis of the tumor. The results suggest the promising application of these metabolites in clinical therapy, and further understanding of the related mechanism warrants further investigation.

  1. Detection and follow-up, after partial liver resection, of the urinary paracetamol metabolites by proton NMR spectroscopy.

    PubMed

    Galinski, Michel; Racine, Stephane-Xavier; Bossard, Anne-Elisabeth; Fleyfel, Maher; Hamza, Lilia; Bouchemal, Nadia; Adnet, Frédéric; Le Moyec, Laurence

    2014-01-01

    Combination drug therapy is often used to achieve optimal analgesia in surgery. Paracetamol can be used as one component of an analgesic regime following hepatic resection. This study was designed to investigate paracetamol and its metabolites by proton NMR spectroscopy in patient urine and to assess whether N-acetyl-p-benzoquinone imine (NAPQI, a hepatotoxic metabolite) formation is increased after liver resection. We studied the excretion of acetaminophen and its metabolites by 5 patients who were operated on for partial liver resection by proton NMR spectroscopy. As an intravenous infusion 1 g of paracetamol was given over 15 min every 6 h during 48 h. The first injection was given in the operating theatre after liver resection was completed. Urine samples were collected before injection (T1) and 24 and 48 h after the first injection (T2 and T3); the samples were frozen and kept at -20°C up to the analysis by NMR spectroscopy. Metabolites of the paracetamol were detected for all patients. Among the discerned metabolites, 4 were identified as metabolites of paracetamol: paracetamol glucuronide, paracetamol sulfate, N-acetyl-L-cysteinyl paracetamol (metabolite of NAPQI) and paracetamol. Their ratios, respectively, were: 46-82.9, 12.6-30.0, 0.5-5.5 and 1.43-3.54%. This study showed that there was no increase in the formation of toxic metabolite (NAPQI) after treatment with paracetamol in these few cases of liver resections. A larger study is necessary to confirm these results.

  2. Secondary metabolite profiling of Curcuma species grown at different locations using GC/TOF and UPLC/Q-TOF MS.

    PubMed

    Lee, Jueun; Jung, Youngae; Shin, Jeoung-Hwa; Kim, Ho Kyoung; Moon, Byeong Cheol; Ryu, Do Hyun; Hwang, Geum-Sook

    2014-07-04

    Curcuma, a genus of rhizomatous herbaceous species, has been used as a spice, traditional medicine, and natural dye. In this study, the metabolite profile of Curcuma extracts was determined using gas chromatography-time of flight mass spectrometry (GC/TOF MS) and ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) to characterize differences between Curcuma aromatica and Curcuma longa grown on the Jeju-do or Jin-do islands, South Korea. Previous studies have performed primary metabolite profiling of Curcuma species grown in different regions using NMR-based metabolomics. This study focused on profiling of secondary metabolites from the hexane extract of Curcuma species. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) plots showed significant differences between the C. aromatica and C. longa metabolite profiles, whereas geographical location had little effect. A t-test was performed to identify statistically significant metabolites, such as terpenoids. Additionally, targeted profiling using UPLC/Q-TOF MS showed that the concentration of curcuminoids differed depending on the plant origin. Based on these results, a combination of GC- and LC-MS allowed us to analyze curcuminoids and terpenoids, the typical bioactive compounds of Curcuma, which can be used to discriminate Curcuma samples according to species or geographical origin.

  3. Application of diffusion-edited NMR spectroscopy for the structural characterization of drug metabolites in mixtures.

    PubMed

    Khera, Smriti; Grillo, Mark; Schnier, Paul; Hollis, Steve

    2010-01-05

    Diffusion-edited NMR spectroscopy is used to enable the structural characterization of low level metabolites in the presence of endogenous compounds, and organic solvents. We compared data from standard one-dimensional (1D) (1)H, 1D NOESY-presaturation, and 1D diffusion-edited experiments run on 20 microg and 100 microg samples of ethacrynic acid glutathione thioether (EASG) and a previously unreported metabolite of mefenamic acid, mefenamic acid glutathione thioester (MSG). The 1D NOESY-presaturation technique gave spectra with the best signal-to-noise (S/N) ratio, approximately three times that observed with the standard (1)H experiment, with respect to the metabolite signals. However, it was not selective for solvent signals as overlapping metabolite signals were also suppressed by this technique. In some cases, these signals were key to determining the site of glutathione attachment on the parent molecule. 1D NOESY-presaturation spectra also produced baseline distortions and inconsistent integration values. By comparison, 1D diffusion-edited experiments were found to selectively and simultaneously remove multiple solvent signals, resolve overlapping metabolite signals, and provide more uniform integration for metabolite signals overlapping with or proximal to solvent peaks, without producing baseline distortions. However, the diffusion-edited experiments caused significant signal attenuation of the metabolite signals when compared with a standard (1)H spectrum. Partially purified metabolites isolated from biological matrices were also characterized by using two-dimensional diffusion-ordered spectroscopy (DOSY). DOSY spectra acquired on a sample of EASG purified from rat bile proved useful in 'separating' the signals of EASG, from those of a co-eluting bile acid and parent drug ethacrynic acid (EA) in the diffusion-dimension in regions where there was no spectral overlap. In the low-field regions of high overlap, the DOSY experiment did not effectively separate

  4. Profiling formulated monoclonal antibodies by (1)H NMR spectroscopy.

    PubMed

    Poppe, Leszek; Jordan, John B; Lawson, Ken; Jerums, Matthew; Apostol, Izydor; Schnier, Paul D

    2013-10-15

    Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

  5. Urine Metabolite Profiles Predictive of Human Kidney Allograft Status.

    PubMed

    Suhre, Karsten; Schwartz, Joseph E; Sharma, Vijay K; Chen, Qiuying; Lee, John R; Muthukumar, Thangamani; Dadhania, Darshana M; Ding, Ruchuang; Ikle, David N; Bridges, Nancy D; Williams, Nikki M; Kastenmüller, Gabi; Karoly, Edward D; Mohney, Robert P; Abecassis, Michael; Friedewald, John; Knechtle, Stuart J; Becker, Yolanda T; Samstein, Benjamin; Shaked, Abraham; Gross, Steven S; Suthanthiran, Manikkam

    2016-02-01

    Noninvasive diagnosis and prognostication of acute cellular rejection in the kidney allograft may help realize the full benefits of kidney transplantation. To investigate whether urine metabolites predict kidney allograft status, we determined levels of 749 metabolites in 1516 urine samples from 241 kidney graft recipients enrolled in the prospective multicenter Clinical Trials in Organ Transplantation-04 study. A metabolite signature of the ratio of 3-sialyllactose to xanthosine in biopsy specimen-matched urine supernatants best discriminated acute cellular rejection biopsy specimens from specimens without rejection. For clinical application, we developed a high-throughput mass spectrometry-based assay that enabled absolute and rapid quantification of the 3-sialyllactose-to-xanthosine ratio in urine samples. A composite signature of ratios of 3-sialyllactose to xanthosine and quinolinate to X-16397 and our previously reported urinary cell mRNA signature of 18S ribosomal RNA, CD3ε mRNA, and interferon-inducible protein-10 mRNA outperformed the metabolite signatures and the mRNA signature. The area under the receiver operating characteristics curve for the composite metabolite-mRNA signature was 0.93, and the signature was diagnostic of acute cellular rejection with a specificity of 84% and a sensitivity of 90%. The composite signature, developed using solely biopsy specimen-matched urine samples, predicted future acute cellular rejection when applied to pristine samples taken days to weeks before biopsy. We conclude that metabolite profiling of urine offers a noninvasive means of diagnosing and prognosticating acute cellular rejection in the human kidney allograft, and that the combined metabolite and mRNA signature is diagnostic and prognostic of acute cellular rejection with very high accuracy.

  6. Potent Antidiabetic Activity and Metabolite Profiling of Melicope Lunu-ankenda Leaves.

    PubMed

    Al-Zuaidy, Mizher Hezam; Hamid, Azizah Abdul; Ismail, Amin; Mohamed, Suhaila; Abdul Razis, Ahmad Faizal; Mumtaz, Muhammad Waseem; Salleh, Syafiq Zikri

    2016-05-01

    Diabetes mellitus is normally characterized by chronic hyperglycemia associated with disturbances in the fat, carbohydrate, and protein metabolism. There is an increasing trend of using natural products instead of synthetic agents as alternative therapy for disorders due to their fewer side effects. In this study, antidiabetic and antioxidant activities of different Melicope lunu-ankenda (ML) ethanolic extracts were evaluated using inhibition of α-glucosidase and 2,2-diphenyl-l-picrylhydrazyl (DPPH) radicals scavenging activity, respectively; whereas, proton nuclear magnetic resonance ((1) H NMR) and ultra-high performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) techniques were used for metabolite profiling of ML leaf extracts at different concentrations of ethanol and water. Sixty percent of ethanolic ML extract showed highest inhibitory effect against α-glucosidase enzyme (IC50 of 37 μg/mL) and DPPH scavenging activity (IC50 of 48 μg/mL). Antidiabetic effect of ML extracts was also evaluated in vivo and it was found that the high doses (400 mg/Kg BW) of ML extract exhibited high suppression in fasting blood glucose level by 62.75%. The metabolites responsible for variation among ML samples with variable ethanolic levels have been evaluated successfully using (1) H-NMR-based metabolomics. The principal component analysis (PCA) and partial least squares(PLS) analysis scores depicted clear and distinct separations into 4 clusters representing the 4 ethanolic concentrations by PC1 and PC2, with an eigenvalue of 69.9%. Various (1) H-NMR chemical shifts related to the metabolites responsible for sample difference were also ascribed. The main bioactive compounds identified attributing toward the separation included: isorhamnetin, skimmianine, scopoletin, and melicarpinone. Hence, ML may be used as promising medicinal plant for the development of new functional foods, new generation antidiabetic drugs, as a single entity phytomedicine or in

  7. Metabolite Content Profiling of Bottlenose Dolphin Exhaled Breath

    PubMed Central

    2014-01-01

    Changing ocean health and the potential impact on marine mammal health are gaining global attention. Direct health assessments of wild marine mammals, however, is inherently difficult. Breath analysis metabolomics is a very attractive assessment tool due to its noninvasive nature, but it is analytically challenging. It has never been attempted in cetaceans for comprehensive metabolite profiling. We have developed a method to reproducibly sample breath from small cetaceans, specifically Atlantic bottlenose dolphins (Tursiops truncatus). We describe the analysis workflow to profile exhaled breath metabolites and provide here a first library of volatile and nonvolatile compounds in cetacean exhaled breath. The described analytical methodology enabled us to document baseline compounds in exhaled breath of healthy animals and to study changes in metabolic content of dolphin breath with regard to a variety of factors. The method of breath analysis may provide a very valuable tool in future wildlife conservation efforts as well as deepen our understanding of marine mammals biology and physiology. PMID:25254551

  8. Evaluation of 1H NMR metabolic profiling using biofluid mixture design.

    PubMed

    Athersuch, Toby J; Malik, Shahid; Weljie, Aalim; Newton, Jack; Keun, Hector C

    2013-07-16

    A strategy for evaluating the performance of quantitative spectral analysis tools in conditions that better approximate background variation in a metabonomics experiment is presented. Three different urine samples were mixed in known proportions according to a {3, 3} simplex lattice experimental design and analyzed in triplicate by 1D (1)H NMR spectroscopy. Fifty-four urinary metabolites were subsequently quantified from the sample spectra using two methods common in metabolic profiling studies: (1) targeted spectral fitting and (2) targeted spectral integration. Multivariate analysis using partial least-squares (PLS) regression showed the latent structure of the spectral set recapitulated the experimental mixture design. The goodness-of-prediction statistic (Q(2)) of each metabolite variable in a PLS model was calculated as a metric for the reliability of measurement, across the sample compositional space. Several metabolites were observed to have low Q(2) values, largely as a consequence of their spectral resonances having low s/n or strong overlap with other sample components. This strategy has the potential to allow evaluation of spectral features obtained from metabolic profiling platforms in the context of the compositional background found in real biological sample sets, which may be subject to considerable variation. We suggest that it be incorporated into metabolic profiling studies to improve the estimation of matrix effects that confound accurate metabolite measurement. This novel method provides a rational basis for exploiting information from several samples in an efficient manner and avoids the use of multiple spike-in authentic standards, which may be difficult to obtain.

  9. Autonomous Metabolomics for Rapid Metabolite Identification in Global Profiling

    DOE PAGES

    Benton, H. Paul; Ivanisevic, Julijana; Mahieu, Nathaniel G.; ...

    2014-12-12

    An autonomous metabolomic workflow combining mass spectrometry analysis with tandem mass spectrometry data acquisition was designed to allow for simultaneous data processing and metabolite characterization. Although previously tandem mass spectrometry data have been generated on the fly, the experiments described herein combine this technology with the bioinformatic resources of XCMS and METLIN. We can analyze large profiling datasets and simultaneously obtain structural identifications, as a result of this unique integration. Furthermore, validation of the workflow on bacterial samples allowed the profiling on the order of a thousand metabolite features with simultaneous tandem mass spectra data acquisition. The tandem mass spectrometrymore » data acquisition enabled automatic search and matching against the METLIN tandem mass spectrometry database, shortening the current workflow from days to hours. Overall, the autonomous approach to untargeted metabolomics provides an efficient means of metabolomic profiling, and will ultimately allow the more rapid integration of comparative analyses, metabolite identification, and data analysis at a systems biology level.« less

  10. Toward better annotation in plant metabolomics: isolation and structure elucidation of 36 specialized metabolites from Oryza sativa (rice) by using MS/MS and NMR analyses.

    PubMed

    Yang, Zhigang; Nakabayashi, Ryo; Okazaki, Yozo; Mori, Tetsuya; Takamatsu, Satoshi; Kitanaka, Susumu; Kikuchi, Jun; Saito, Kazuki

    2014-01-01

    Metabolomics plays an important role in phytochemical genomics and crop breeding; however, metabolite annotation is a significant bottleneck in metabolomic studies. In particular, in liquid chromatography-mass spectrometry (MS)-based metabolomics, which has become a routine technology for the profiling of plant-specialized metabolites, a substantial number of metabolites detected as MS peaks are still not assigned properly to a single metabolite. Oryza sativa (rice) is one of the most important staple crops in the world. In the present study, we isolated and elucidated the structures of specialized metabolites from rice by using MS/MS and NMR. Thirty-six compounds, including five new flavonoids and eight rare flavonolignan isomers, were isolated from the rice leaves. The MS/MS spectral data of the isolated compounds, with a detailed interpretation of MS fragmentation data, will facilitate metabolite annotation of the related phytochemicals by enriching the public mass spectral data depositories, including the plant-specific MS/MS-based database, ReSpect.

  11. NMR Spectroscopy Identifies Metabolites Translocated from Powdery Mildew Resistant Rootstocks to Susceptible Watermelon Scions.

    PubMed

    Mahmud, Iqbal; Kousik, Chandrasekar; Hassell, Richard; Chowdhury, Kamal; Boroujerdi, Arezue F

    2015-09-16

    Powdery mildew (PM) disease causes significant loss in watermelon. Due to the unavailability of a commercial watermelon variety that is resistant to PM, grafting susceptible cultivars on wild resistant rootstocks is being explored as a short-term management strategy to combat this disease. Nuclear magnetic resonance-based metabolic profiles of susceptible and resistant rootstocks of watermelon and their corresponding susceptible scions (Mickey Lee) were compared to screen for potential metabolites related to PM resistance using multivariate principal component analysis. Significant score plot differences between the susceptible and resistant groups were revealed through Mahalanobis distance analysis. Significantly different spectral buckets and their corresponding metabolites (including choline, fumarate, 5-hydroxyindole-3-acetate, and melatonin) have been identified quantitatively using multivariate loading plots and verified by volcano plot analyses. The data suggest that these metabolites were translocated from the powdery mildew resistant rootstocks to their corresponding powdery mildew susceptible scions and can be related to PM disease resistance.

  12. Metabolic Profiling of Intact Arabidopsis thaliana Leaves during Circadian Cycle Using 1H High Resolution Magic Angle Spinning NMR

    PubMed Central

    van Schadewijk, R.; de Groot, H. J. M.; Alia, A.

    2016-01-01

    Arabidopsis thaliana is the most widely used model organism for research in plant biology. While significant advances in understanding plant growth and development have been made by focusing on the molecular genetics of Arabidopsis, extracting and understanding the functional framework of metabolism is challenging, both from a technical perspective due to losses and modification during extraction of metabolites from the leaves, and from the biological perspective, due to random variation obscuring how well the function is performed. The purpose of this work is to establish the in vivo metabolic profile directly from the Arabidopsis thaliana leaves without metabolite extraction, to reduce the complexity of the results by multivariate analysis, and to unravel the mitigation of cellular complexity by predominant functional periodicity. To achieve this, we use the circadian cycle that strongly influences metabolic and physiological processes and exerts control over the photosynthetic machinery. High resolution-magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to obtain the metabolic profile directly from intact Arabidopsis leaves. Combining one- and two-dimensional 1H HR-MAS NMR allowed the identification of several metabolites including sugars and amino acids in intact leaves. Multivariate analysis on HR-MAS NMR spectra of leaves throughout the circadian cycle revealed modules of primary metabolites with significant and consistent variations of their molecular components at different time points of the circadian cycle. Since robust photosynthetic performance in plants relies on the functional periodicity of the circadian rhythm, our results show that HR-MAS NMR promises to be an important non-invasive method that can be used for metabolomics of the Arabidopsis thaliana mutants with altered physiology and photosynthetic efficiency. PMID:27662620

  13. Tissue metabolic profiling of human gastric cancer assessed by (1)H NMR.

    PubMed

    Wang, Huijuan; Zhang, Hailong; Deng, Pengchi; Liu, Chunqi; Li, Dandan; Jie, Hui; Zhang, Hu; Zhou, Zongguang; Zhao, Ying-Lan

    2016-06-29

    Gastric cancer is the fourth most common cancer and the second most deadly cancer worldwide. Study on molecular mechanisms of carcinogenesis will play a significant role in diagnosing and treating gastric cancer. Metabolic profiling may offer the opportunity to understand the molecular mechanism of carcinogenesis and help to identify the potential biomarkers for the early diagnosis of gastric cancer. In this study, we reported the metabolic profiling of tissue samples on a large cohort of human gastric cancer subjects (n = 125) and normal controls (n = 54) based on (1)H nuclear magnetic resonance ((1)H NMR) together with multivariate statistical analyses (PCA, PLS-DA, OPLS-DA and ROC curve). The OPLS-DA model showed adequate discrimination between cancer tissues and normal controls, and meanwhile, the model excellently discriminated the stage-related of tissue samples (stage I, 30; stage II, 46; stage III, 37; stage IV, 12) and normal controls. A total of 48 endogenous distinguishing metabolites (VIP > 1 and p < 0.05) were identified, 13 of which were changed with the progression of gastric cancer. These modified metabolites revealed disturbance of glycolysis, glutaminolysis, TCA, amino acids and choline metabolism, which were correlated with the occurrence and development of human gastric cancer. The receiver operating characteristic diagnostic AUC of OPLS-DA model between cancer tissues and normal controls was 0.945. And the ROC curves among different stages cancer subjects and normal controls were gradually improved, the corresponding AUC values were 0.952, 0.994, 0.998 and 0.999, demonstrating the robust diagnostic power of this metabolic profiling approach. As far as we know, the present study firstly identified the differential metabolites in various stages of gastric cancer tissues. And the AUC values were relatively high. So these results suggest that the metabolic profiling of gastric cancer tissues has great potential in detecting this

  14. Intermolecular interaction of voriconazole analogues with model membrane by DSC and NMR, and their antifungal activity using NMR based metabolic profiling.

    PubMed

    Kalamkar, Vaibhav; Joshi, Mamata; Borkar, Varsha; Srivastava, Sudha; Kanyalkar, Meena

    2013-11-01

    The development of novel antifungal agents with high susceptibility and increased potency can be achieved by increasing their overall lipophilicity. To enhance the lipophilicity of voriconazole, a second generation azole antifungal agent, we have synthesized its carboxylic acid ester analogues, namely p-methoxybenzoate (Vpmb), toluate (Vtol), benzoate (Vbz) and p-nitrobenzoate (Vpnb). The intermolecular interactions of these analogues with model membrane have been investigated using nuclear magnetic resonance (NMR) and differential scanning calorimetric (DSC) techniques. The results indicate varying degree of changes in the membrane bilayer's structural architecture and physico-chemical characteristics which possibly can be correlated with the antifungal effects via fungal membrane. Rapid metabolite profiling of chemical entities using cell preparations is one of the most important steps in drug discovery. We have evaluated the effect of synthesized analogues on Candida albicans. The method involves real time (1)H NMR measurement of intact cells monitoring NMR signals from fungal metabolites which gives Metabolic End Point (MEP). This is then compared with Minimum Inhibitory Concentration (MIC) determined using conventional methods. Results indicate that one of the synthesized analogues, Vpmb shows reasonably good activity.

  15. Acetate and bicarbonate assimilation and metabolite formation in Chlamydomonas reinhardtii: a 13C-NMR study.

    PubMed

    Singh, Himanshu; Shukla, Manish R; Chary, Kandala V R; Rao, Basuthkar J

    2014-01-01

    Cellular metabolite analyses by (13)C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly (13)C-labelled acetate ((13)CH(3)-COOH or CH(3)-(13)COOH) supported that both the (13)C nuclei give rise to bicarbonate and CO2(aq). The observed metabolite(s) upon further incubation led to the production of starch and triacylglycerol (TAG) in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2(aq) in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2(aq), which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2(aq) pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.

  16. Acetate and Bicarbonate Assimilation and Metabolite Formation in Chlamydomonas reinhardtii: A 13C-NMR Study

    PubMed Central

    Singh, Himanshu; Shukla, Manish R.; Chary, Kandala V. R.; Rao, Basuthkar J.

    2014-01-01

    Cellular metabolite analyses by 13C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly 13C-labelled acetate (13CH3-COOH or CH3-13COOH) supported that both the 13C nuclei give rise to bicarbonate and CO2aq. The observed metabolite(s) upon further incubation led to the production of starch and triacylglycerol (TAG) in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2aq in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2aq, which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2aq pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii. PMID:25207648

  17. 1H NMR metabolite fingerprinting as a new tool for body fluid identification in forensic science.

    PubMed

    Scano, Paola; Locci, Emanuela; Noto, Antonio; Navarra, Gabriele; Murgia, Federica; Lussu, Milena; Barberini, Luigi; Atzori, Luigi; De Giorgio, Fabio; Rosa, Maria Francesca; d'Aloja, Ernesto

    2013-08-01

    In this feasibility study, we propose, for the first time, (1)H NMR spectroscopy coupled with mathematical strategies as a valid tool for body fluid (BF) trace identification in forensic science. In order to assess the ability of this approach to identify traces composed either by a single or by two different BFs, samples of blood, urine, saliva, and semen were collected from different donors, and binary mixtures were prepared. (1)H NMR analyses were carried out for all samples. Spectral data of the whole set were firstly submitted to unsupervised principal component analysis (PCA); it showed that samples of the same BF cluster well on the basis of their characterizing molecular components and that mixtures exhibit intermediate characteristics among BF typologies. Furthermore, samples were divided into a training set and a test set. An average NMR spectral profile for each typology of BF was obtained from the training set and validated as representative of each BF class. Finally, a fitting procedure, based on a system of linear equations with the four obtained average spectral profiles, was applied to the test set and the mixture samples; it showed that BFs can be unambiguously identified, even as components of a mixture. The successful use of this mathematical procedure has the advantage, in forensics, of overcoming bias due to the analyst's personal judgment. We therefore propose this combined approach as a valid, fast, and non-destructive tool for addressing the challenges in the identification of composite traces in forensics. Copyright © 2013 John Wiley & Sons, Ltd.

  18. 1H-NMR analysis provides a metabolomic profile of patients with multiple sclerosis

    PubMed Central

    Cocco, Eleonora; Murgia, Federica; Lorefice, Lorena; Barberini, Luigi; Poddighe, Simone; Frau, Jessica; Fenu, Giuseppe; Coghe, Giancarlo; Murru, Maria Rita; Murru, Raffaele; Del Carratore, Francesco; Atzori, Luigi

    2015-01-01

    Objective: To investigate the metabolomic profiles of patients with multiple sclerosis (MS) and to define the metabolic pathways potentially related to MS pathogenesis. Methods: Plasma samples from 73 patients with MS (therapy-free for at least 90 days) and 88 healthy controls (HC) were analyzed by 1H-NMR spectroscopy. Data analysis was conducted with principal components analysis followed by a supervised analysis (orthogonal partial least squares discriminant analysis [OPLS-DA]). The metabolites were identified and quantified using Chenomx software, and the receiver operating characteristic (ROC) curves were calculated. Results: The model obtained with the OPLS-DA identified predictive metabolic differences between the patients with MS and HC (R2X = 0.615, R2Y = 0.619, Q2 = 0.476; p < 0.001). The differential metabolites included glucose, 5-OH-tryptophan, and tryptophan, which were lower in the MS group, and 3-OH-butyrate, acetoacetate, acetone, alanine, and choline, which were higher in the MS group. The suitability of the model was evaluated using an external set of samples. The values returned by the model were used to build the corresponding ROC curve (area under the curve of 0.98). Conclusion: NMR metabolomic analysis was able to discriminate different metabolic profiles in patients with MS compared with HC. With the exception of choline, the main metabolic changes could be connected to 2 different metabolic pathways: tryptophan metabolism and energy metabolism. Metabolomics appears to represent a promising noninvasive approach for the study of MS. PMID:26740964

  19. Endometriosis is associated with aberrant metabolite profiles in plasma.

    PubMed

    Letsiou, Sophia; Peterse, Dirkje P; Fassbender, Amelie; Hendriks, Margriet M; van den Broek, Niels J; Berger, Rudolf; O, Dorien F; Vanhie, Arne; Vodolazkaia, Alexandra; Van Langendonckt, Anne; Donnez, Jacques; Harms, Amy C; Vreeken, Rob J; Groothuis, Patrick G; Dolmans, Marie-Madeleine; Brenkman, Arjan B; D'Hooghe, Thomas M

    2017-03-01

    To identify metabolites that are associated with and predict the presence of endometriosis. Metabolomics study using state-of-the-art mass spectrometry approaches. University hospital and universities. Twenty-five women with laparoscopically confirmed endometriosis (cases) and 19 women with laparoscopically documented absence of endometriosis (controls). None of the women included in this study had received oral contraception or GnRH agonists for a minimum of 1 month before blood collection. Plasma collection. Metabolite profiles were generated and interrogated using multiple mass spectrometry methods, that is, high performance liquid chromatography coupled with negative mode electrospray ionization tandem mass spectrometry, UPLC-MS/MS, and ultra performance liquid chromatography-electroSpray ionization-quadrupole time-of-flight (UPLC-ESI-Q-TOF). Metabolite groups investigated included phospholipids, glycerophospholipids, ether-phospholipids, cholesterol-esters, triacylglycerol, sphingolipids, free fatty acids, steroids, eicosanoids, and acylcarnitines. A panel of acylcarnitines predicted the presence of endometriosis with 88.9% specificity and 81.5% sensitivity in human plasma, with a positive predictive value of 75%. However, due to data limitations the outcome of the receiver operating characteristic curve analysis was not significant. A diagnostic model based on acylcarnitines has the potential to predict the presence and stage of endometriosis. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  20. Intracellular metabolite profiling of Saccharomyces cerevisiae evolved under furfural.

    PubMed

    Jung, Young Hoon; Kim, Sooah; Yang, Jungwoo; Seo, Jin-Ho; Kim, Kyoung Heon

    2017-03-01

    Furfural, one of the most common inhibitors in pre-treatment hydrolysates, reduces the cell growth and ethanol production of yeast. Evolutionary engineering has been used as a selection scheme to obtain yeast strains that exhibit furfural tolerance. However, the response of Saccharomyces cerevisiae to furfural at the metabolite level during evolution remains unknown. In this study, evolutionary engineering and metabolomic analyses were applied to determine the effects of furfural on yeasts and their metabolic response to continuous exposure to furfural. After 50 serial transfers of cultures in the presence of furfural, the evolved strains acquired the ability to stably manage its physiological status under the furfural stress. A total of 98 metabolites were identified, and their abundance profiles implied that yeast metabolism was globally regulated. Under the furfural stress, stress-protective molecules and cofactor-related mechanisms were mainly induced in the parental strain. However, during evolution under the furfural stress, S. cerevisiae underwent global metabolic allocations to quickly overcome the stress, particularly by maintaining higher levels of metabolites related to energy generation, cofactor regeneration and recovery from cellular damage. Mapping the mechanisms of furfural tolerance conferred by evolutionary engineering in the present study will be led to rational design of metabolically engineered yeasts. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  1. Perilla frutescens (L.) Britton: honeybee forage and preliminary results on the metabolic profiling by NMR spectroscopy.

    PubMed

    Consonni, Roberto; Cagliani, Laura Ruth; Docimo, Teresa; Romane, Abderrahmane; Ferrazzi, Paola

    2013-01-01

    Nuclear magnetic resonance (NMR) spectroscopy has emerged as a technology for metabolite characterisation of both foods and plants. NMR technique allows to analyse metabolite content in a single experiment, in a non-destructive way and with a very simple sample preparation. This study characterises the metabolites of Perilla frutescens var. crispa leaf and flower for the first time by NMR. Our results showed higher metabolite content in leaves compared to flowers, highlighting the presence of amino acids, organic acids, saccharides and large amounts of aromatic compounds, mainly in the form of rosmarinic acid. Moreover, we cultivated Perilla, an important medicinal plant native to Asia, in a low mountain environment in Italy, to continue its evaluation as a honeybee attractive species. Interestingly, even in this type of environment, Perilla has been confirmed to be a good bee plant for both nectar and pollen.

  2. Quantitative profiling of polar metabolites in herbal medicine injections for multivariate statistical evaluation based on independence principal component analysis.

    PubMed

    Jiang, Miaomiao; Jiao, Yujiao; Wang, Yuefei; Xu, Lei; Wang, Meng; Zhao, Buchang; Jia, Lifu; Pan, Hao; Zhu, Yan; Gao, Xiumei

    2014-01-01

    Botanical primary metabolites extensively exist in herbal medicine injections (HMIs), but often were ignored to control. With the limitation of bias towards hydrophilic substances, the primary metabolites with strong polarity, such as saccharides, amino acids and organic acids, are usually difficult to detect by the routinely applied reversed-phase chromatographic fingerprint technology. In this study, a proton nuclear magnetic resonance (1H NMR) profiling method was developed for efficient identification and quantification of small polar molecules, mostly primary metabolites in HMIs. A commonly used medicine, Danhong injection (DHI), was employed as a model. With the developed method, 23 primary metabolites together with 7 polyphenolic acids were simultaneously identified, of which 13 metabolites with fully separated proton signals were quantified and employed for further multivariate quality control assay. The quantitative 1H NMR method was validated with good linearity, precision, repeatability, stability and accuracy. Based on independence principal component analysis (IPCA), the contents of 13 metabolites were characterized and dimensionally reduced into the first two independence principal components (IPCs). IPC1 and IPC2 were then used to calculate the upper control limits (with 99% confidence ellipsoids) of χ2 and Hotelling T2 control charts. Through the constructed upper control limits, the proposed method was successfully applied to 36 batches of DHI to examine the out-of control sample with the perturbed levels of succinate, malonate, glucose, fructose, salvianic acid and protocatechuic aldehyde. The integrated strategy has provided a reliable approach to identify and quantify multiple polar metabolites of DHI in one fingerprinting spectrum, and it has also assisted in the establishment of IPCA models for the multivariate statistical evaluation of HMIs.

  3. Profiling of plasma metabolites in postmenopausal women with metabolic syndrome

    PubMed Central

    Iida, Miho; Harada, Sei; Kurihara, Ayako; Fukai, Kota; Kuwabara, Kazuyo; Sugiyama, Daisuke; Takeuchi, Ayano; Okamura, Tomonori; Akiyama, Miki; Nishiwaki, Yuji; Suzuki, Asako; Hirayama, Akiyoshi; Sugimoto, Masahiro; Soga, Tomoyoshi; Tomita, Masaru; Banno, Kouji; Aoki, Daisuke; Takebayashi, Toru

    2016-01-01

    Abstract Objective: The aim of the study was to investigate the associations of amino acids and other polar metabolites with metabolic syndrome (MetS) in postmenopausal women in a lean Asian population. Methods: The participants were 1,422 female residents enrolled in a cohort study from April to August 2012. MetS was defined according to the National Cholesterol Education Program Adult Treatment Panel III modified for Japanese women. Associations were examined between MetS and 78 metabolites assayed in fasting plasma samples using capillary electrophoresis-mass spectrometry. Replication analysis was performed to confirm the robustness of the results in a separate population created by random allocation. Results: Analysis was performed for 877 naturally postmenopausal women, including 594 in the original population and 283 in the replication population. The average age, body mass index, and levels of high- and low-density lipoprotein cholesterol of the entire population were 64.6 years, 23.0 kg/m2, 72.1 mg/dL, and 126.1 mg/dL, respectively. There was no significant difference in low-density lipoprotein cholesterol levels between women with and without MetS. Thirteen metabolites were significantly related to MetS: multiple plasma amino acids were elevated in women with MetS, including branched-chain amino acids, alanine, glutamate, and proline; and alpha-aminoadipate, which is generated by lysine degradation, was also significantly increased. Conclusions: Our large-scale metabolomic profiling indicates that Japanese postmenopausal women with MetS have abnormal polar metabolites, suggesting altered catabolic pathways. These results may help to understand metabolic disturbance, including in persons with normal body mass index and relatively high levels of high-density lipoprotein cholesterol, and may have clinical utility based on further studies. PMID:27070805

  4. Metabolite Profiling Identifies Pathways Associated with Metabolic Risk in Humans

    PubMed Central

    Cheng, Susan; Rhee, Eugene P.; Larson, Martin G.; Lewis, Gregory D.; McCabe, Elizabeth L.; Shen, Dongxiao; Palma, Melinda J.; Roberts, Lee D.; Dejam, Andre; Souza, Amanda L.; Deik, Amy A.; Magnusson, Martin; Fox, Caroline S.; O'Donnell, Christopher J.; Vasan, Ramachandran S.; Melander, Olle; Clish, Clary B.; Gerszten, Robert E.; Wang, Thomas J.

    2012-01-01

    Background Although metabolic risk factors are known to cluster in individuals who are prone to developing diabetes and cardiovascular disease, the underlying biological mechanisms remain poorly understood. Methods and Results To identify pathways associated with cardiometabolic risk, we used liquid chromatography/mass spectrometry to determine the plasma concentrations of 45 distinct metabolites and examine their relation to cardiometabolic risk in the Framingham Heart Study (FHS; N=1015) and the Malmö Diet and Cancer Study (MDC; N=746). We then interrogated significant findings in experimental models of cardiovascular and metabolic disease. We observed that metabolic risk factors (obesity, insulin resistance, high blood pressure, dyslipidemia) were associated with multiple metabolites including branched-chain amino acids, other hydrophobic amino acids, tryptophan breakdown products, and nucleotide metabolites. We observed strong associations of insulin resistance traits with glutamine (standardized regression coefficients −0.04 to −0.22, per 1-SD change in log-glutamine, P<0.001), glutamate (0.05 to 0.14, P<0.001), and glutamine-glutamate ratio (−0.05 to −0.20, P<0.001) in the discovery sample (FHS); similar associations were observed in the replication sample (MDC). High glutamine-glutamate ratio was associated with lower risk of incident diabetes in FHS (OR 0.79; adjusted P=0.03), but not in MDC. In experimental models, administration of glutamine in mice led to both increased glucose tolerance (P=0.01) and to lower blood pressure (P<0.05). Conclusions Biochemical profiling identified circulating metabolites not previously associated with metabolic traits. Experimentally interrogating one of these pathways demonstrated that excess glutamine relative to glutamate, resulting from exogenous administration, is associated with reduced metabolic risk in mice. PMID:22496159

  5. Metabolite profiling identifies pathways associated with metabolic risk in humans.

    PubMed

    Cheng, Susan; Rhee, Eugene P; Larson, Martin G; Lewis, Gregory D; McCabe, Elizabeth L; Shen, Dongxiao; Palma, Melinda J; Roberts, Lee D; Dejam, Andre; Souza, Amanda L; Deik, Amy A; Magnusson, Martin; Fox, Caroline S; O'Donnell, Christopher J; Vasan, Ramachandran S; Melander, Olle; Clish, Clary B; Gerszten, Robert E; Wang, Thomas J

    2012-05-08

    Although metabolic risk factors are known to cluster in individuals who are prone to developing diabetes mellitus and cardiovascular disease, the underlying biological mechanisms remain poorly understood. To identify pathways associated with cardiometabolic risk, we used liquid chromatography/mass spectrometry to determine the plasma concentrations of 45 distinct metabolites and to examine their relation to cardiometabolic risk in the Framingham Heart Study (FHS; n=1015) and the Malmö Diet and Cancer Study (MDC; n=746). We then interrogated significant findings in experimental models of cardiovascular and metabolic disease. We observed that metabolic risk factors (obesity, insulin resistance, high blood pressure, and dyslipidemia) were associated with multiple metabolites, including branched-chain amino acids, other hydrophobic amino acids, tryptophan breakdown products, and nucleotide metabolites. We observed strong associations of insulin resistance traits with glutamine (standardized regression coefficients, -0.04 to -0.22 per 1-SD change in log-glutamine; P<0.001), glutamate (0.05 to 0.14; P<0.001), and the glutamine-to-glutamate ratio (-0.05 to -0.20; P<0.001) in the discovery sample (FHS); similar associations were observed in the replication sample (MDC). High glutamine-to-glutamate ratio was associated with lower risk of incident diabetes mellitus in FHS (odds ratio, 0.79; adjusted P=0.03) but not in MDC. In experimental models, administration of glutamine in mice led to both increased glucose tolerance (P=0.01) and decreased blood pressure (P<0.05). Biochemical profiling identified circulating metabolites not previously associated with metabolic traits. Experimentally interrogating one of these pathways demonstrated that excess glutamine relative to glutamate, resulting from exogenous administration, is associated with reduced metabolic risk in mice.

  6. Metabolic Profiling and Population Screening of Analgesic Usage in NMR-Spectroscopy-based Large-scale Epidemiologic Studies

    PubMed Central

    Loo, Ruey Leng; Coen, Muireann; Ebbels, Timothy; Cloarec, Olivier; Maibaum, Elaine; Bictash, Magda; Yap, Ivan; Elliott, Paul; Stamler, Jeremiah; Nicholson, Jeremy K.; Holmes, Elaine

    2009-01-01

    The application of a 1H NMR spectroscopy based screening method for determining the use of two widely available analgesics (acetaminophen and ibuprofen) in epidemiologic studies has been investigated. We used samples and data from the cross-sectional INTERMAP Study involving participants from Japan (n=1,145), China (n=839), UK (n=501) and USA (n=2,195). An orthogonal projection to latent structures discriminant analysis (OPLS-DA) algorithm with an incorporated Monte Carlo re-sampling function was applied to the NMR dataset to determine which spectra contained analgesic metabolites. OPLS-DA pre-processing parameters (normalization, bin width, scaling and input parameters) were assessed systematically to identify an optimal acetaminophen prediction model. Subsets of INTERMAP spectra were examined to verify and validate the presence/absence of acetaminophen/ibuprofen based on known chemical shift and coupling patterns. The optimized and validated acetaminophen model correctly predicted 98.2% and the ibuprofen model correctly predicted 99.0 % of the urine specimens containing these drug metabolites. The acetaminophen and ibuprofen models were subsequently used to predict the presence/absence of these drug metabolites for the remaining INTERMAP specimens. The acetaminophen model identified 415 of 8,436 spectra containing acetaminophen metabolites while the ibuprofen model identified 245 of 8,604 spectra containing ibuprofen metabolites from the global dataset. The NMR-based metabolic screening strategy provides a new objective approach for evaluation of self-reported medication data and is extendable to other aspects of population xenometabolome profiling. PMID:19489597

  7. Spatial mapping and profiling of metabolite distributions during germination

    DOE PAGES

    Feenstra, Adam D.; Alexander, Liza E.; Song, Zhihong; ...

    2017-06-20

    Germination is a highly complex process by which seeds begin to develop and establish themselves as viable organisms. In this paper, we utilize a combination of GC-MS, LC-fluorescence, and mass spectrometry imaging (MSI) approaches to profile and visualize the metabolic distributions of germinating seeds from two different inbreds of maize seeds, B73 and Mo17. GC and LC analyses demonstrate that the two inbreds are highly differentiated in their metabolite profiles throughout the course of germination, especially with regard to amino acids, sugar alcohols, and small organic acids. Crude dissection of the seed followed by GC-MS analysis of polar metabolites alsomore » revealed that many compounds were highly sequestered among the various seed tissue types. To further localize compounds, matrix-assisted laser desorption/ionization MSI is utilized to visualize compounds in fine detail in their native environments over the course of germination. Most notably, the fatty acyl chain-dependent differential localization of phospholipids and TAGs were observed within the embryo and radicle, showing correlation with the heterogeneous distribution of fatty acids. Furthermore, other interesting observations include unusual localization of ceramides on the endosperm/scutellum boundary, and subcellular localization of ferulate in the aleurone.« less

  8. Cherry tomatoes metabolic profile determined by ¹H-High Resolution-NMR spectroscopy as influenced by growing season.

    PubMed

    Masetti, Olimpia; Ciampa, Alessandra; Nisini, Luigi; Valentini, Massimiliano; Sequi, Paolo; Dell'Abate, Maria Teresa

    2014-11-01

    The content of the most valuable metabolites present in the lipophilic fraction of Protected Geographical Indication cherry tomatoes produced in Pachino (Italy) was observed for 2 cultivated varieties, i.e. cv. Naomi and cv. Shiren, over a period of 3 years in order to observe variations due to relevant climatic parameters, e.g. solar radiation and average temperature, characterising different seasons. (1)H-NMR spectroscopy was applied and spectral data were processed by means of Principal Component Analysis (PCA). We found that the metabolic profile was different for the two considered cultivated varieties and they were differently affected by climatic conditions. Major metabolites influenced by cropping period were α-tocopherol and the unsaturated lipid fraction in Naomi cherry tomatoes, and chlorophylls and phospholipids in Shiren variety, respectively. These results furnished useful information on seasonal dynamics of such important nutritional metabolites contained in tomatoes, confirming also NMR spectroscopy as powerful tool to define a complete metabolic profiling. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Metabolomic profiling of the phytomedicinal constituents of Carica papaya L. leaves and seeds by 1H NMR spectroscopy and multivariate statistical analysis.

    PubMed

    Gogna, Navdeep; Hamid, Neda; Dorai, Kavita

    2015-11-10

    Extracts from the Carica papaya L. plant are widely reported to contain metabolites with antibacterial, antioxidant and anticancer activity. This study aims to analyze the metabolic profiles of papaya leaves and seeds in order to gain insights into their phytomedicinal constituents. We performed metabolite fingerprinting using 1D and 2D 1H NMR experiments and used multivariate statistical analysis to identify those plant parts that contain the most concentrations of metabolites of phytomedicinal value. Secondary metabolites such as phenyl propanoids, including flavonoids, were found in greater concentrations in the leaves as compared to the seeds. UPLC-ESI-MS verified the presence of significant metabolites in the papaya extracts suggested by the NMR analysis. Interestingly, the concentration of eleven secondary metabolites namely caffeic, cinnamic, chlorogenic, quinic, coumaric, vanillic, and protocatechuic acids, naringenin, hesperidin, rutin, and kaempferol, were higher in young as compared to old papaya leaves. The results of the NMR analysis were corroborated by estimating the total phenolic and flavonoid content of the extracts. Estimation of antioxidant activity in leaves and seed extracts by DPPH and ABTS in-vitro assays and antioxidant capacity in C2C12 cell line also showed that papaya extracts exhibit high antioxidant activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. 1H NMR Metabolomics Reveals Association of High Expression of Inositol 1, 4, 5 Trisphosphate Receptor and Metabolites in Breast Cancer Patients

    PubMed Central

    Chagtoo, Megha; Agarwal, Gaurav; George, Nelson; Sinha, Neeraj; Godbole, Madan M.

    2017-01-01

    1H NMR is used to detect alterations in metabolites and their linkage to metabolic processes in a number of pathological conditions including breast cancer. Inositol 1, 4, 5 trisphosphate (IP3R) receptor is an intracellular calcium channel known to regulate metabolism and cellular bioenergetics. Its expression is up regulated in a number of cancers. However, its linkage to metabolism in disease conditions has not been evaluated. This study was designed to determine the association if any, of these metabolites with altered expression of IP3R in breast cancer. We used 1H NMR to identify metabolites in the serum of breast cancer patients (n = 27) and performed Real-time Polymerase Chain Reaction analysis for quantifying the expression of IP3R type 3 and type 2 in tissues from breast cancer patients (n = 40). Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) clearly distinguished patients with high/low IP3R expression from healthy subjects. The present study revealed high expression of IP3R type 2 and type 3 in human breast tumor tissue compared to adjacent non-tumorous tissue. Moreover, patients with ≥ 2-fold increase in IP3R (high IP3R group) had significantly higher concentration of metabolic intermediates compared to those with < 2-fold increase in IP3R (low IP3R group). We observed an increase in lipoprotein content and the levels of metabolites like lactate, lysine and alanine and a decrease in the levels of pyruvate and glucose in serum of high IP3R group patients when compared to those in healthy subjects. Receiver operating characteristic (ROC) curve analysis was performed to show the clinical utility of metabolites. In addition to the human studies, functional relevance of IP3Rs in causing metabolic disruption was observed in MCF-7 and MDA MB-231 cells. Results from our studies bring forth the importance of metabolic (or metabolomics) profiling of serum by 1H NMR in conjunction with tissue expression studies for

  11. Biochemical effects of venlafaxine on astrocytes as revealed by (1)H NMR-based metabolic profiling.

    PubMed

    Sun, Lu; Fang, Liang; Lian, Bin; Xia, Jin-Jun; Zhou, Chan-Juan; Wang, Ling; Mao, Qiang; Wang, Xin-Fa; Gong, Xue; Liang, Zi-Hong; Bai, Shun-Jie; Liao, Li; Wu, Yu; Xie, Peng

    2017-01-31

    As a serotonin-norepinephrine reuptake inhibitor [SNRI], venlafaxine is one of the most commonly prescribed clinical antidepressants, with a broad range of antidepressant effects. Accumulating evidence shows that venlafaxine may target astrocytes to exert its antidepressant activity, although the underlying pharmacological mechanisms remained largely unknown. Here, we used a (1)H nuclear magnetic resonance (NMR)-based metabonomics method coupled with multivariate statistical analysis to characterize the metabolic profiling of astrocytes treated with venlafaxine to explore the potential mechanism of its antidepressant effect. In total, 31 differential metabolites involved in energy, amino acid and lipid metabolism were identified. Ingenuity pathway analysis was used to identify the predicted pathways and biological functions with venlafaxine and fluoxetine. The most significantly altered network was "amino acid metabolism, cellular growth and proliferation", with a score above 20. Certain metabolites (lysine, tyrosine, glutamate, methionine, ethanolamine, fructose-6-phosphate, and phosphorylethanolamine) are involved in and play a central role in this network. Collectively, the biological effects of venlafaxine on astrocytes provide us with the further understanding of the mechanisms by which venlafaxine treats major depressive disorder.

  12. Investigation of chemomarkers of astragali radix of different ages and geographical origin by NMR profiling.

    PubMed

    Zheng, Lu; Wang, Mei; Ibarra-Estrada, Emmanuel; Wu, Changsheng; Wilson, Erica Georgina; Verpoorte, Robert; Klinkhamer, Petrus Gerardus Leonardus; Choi, Young Hae

    2015-02-17

    Astragalus roots from Astragalus membranaceus Bunge or Astragalus membranaceus var. mongholicus (Bunge) Hsiao are among the most popular traditional medicinal plants due to their diverse therapeutic uses based on their tonic, antinephritic, immunostimulant, hepatoprotectant, diuretic, antidiabetic, analgesic, expectorant and sedative properties. Currently, the herb is produced or cultivated in various sites, including 10 different locations in China with very diverse environmental conditions. These differences affect their metabolic pools and consequently their medicinal properties. The comparative metabolic profiling of plants of different geographical origins or ages could contribute to detect biomarkers for their quality control and thus guarantee the efficacy of the herbal medicines produced with this drug. In this paper nuclear magnetic resonance spectroscopy (NMR)-based metabolomics was applied for to plants of different origins and age for this purpose. The results of this study show that in the set of samples evaluated, age is more discriminating than geographical location. The quantity of individual flavonoids and some primary metabolites contributed most to this age differentiation. On the other hand, based on the analysis of orthogonal partial least square (OPLS) modeling, the marker metabolites for the geographical origin were saponins and isoflavonoids.

  13. Pharmacokinetic profiles of the active metamizole metabolites in healthy horses.

    PubMed

    Giorgi, M; Aupanun, S; Lee, H-K; Poapolathep, A; Rychshanova, R; Vullo, C; Faillace, V; Laus, F

    2017-04-01

    Metamizole (MT) is an analgesic and antipyretic drug labelled for use in humans, horses, cattle, swine and dogs. MT is rapidly hydrolysed to the active primary metabolite 4-methylaminoantipyrine (MAA). MAA is formed in much larger amounts compared with other minor metabolites. Among the other secondary metabolites, 4-aminoantipyrine (AA) is also relatively active. The aim of this research was to evaluate the pharmacokinetic profiles of MAA and AA after dose of 25 mg/kg MT by intravenous (i.v.) and intramuscular (i.m.) routes in healthy horses. Six horses were randomly allocated to two equally sized treatment groups according to a 2 × 2 crossover study design. Blood was collected at predetermined times within 24 h, and plasma was analysed by a validated HPLC-UV method. No behavioural changes or alterations in health parameters were observed in the i.v. or i.m. groups of animals during or after (up to 7 days) drug administration. Plasma concentrations of MAA after i.v. and i.m. administrations of MT were detectable from 5 min to 10 h in all the horses. Plasma concentrations of AA were detectable in the same range of time, but in smaller amounts. Maximum concentration (Cmax ), time to maximum concentration (Tmax ) and AUMC0-last of MAA were statistically different between the i.v. and i.m. groups. The AUCIM /AUCIV ratio of MAA was 1.06. In contrast, AUC0-last of AA was statistically different between the groups (P < 0.05) with an AUCIM /AUCIV ratio of 0.54. This study suggested that the differences in the MAA and AA plasma concentrations found after i.m. and i.v. administrations of MT might have minor consequences on the pharmacodynamics of the drug.

  14. Differences in metabolite profile between blood plasma and serum.

    PubMed

    Liu, Linsheng; Aa, Jiye; Wang, Guangji; Yan, Bei; Zhang, Ying; Wang, Xinwen; Zhao, Chunyan; Cao, Bei; Shi, Jian; Li, Mengjie; Zheng, Tian; Zheng, Yuanting; Hao, Gang; Zhou, Fang; Sun, Jianguo; Wu, Zimei

    2010-11-15

    In metabolomic research, blood plasma and serum have been considered to possess similar compositions and properties. Their perceived equivalence has resulted in researchers choosing arbitrarily between serum and plasma for analysis. Here, routine serum and plasma were prepared and their low-molecular-weight compounds were determined using gas chromatography/time-of-flight mass spectrometry. Principal components analysis was applied to process the acquired data, and marked differences in metabolite profiles were observed between serum and plasma. Of the 72 identified compounds, 36 (50%) discriminate serum from plasma, with 29 and 7 metabolites showing a significantly higher abundance (t test, P<0.05) in serum and plasma, respectively. Incubation of blood had distinct effects on the analyte peak areas, with the effects being more pronounced for plasma than for serum and more pronounced for a shorter incubation than for a longer incubation. These results highlight the importance in choosing serum or plasma as the analytical sample and in stipulating the incubation time. Because incubation affected the analyte peak areas less in serum than in plasma, we recommend serum as the sample of choice in metabolomic studies.

  15. Metabolite profiling of the carnivorous pitcher plants Darlingtonia and Sarracenia

    PubMed Central

    Seppänen-Laakso, Tuulikki

    2017-01-01

    Sarraceniaceae is a New World carnivorous plant family comprising three genera: Darlingtonia, Heliamphora, and Sarracenia. The plants occur in nutrient-poor environments and have developed insectivorous capability in order to supplement their nutrient uptake. Sarracenia flava contains the alkaloid coniine, otherwise only found in Conium maculatum, in which its biosynthesis has been studied, and several Aloe species. Its ecological role and biosynthetic origin in S. flava is speculative. The aim of the current research was to investigate the occurrence of coniine in Sarracenia and Darlingtonia and to identify common constituents of both genera, unique compounds for individual variants and floral scent chemicals. In this comprehensive metabolic profiling study, we looked for compound patterns that are associated with the taxonomy of Sarracenia species. In total, 57 different Sarracenia and D. californica accessions were used for metabolite content screening by gas chromatography-mass spectrometry. The resulting high-dimensional data were studied using a data mining approach. The two genera are characterized by a large number of metabolites and huge chemical diversity between different species. By applying feature selection for clustering and by integrating new biochemical data with existing phylogenetic data, we were able to demonstrate that the chemical composition of the species can be explained by their known classification. Although transcriptome analysis did not reveal a candidate gene for coniine biosynthesis, the use of a sensitive selected ion monitoring method enabled the detection of coniine in eight Sarracenia species, showing that it is more widespread in this genus than previously believed. PMID:28222171

  16. Metabolite profiling of the carnivorous pitcher plants Darlingtonia and Sarracenia.

    PubMed

    Hotti, Hannu; Gopalacharyulu, Peddinti; Seppänen-Laakso, Tuulikki; Rischer, Heiko

    2017-01-01

    Sarraceniaceae is a New World carnivorous plant family comprising three genera: Darlingtonia, Heliamphora, and Sarracenia. The plants occur in nutrient-poor environments and have developed insectivorous capability in order to supplement their nutrient uptake. Sarracenia flava contains the alkaloid coniine, otherwise only found in Conium maculatum, in which its biosynthesis has been studied, and several Aloe species. Its ecological role and biosynthetic origin in S. flava is speculative. The aim of the current research was to investigate the occurrence of coniine in Sarracenia and Darlingtonia and to identify common constituents of both genera, unique compounds for individual variants and floral scent chemicals. In this comprehensive metabolic profiling study, we looked for compound patterns that are associated with the taxonomy of Sarracenia species. In total, 57 different Sarracenia and D. californica accessions were used for metabolite content screening by gas chromatography-mass spectrometry. The resulting high-dimensional data were studied using a data mining approach. The two genera are characterized by a large number of metabolites and huge chemical diversity between different species. By applying feature selection for clustering and by integrating new biochemical data with existing phylogenetic data, we were able to demonstrate that the chemical composition of the species can be explained by their known classification. Although transcriptome analysis did not reveal a candidate gene for coniine biosynthesis, the use of a sensitive selected ion monitoring method enabled the detection of coniine in eight Sarracenia species, showing that it is more widespread in this genus than previously believed.

  17. NMR-based evaluation of the metabolic profile and response to dichloroacetate of human prostate cancer cells

    PubMed Central

    Kailavasan, Mithun; Rehman, Ishtiaq; Reynolds, Steven; Bucur, Adriana; Tozer, Gillian; Paley, Martyn

    2014-01-01

    The aim of this study was to evaluate the metabolic profile of human prostate cancer cells that have different metastatic potential and to determine their response to dichloroacetate (DCA) using NMR technology. Two isogenic human prostate cancer cell lines, differing in their metastatic potential [LNCaP (poorly metastatic) and LNCaP-LN3 (highly metastatic)], were studied. Metabolite ratios from NMR spectral integrals acquired at a field strength of 9.4 T using a 5-mm broadband probe with an NMR-compatible bioreactor were compared in the presence and absence of the pyruvate dehydrogenase kinase inhibitor DCA. Lactate dehydrogenase (LDH) isoenzymes were assessed by zymography. Following the treatment of cells with 50 mm DCA, there was a significant reduction in the lactate/choline, lactate/creatine, lactate/alanine and the combined lactate/(choline + creatine + alanine) ratios in LNCaP-LN3 cells relative to LNCaP cells. No significant changes in metabolite ratios were found in LNCaP cells following DCA treatment. As expected, LDH zymography assays showed an absence of the LDH-B subunit in LNCaP-LN3 cells, whereas both LDH-A and LDH-B subunits were present in LNCaP cells. DCA was shown to significantly modify the metabolite ratios in highly metastatic LNCaP-LN3 cells, but not in poorly metastatic LNCaP cells. This effect was probably related to the absence of the LDH-B subunit in LNCaP-LN3 cells, and could have a bearing on cancer treatment with DCA and related compounds. © 2014 The Authors. NMR in Biomedicine published by John Wiley & Sons, Ltd. PMID:24639007

  18. Unambiguous metabolite identification in high-throughput metabolomics by hybrid 1D 1 H NMR/ESI MS 1 approach: Hybrid 1D 1 H NMR/ESI MS 1 metabolomics method

    SciTech Connect

    Walker, Lawrence R.; Hoyt, David W.; Walker, S. Michael; Ward, Joy K.; Nicora, Carrie D.; Bingol, Kerem

    2016-09-16

    We present a novel approach to improve accuracy of metabolite identification by combining direct infusion ESI MS1 with 1D 1H NMR spectroscopy. The new approach first applies standard 1D 1H NMR metabolite identification protocol by matching the chemical shift, J-coupling and intensity information of experimental NMR signals against the NMR signals of standard metabolites in metabolomics library. This generates a list of candidate metabolites. The list contains false positive and ambiguous identifications. Next, we constrained the list with the chemical formulas derived from high-resolution direct infusion ESI MS1 spectrum of the same sample. Detection of the signals of a metabolite both in NMR and MS significantly improves the confidence of identification and eliminates false positive identification. 1D 1H NMR and direct infusion ESI MS1 spectra of a sample can be acquired in parallel in several minutes. This is highly beneficial for rapid and accurate screening of hundreds of samples in high-throughput metabolomics studies. In order to make this approach practical, we developed a software tool, which is integrated to Chenomx NMR Suite. The approach is demonstrated on a model mixture, tomato and Arabidopsis thaliana metabolite extracts, and human urine.

  19. /sup 13/C NMR spectroscopy in the analysis of conjugate metabolites in the bile of fish exposed to petroleum

    SciTech Connect

    Hellou, J.; Banoub, J.H.; Payne, J.F.

    1986-01-01

    The first natural abundance /sup 13/C NMR investigation of a complex mixture of conjugate metabolites obtained from the gall bladder bile of fish exposed to hydrocarbons is presented. Cunners were exposed to water accommodated No. 2 fuel oil containing about 68% saturates and 22% aromatics. Spectral analysis indicated that the hydrocarbon derivatives were present predominantly as ..beta..-glucuronides, with the oxygen at carbon-1 of glucuronic acid preferentially attached to an aliphatic carbon. The conjugate metabolites were enriched in aromatic-type carbons when compared to the fuel oil or the aromatic fraction of oil.

  20. Microbial metabolites are associated with a high adherence to a Mediterranean dietary pattern using a (1)H-NMR-based untargeted metabolomics approach.

    PubMed

    Almanza-Aguilera, Enrique; Urpi-Sarda, Mireia; Llorach, Rafael; Vázquez-Fresno, Rosa; Garcia-Aloy, Mar; Carmona, Francesc; Sanchez, Alex; Madrid-Gambin, Francisco; Estruch, Ramon; Corella, Dolores; Andres-Lacueva, Cristina

    2017-10-01

    The study of biomarkers of dietary patterns including the Mediterranean diet (MedDiet) is scarce and could improve the assessment of these patterns. Moreover, it could provide a better understanding of health benefits of dietary patterns in nutritional epidemiology. We aimed to determine a robust and accurate biomarker associated with a high adherence to a MedDiet pattern that included dietary assessment and its biological effect. In this cross-sectional study, we included 56 and 63 individuals with high (H-MDA) and low (L-MDA) MedDiet adherence categories, respectively, all from the Prevención con Dieta Mediterránea trial. A (1)H-NMR-based untargeted metabolomics approach was applied to urine samples. Multivariate statistical analyses were conducted to determine the metabolite differences between groups. A stepwise logistic regression and receiver operating characteristic curves were used to build and evaluate the prediction model for H-MDA. Thirty-four metabolites were identified as discriminant between H-MDA and L-MDA. The fingerprint associated with H-MDA included higher excretion of proline betaine and phenylacetylglutamine, among others, and decreased amounts of metabolites related to glucose metabolism. Three microbial metabolites - phenylacetylglutamine, p-cresol and 4-hydroxyphenylacetate - were included in the prediction model of H-MDA (95% specificity, 95% sensitivity and 97% area under the curve). The model composed of microbial metabolites was the biomarker that defined high adherence to a Mediterranean dietary pattern. The overall metabolite profiling identified reflects the metabolic modulation produced by H-MDA. The proposed biomarker may be a better tool for assessing and aiding nutritional epidemiology in future associations between H-MDA and the prevention or amelioration of chronic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. NMR studies of renal phosphate metabolites in vivo: Effects of hydration and dehydration

    SciTech Connect

    Wolff, S.D.; Eng, C.; Balaban, R.S. )

    1988-10-01

    The present study characterizes the {sup 31}P-nuclear magnetic resonance (NMR) spectrum of rabbit kidneys in vivo and evaluates the effect of hydration on phosphorous metabolites including the organic solute glycerophosphorylcholine (GPC). Cortical phosphorylethanolamine is the predominant component of the phosphomonoester region of the {sup 31}P spectrum. The contribution of blood to the spectrum is mainly from 2,3 diphosphoglycerate, which comprises {approximately}30% of the inorganic phosphate region. Acute infusion of 0.9% saline decreases the sodium content of the inner medulla by >50% in 15 min as shown by {sup 23}Na imaging. Despite this medullary Na dilution, no change in renal GPC content was observed for >1 h even with the addition of furosemide or furosemide and antidiuretic hormone. However, 20 h of chronic dehydration with 0.45% saline did result in a 30% decrease in renal GPC content when compared with dehydrated animals. These findings are consistent with GPC not playing a role in the short-term regulation of the medullary intracellular milieu in response to acute reductions in medullary Na content.

  2. Spatially localized sup 1 H NMR spectra of metabolites in the human brain

    SciTech Connect

    Hanstock, C.C. ); Rothman, D.L.; Jue, T.; Shulman, R.G. ); Prichard, J.W. )

    1988-03-01

    Using a surface coil, the authors have obtained {sup 1}H NMR spectra from metabolites in the human brain. Localization was achieved by combining depth pulses with image-selected in vivo spectroscopy magnetic field gradient methods. {sup 1}H spectra in which total creatine (3.03 ppm) has a signal/noise ratio of 95:1 were obtained in 4 min from 14 ml of brain. A resonance at 2.02 ppm consisting predominantly of N-acetylaspartate was measured relative to the creatine peak in gray and white matter, and the ratio was lower in the white matter. The spin-spin relaxation times of N-acetylaspartate and creatine were measured in white and gray matter and while creatine relaxation times were the same in both, the N-acetylaspartate relaxation time was longer in white matter. Lactate was detected in the normoxic brain and the average of three measurements was {approx}0.5 mM from comparison with the creatine plus phosphocreatine peak, which was assumed to be 10.5 mM.

  3. Quantification of Human Brain Metabolites from in Vivo1H NMR Magnitude Spectra Using Automated Artificial Neural Network Analysis

    NASA Astrophysics Data System (ADS)

    Hiltunen, Yrjö; Kaartinen, Jouni; Pulkkinen, Juhani; Häkkinen, Anna-Maija; Lundbom, Nina; Kauppinen, Risto A.

    2002-01-01

    Long echo time (TE=270 ms) in vivo proton NMR spectra resembling human brain metabolite patterns were simulated for lineshape fitting (LF) and quantitative artificial neural network (ANN) analyses. A set of experimental in vivo1H NMR spectra were first analyzed by the LF method to match the signal-to-noise ratios and linewidths of simulated spectra to those in the experimental data. The performance of constructed ANNs was compared for the peak area determinations of choline-containing compounds (Cho), total creatine (Cr), and N-acetyl aspartate (NAA) signals using both manually phase-corrected and magnitude spectra as inputs. The peak area data from ANN and LF analyses for simulated spectra yielded high correlation coefficients demonstrating that the peak areas quantified with ANN gave similar results as LF analysis. Thus, a fully automated ANN method based on magnitude spectra has demonstrated potential for quantification of in vivo metabolites from long echo time spectroscopic imaging.

  4. Metabolite Profiling of Justicia gendarussa Burm. f. Leaves Using UPLC-UHR-QTOF-MS.

    PubMed

    Ningsih, Indah Yulia; Purwanti, Diah Intan; Wongso, Suwidji; Prajogo, Bambang E W; Indrayanto, Gunawan

    2015-01-01

    An ultra-performance liquid chromatography ultra-high-resolution quadrupole-time-of-flight-mass spectrometry (UPLC-UHR-QTOF-MS) metabolite profiling ofxs Justicia gendarussa Burm. f. leaves was performed. PCA and HCA analyses were applied to observe the clustering patterns and inter-sample relationships. It seemed that the concentrations of Ca, P, and Cu in the soil could affect the metabolite profiles of Justicia gendarussa. Six significant metabolites were proposed.

  5. Metabolite Profiling of Justicia gendarussa Burm. f. Leaves Using UPLC-UHR-QTOF-MS

    PubMed Central

    Ningsih, Indah Yulia; Purwanti, Diah Intan; Wongso, Suwidji; Prajogo, Bambang E. W.; Indrayanto, Gunawan

    2015-01-01

    An ultra-performance liquid chromatography ultra-high-resolution quadrupole-time-of-flight-mass spectrometry (UPLC-UHR-QTOF-MS) metabolite profiling ofxs Justicia gendarussa Burm. f. leaves was performed. PCA and HCA analyses were applied to observe the clustering patterns and inter-sample relationships. It seemed that the concentrations of Ca, P, and Cu in the soil could affect the metabolite profiles of Justicia gendarussa. Six significant metabolites were proposed. PMID:26839833

  6. Optimization of cell disruption methods for efficient recovery of bioactive metabolites via NMR of three freshwater microalgae (chlorophyta).

    PubMed

    Ma, Nyuk Ling; Teh, Kit Yinn; Lam, Su Shiung; Kaben, Anne Marie; Cha, Thye San

    2015-08-01

    This study demonstrates the use of NMR techniques coupled with chemometric analysis as a high throughput data mining method to identify and examine the efficiency of different disruption techniques tested on microalgae (Chlorella variabilis, Scenedesmus regularis and Ankistrodesmus gracilis). The yield and chemical diversity from the disruptions together with the effects of pre-oven and pre-freeze drying prior to disruption techniques were discussed. HCl extraction showed the highest recovery of oil compounds from the disrupted microalgae (up to 90%). In contrast, NMR analysis showed the highest intensity of bioactive metabolites obtained for homogenized extracts pre-treated with freeze-drying, indicating that homogenizing is a more favorable approach to recover bioactive substances from the disrupted microalgae. The results show the potential of NMR as a useful metabolic fingerprinting tool for assessing compound diversity in complex microalgae extracts.

  7. Influence of growing conditions on metabolite profile of Ammi visnaga umbels with special reference to bioactive furanochromones and pyranocoumarins.

    PubMed

    Sellami, Hela Kallel; Napolitano, Assunta; Masullo, Milena; Smiti, Samira; Piacente, Sonia; Pizza, Cosimo

    2013-11-01

    The medicinal plant Ammi visnaga is a valuable source of furanochromones and pyranocoumarins used as vasodilator agents. Its ability to germinate under unfavourable growth conditions, such as saline soil and hypoxia characterizing clay soils and marshes ecosystems, prompted us to qualitatively characterize secondary metabolites in umbels of A. visnaga plants grown under different conditions (in field, hydroponically controlled, and contrasted by salinity and/or hypoxia) by HPLC-ESI/IT/MS(n) analysis. Subsequently, the quantitative analysis of the bioactive compounds, above all furanochromones and pyranocoumarins, was carried out by HPLC-ESI/QqQ/MS/MS. The results show the influence of growing conditions on the quali-quantitative profile of A. visnaga secondary metabolites and evidence that hydroponic culture leads to increased level of A. visnaga active principles. Furthermore, two furanochromones never reported before were identified and characterized by 1D- and 2D-NMR analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Defining genetic and chemical diversity in wheat grain by 1H‐NMR spectroscopy of polar metabolites

    PubMed Central

    Corol, Delia I.; Jones, Huw D.; Beale, Michael H.; Ward, Jane L.

    2017-01-01

    Scope The application of high‐throughput 1H nuclear magnetic resonance (1H‐NMR) of unpurified extracts to determine genetic diversity and the contents of polar components in grain of wheat. Methods and results Milled whole wheat grain was extracted with 80:20 D2O:CD3OD containing 0.05% d4–trimethylsilylpropionate. 1H‐NMR spectra were acquired under automation at 300°K using an Avance Spectrometer operating at 600.0528 MHz. Regions for individual metabolites were identified by comparison to a library of known standards run under identical conditions. The individual 1H‐NMR peaks or levels of known metabolites were then compared by Principal Component Analysis using SIMCA‐P software. Conclusions High‐throughput 1H‐NMR is an excellent tool to compare the extent of genetic diversity within and between wheat species, and to quantify specific components (including glycine betaine, choline, and asparagine) in individual genotypes. It can also be used to monitor changes in composition related to environmental factors and to support comparisons of the substantial equivalence of transgenic lines. PMID:28087883

  9. Defining genetic and chemical diversity in wheat grain by 1H-NMR spectroscopy of polar metabolites.

    PubMed

    Shewry, Peter R; Corol, Delia I; Jones, Huw D; Beale, Michael H; Ward, Jane L

    2017-07-01

    The application of high-throughput 1H nuclear magnetic resonance (1H-NMR) of unpurified extracts to determine genetic diversity and the contents of polar components in grain of wheat. Milled whole wheat grain was extracted with 80:20 D2 O:CD3 OD containing 0.05% d4 -trimethylsilylpropionate. 1H-NMR spectra were acquired under automation at 300°K using an Avance Spectrometer operating at 600.0528 MHz. Regions for individual metabolites were identified by comparison to a library of known standards run under identical conditions. The individual 1H-NMR peaks or levels of known metabolites were then compared by Principal Component Analysis using SIMCA-P software. High-throughput 1H-NMR is an excellent tool to compare the extent of genetic diversity within and between wheat species, and to quantify specific components (including glycine betaine, choline, and asparagine) in individual genotypes. It can also be used to monitor changes in composition related to environmental factors and to support comparisons of the substantial equivalence of transgenic lines. © 2017 Rothamsted Research. Molecular Nutrition & Food Research Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Using radial NMR profiles to characterize pore size distributions

    NASA Astrophysics Data System (ADS)

    Deriche, Rachid; Treilhard, John

    2012-02-01

    Extracting information about axon diameter distributions in the brain is a challenging task which provides useful information for medical purposes; for example, the ability to characterize and monitor axon diameters would be useful in diagnosing and investigating diseases like amyotrophic lateral sclerosis (ALS)1 or autism.2 Three families of operators are defined by Ozarslan,3 whose action upon an NMR attenuation signal extracts the moments of the pore size distribution of the ensemble under consideration; also a numerical method is proposed to continuously reconstruct a discretely sampled attenuation profile using the eigenfunctions of the simple harmonic oscillator Hamiltonian: the SHORE basis. The work presented here extends Ozarlan's method to other bases that can offer a better description of attenuation signal behaviour; in particular, we propose the use of the radial Spherical Polar Fourier (SPF) basis. Testing is performed to contrast the efficacy of the radial SPF basis and SHORE basis in practical attenuation signal reconstruction. The robustness of the method to additive noise is tested and analysed. We demonstrate that a low-order attenuation signal reconstruction outperforms a higher-order reconstruction in subsequent moment estimation under noisy conditions. We propose the simulated annealing algorithm for basis function scale parameter estimation. Finally, analytic expressions are derived and presented for the action of the operators on the radial SPF basis (obviating the need for numerical integration, thus avoiding a spectrum of possible sources of error).

  11. Targeting high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance analysis with high-resolution radical scavenging profiles-Bioactive secondary metabolites from the endophytic fungus Penicillium namyslowskii.

    PubMed

    Wubshet, Sileshi G; Nyberg, Nils T; Tejesvi, Mysore V; Pirttilä, Anna Maria; Kajula, Marena; Mattila, Sampo; Staerk, Dan

    2013-08-09

    The high-resolution radical scavenging profile of an extract of the endophytic fungus Penicillium namyslowskii was used to target analysis by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-HRMS-SPE-NMR, for identification of anti-oxidative secondary metabolites. This revealed the two chromatographic peaks with the highest relative response in the radical scavenging profile to be griseophenone C and peniprequinolone. The HPLC-HRMS-SPE-NMR analysis was performed in the tube-transfer mode using a cryogenically cooled NMR probe designed for 1.7mm NMR tubes. To further explore the potential of the above HPLC-HRMS-SPE-NMR platform for analysis of endophytic extracts, six peaks displaying no radical scavenging activity were also analyzed. This allowed unambiguous identification of six metabolites, i.e., dechlorogriseofulvin, dechlorodehydrogriseofulvin, griseofulvin, dehydrogriseofulvin, mevastatin acid, and mevastatin. The high mass sensitivity of the 1.7mm cryogenically cooled NMR probe allowed for the first time acquisition of direct detected (13)C NMR spectra of fungal metabolites, i.e., dechlorogriseofulvin and griseofulvin, directly from crude extract via HPLC-HRMS-SPE-NMR. Dechlorodehydrogriseofulvin was reported for the first time from nature.

  12. Metabolite profiling of two low phytic acid (lpa) rice mutants.

    PubMed

    Frank, Thomas; Meuleye, Bertrand Seumo; Miller, Andreas; Shu, Qing-Yao; Engel, Karl-Heinz

    2007-12-26

    Two low phytic acid (lpa) rice mutant lines, Os-lpa-XS110-1 and Os-lpa-XS110-2, were grown together with their parent wild-type variety Xiushui 110 in four field trials. HPLC analysis of inositol phosphates in the seeds produced demonstrated that compared to the wild-type, the reduction in phytic acid content in Os-lpa-XS110-1 (-46%) was more pronounced than that in Os-lpa-XS110-2 (-23%). Lower inositol phosphates (InsP 3, InsP 4, InsP 5) were not detected in the mutants. The lpa mutants and the wild-type rice were subjected to comparative metabolite profiling by capillary gas chromatography. On average, 34% (Os-lpa-XS110-1) and 42% (Os-lpa-XS110-2) of the detected peaks were statistically significantly different between wild-type and mutants. However, only a few of these differences could be consistently observed for all field trials. Identification and quantification of the consistently different metabolites revealed that contents of myo-inositol and raffinose were increased in Os-lpa-XS110-1 but decreased in Os-lpa-XS110-2 compared to the wild-type. In addition, Os-lpa-XS110-1 exhibited increased levels of galactose and galactinol. Consideration of these metabolic changes in light of the routes involved in the biosynthesis of phytic acid indicated a disturbance in the early biosynthetic pathway of phytic acid in Os-lpa-XS110-2 (similar to the lpa-1 type mutation in maize) and a mutation event affecting phosphorylation of myo-inositol in Os-lpa-XS110-1 (similar to the lpa-3-type mutation).

  13. Further insight into the latex metabolite profile of Ficus carica.

    PubMed

    Oliveira, Andreia P; Silva, Luís R; Andrade, Paula B; Valentão, Patrícia; Silva, Branca M; Gonçalves, Rui F; Pereira, José A; Guedes de Pinho, Paula

    2010-10-27

    Latex is a sticky emulsion that exudes upon damage from specialized canals from several plants. It contains several biologically active compounds, such as phytosterols, fatty acids, and amino acids. In plants, these compounds are involved in the interaction between plants, insects, and the environment. Despite its chemical, biological, and ecological importance, Ficus carica latex is still poorly studied. To improve the knowledge on the metabolite profile of this matrix, a targeted metabolite analysis was performed in a representative sample from F. carica latex. Seven phytosterols were determined by gas chromatography-ion trap mass spectrometry (GC-ITMS) and high-performance liquid chromatography coupled to diode array detection (HPLC-DAD), with β-sitosterol and lupeol being the compounds present in higher concentrations (ca. 54 and 14%, respectively). A total of 18 fatty acids were characterized by GC-ITMS, being essentially represented by saturated fatty acids (ca. 86.4% of total fatty acids). A total of 13 free amino acids were also identified by high-performance liquid chromatography coupled to ultraviolet-visible spectroscopy (HPLC/UV-vis), and cysteine and tyrosine were the major ones (ca. 38.7 and 31.4%, respectively). In humans, phytosterols and some polyunsaturated fatty acids, such as linoleic acid, are known for their anticarcinogenic properties. With regard to amino acids, some of them, such as glycine, are neurotransmitters. Our results reveal the presence of a wide diversity of compounds, from distinct classes, in F. carica latex, possessing various potential pharmacological activities; thus, its biological potential appears to be worth further exploring.

  14. Metabolic profiling of kidney and urine in rats with lithium-induced nephrogenic diabetes insipidus by (1)H-NMR-based metabonomics.

    PubMed

    Hwang, Geum-Sook; Yang, Ji-Young; Ryu, Do Hyun; Kwon, Tae-Hwan

    2010-02-01

    Lithium (Li) treatment for bipolar affective disorders is associated with a variety of renal side effects. The metabolic response of the kidney to chronic Li treatment has rarely been studied. We applied a novel method of (1)H-nuclear magnetic resonance (NMR)-based metabonomics to integrate metabolic profiling and to identify the changes in the levels of metabolites in the kidney and urine from rats with Li-induced NDI. Metabolic profiles of urine and kidney homogenate [3 different zones (cortex, outer medulla, and inner medulla) or whole kidney] were investigated using high-resolution NMR spectroscopy coupled with pattern recognition methods. The accurate concentrations of metabolites in kidney homogenates and urine were rapidly measured using the target-profiling procedure, and the difference in the levels of metabolites was compared using multivariate analysis, such as principal component analysis and orthogonal partial least squares-discriminant analysis. Major endogenous metabolites for kidney homogenates contained products of glycolysis (glucose, lactate) and amino acids, as well as organic osmolytes (e.g., betaine, myo-inositol, taurine, and glycerophosphocholine). Many metabolites revealed changes in their levels, including decreased levels of organic osmolytes and amino acids in the inner medulla. A number of urinary metabolites were changed in Li-induced NDI, and in particular, elevated urinary levels of acetate, lactate, allantoin, trimethylamine, and creatine could suggest Li-induced renal cell stress or injury. Taken together, metabonomics of kidney tissue and urine based on (1)H-NMR spectroscopy could provide insight into the effects of Li-induced renal effects and cell injury.

  15. Metabolite Profiling and Cardiovascular Event Risk: A Prospective Study of Three Population-Based Cohorts

    PubMed Central

    Würtz, Peter; Havulinna, Aki S; Soininen, Pasi; Tynkkynen, Tuulia; Prieto-Merino, David; Tillin, Therese; Ghorbani, Anahita; Artati, Anna; Wang, Qin; Tiainen, Mika; Kangas, Antti J; Kettunen, Johannes; Kaikkonen, Jari; Mikkilä, Vera; Jula, Antti; Kähönen, Mika; Lehtimäki, Terho; Lawlor, Debbie A; Gaunt, Tom R; Hughes, Alun D; Sattar, Naveed; Illig, Thomas; Adamski, Jerzy; Wang, Thomas J; Perola, Markus; Ripatti, Samuli; Vasan, Ramachandran S; Raitakari, Olli T; Gerszten, Robert E; Casas, Juan-Pablo; Chaturvedi, Nish; Ala-Korpela, Mika; Salomaa, Veikko

    2015-01-01

    Background High-throughput profiling of circulating metabolites may improve cardiovascular risk prediction over established risk factors. Methods and Results We applied quantitative NMR metabolomics to identify biomarkers for incident cardiovascular disease during long-term follow-up. Biomarker discovery was conducted in the FINRISK study (n=7256; 800 events). Replication and incremental risk prediction was assessed in the SABRE study (n=2622; 573 events) and British Women’s Health and Heart Study (n=3563; 368 events). In targeted analyses of 68 lipids and metabolites, 33 measures were associated with incident cardiovascular events at P<0.0007 after adjusting for age, sex, blood pressure, smoking, diabetes and medication. When further adjusting for routine lipids, four metabolites were associated with future cardiovascular events in meta-analyses: higher serum phenylalanine (hazard ratio per standard deviation: 1.18 [95%CI 1.12–1.24]; P=4×10−10) and monounsaturated fatty acid levels (1.17 [1.11–1.24]; P=1×10−8) were associated with increased cardiovascular risk, while higher omega-6 fatty acids (0.89 [0.84–0.94]; P=6×10−5) and docosahexaenoic acid levels (0.90 [0.86–0.95]; P=5×10−5) were associated with lower risk. A risk score incorporating these four biomarkers was derived in FINRISK. Risk prediction estimates were more accurate in the two validation cohorts (relative integrated discrimination improvement 8.8% and 4.3%), albeit discrimination was not enhanced. Risk classification was particularly improved for persons in the 5–10% risk range (net reclassification 27.1% and 15.5%). Biomarker associations were further corroborated with mass spectrometry in FINRISK (n=671) and the Framingham Offspring Study (n=2289). Conclusions Metabolite profiling in large prospective cohorts identified phenylalanine, monounsaturated and polyunsaturated fatty acids as biomarkers for cardiovascular risk. This study substantiates the value of high

  16. Solving the Jigsaw Puzzle of Wound-Healing Potato Cultivars: Metabolite Profiling and Antioxidant Activity of Polar Extracts

    PubMed Central

    2015-01-01

    Potato (Solanum tuberosum L.) is a worldwide food staple, but substantial waste accompanies the cultivation of this crop due to wounding of the outer skin and subsequent unfavorable healing conditions. Motivated by both economic and nutritional considerations, this metabolite profiling study aims to improve understanding of closing layer and wound periderm formation and guide the development of new methods to ensure faster and more complete healing after skin breakage. The polar metabolites of wound-healing tissues from four potato cultivars with differing patterns of tuber skin russeting (Norkotah Russet, Atlantic, Chipeta, and Yukon Gold) were analyzed at three and seven days after wounding, during suberized closing layer formation and nascent wound periderm development, respectively. The polar extracts were assessed using LC-MS and NMR spectroscopic methods, including multivariate analysis and tentative identification of 22 of the 24 biomarkers that discriminate among the cultivars at a given wound-healing time point or between developmental stages. Differences among the metabolites that could be identified from NMR- and MS-derived biomarkers highlight the strengths and limitations of each method, also demonstrating the complementarity of these approaches in terms of assembling a complete molecular picture of the tissue extracts. Both methods revealed that differences among the cultivar metabolite profiles diminish as healing proceeds during the period following wounding. The biomarkers included polyphenolic amines, flavonoid glycosides, phenolic acids and glycoalkaloids. Because wound healing is associated with oxidative stress, the free radical scavenging activities of the extracts from different cultivars were measured at each wounding time point, revealing significantly higher scavenging activity of the Yukon Gold periderm especially after 7 days of wounding. PMID:24998264

  17. Solving the jigsaw puzzle of wound-healing potato cultivars: metabolite profiling and antioxidant activity of polar extracts.

    PubMed

    Dastmalchi, Keyvan; Cai, Qing; Zhou, Kevin; Huang, Wenlin; Serra, Olga; Stark, Ruth E

    2014-08-06

    Potato (Solanum tuberosum L.) is a worldwide food staple, but substantial waste accompanies the cultivation of this crop due to wounding of the outer skin and subsequent unfavorable healing conditions. Motivated by both economic and nutritional considerations, this metabolite profiling study aims to improve understanding of closing layer and wound periderm formation and guide the development of new methods to ensure faster and more complete healing after skin breakage. The polar metabolites of wound-healing tissues from four potato cultivars with differing patterns of tuber skin russeting (Norkotah Russet, Atlantic, Chipeta, and Yukon Gold) were analyzed at three and seven days after wounding, during suberized closing layer formation and nascent wound periderm development, respectively. The polar extracts were assessed using LC-MS and NMR spectroscopic methods, including multivariate analysis and tentative identification of 22 of the 24 biomarkers that discriminate among the cultivars at a given wound-healing time point or between developmental stages. Differences among the metabolites that could be identified from NMR- and MS-derived biomarkers highlight the strengths and limitations of each method, also demonstrating the complementarity of these approaches in terms of assembling a complete molecular picture of the tissue extracts. Both methods revealed that differences among the cultivar metabolite profiles diminish as healing proceeds during the period following wounding. The biomarkers included polyphenolic amines, flavonoid glycosides, phenolic acids and glycoalkaloids. Because wound healing is associated with oxidative stress, the free radical scavenging activities of the extracts from different cultivars were measured at each wounding time point, revealing significantly higher scavenging activity of the Yukon Gold periderm especially after 7 days of wounding.

  18. Comprehensive MS and Solid-State NMR Metabolomic Profiling Reveals Molecular Variations in Native Periderms from Four Solanum tuberosum Potato Cultivars.

    PubMed

    Huang, Wenlin; Serra, Olga; Dastmalchi, Keyvan; Jin, Liqing; Yang, Lijia; Stark, Ruth E

    2017-03-15

    The potato (Solanum tuberosum L.) ranks third in worldwide consumption among food crops. Whereas disposal of potato peels poses significant challenges for the food industry, secondary metabolites in these tissues are also bioactive and essential to crop development. The diverse primary and secondary metabolites reported in whole tubers and wound-healing tissues prompted a comprehensive profiling study of native periderms from four cultivars with distinctive skin morphologies and commercial food uses. Polar and nonpolar soluble metabolites were extracted concurrently, analyzed chromatographically, and characterized with mass spectrometry; the corresponding solid interfacial polymeric residue was examined by solid-state (13)C NMR. In total, 112 secondary metabolites were found in the phellem tissues; multivariate analysis identified 10 polar and 30 nonpolar potential biomarkers that distinguish a single cultivar among Norkotah Russet, Atlantic, Chipeta, and Yukon Gold cultivars which have contrasting russeting features. Compositional trends are interpreted in the context of periderm protective function.

  19. NMR-based evaluation of the metabolic profile and response to dichloroacetate of human prostate cancer cells.

    PubMed

    Kailavasan, Mithun; Rehman, Ishtiaq; Reynolds, Steven; Bucur, Adriana; Tozer, Gillian; Paley, Martyn

    2014-05-01

    The aim of this study was to evaluate the metabolic profile of human prostate cancer cells that have different metastatic potential and to determine their response to dichloroacetate (DCA) using NMR technology. Two isogenic human prostate cancer cell lines, differing in their metastatic potential [LNCaP (poorly metastatic) and LNCaP-LN3 (highly metastatic)], were studied. Metabolite ratios from NMR spectral integrals acquired at a field strength of 9.4 T using a 5-mm broadband probe with an NMR-compatible bioreactor were compared in the presence and absence of the pyruvate dehydrogenase kinase inhibitor DCA. Lactate dehydrogenase (LDH) isoenzymes were assessed by zymography. Following the treatment of cells with 50 mm DCA, there was a significant reduction in the lactate/choline, lactate/creatine, lactate/alanine and the combined lactate/(choline + creatine + alanine) ratios in LNCaP-LN3 cells relative to LNCaP cells. No significant changes in metabolite ratios were found in LNCaP cells following DCA treatment. As expected, LDH zymography assays showed an absence of the LDH-B subunit in LNCaP-LN3 cells, whereas both LDH-A and LDH-B subunits were present in LNCaP cells. DCA was shown to significantly modify the metabolite ratios in highly metastatic LNCaP-LN3 cells, but not in poorly metastatic LNCaP cells. This effect was probably related to the absence of the LDH-B subunit in LNCaP-LN3 cells, and could have a bearing on cancer treatment with DCA and related compounds.

  20. Hepatic metabolite profiles in mice with a suboptimal selenium status.

    PubMed

    Geillinger, Kerstin E; Rathmann, Daniel; Köhrle, Josef; Fiamoncini, Jarlei; Daniel, Hannelore; Kipp, Anna P

    2014-09-01

    Selenium is an essential trace element and mediates its functions via various selenoproteins such as glutathione peroxidases or thioredoxin reductases. A suboptimal selenium supply causes metabolic disturbances and is associated with an increased risk to develop different disorders, including cancer or cardiovascular diseases. This study aimed to assess the impact of a suboptimal selenium status on the hepatic metabolome of male mice analyzed by a targeted liquid chromatography/tandem mass spectrometry and a method based on non-targeted gas chromatography hyphenated with mass spectrometry. Feeding animals a diet with about half of the recommended selenium content supplied as selenomethionine caused liver glutathione peroxidase and thioredoxin reductase activities to decline and lipid peroxidation to increase. Serum T3 thyroid hormone concentration also declined via a reduced hepatic deiodinase activity. Metabolite profiling revealed predominantly changes in cysteine and carbon-1 metabolism as well as in selected lipid subclasses. In particular the concentrations of palmitoylcarnitines and oleoylcarnitines (C18:1 and C16:1) and various phosphatidylcholine species containing saturated fatty acids were elevated. Increased taurine levels suggested an enhanced cysteine flux through the salvage pathway whereas increased homocysteine levels appeared to be a consequence of a massive down-regulation of cystathionine β lyase (cystathionine β synthase) and a reduced flux through the transsulfuration pathway. The findings demonstrate that a suboptimal selenium status causes alterations in lipid and carbon-1 metabolism in mouse liver. These changes may contribute to the development of diseases associated with a suboptimal selenium status.

  1. Qualitative and spatial metabolite profiling of lichens by a LC-MS approach combined with optimised extraction.

    PubMed

    Parrot, Delphine; Peresse, Tiphaine; Hitti, Eric; Carrie, Daniel; Grube, Martin; Tomasi, Sophie

    2015-01-01

    Lichens are self-sustaining partnerships comprising fungi as shape-forming partners for their enclosed symbiotic algae. They produce a tremendous diversity of metabolites (1050 metabolites described so far). A comparison of metabolic profiles in nine lichen species belonging to three genera (Lichina, Collema and Roccella) by using an optimised extraction protocol, determination of the fragmentation pathway and the in situ localisation for major compounds in Roccella species. Chemical analysis was performed using a complementary study combining a Taguchi experimental design with qualitative analysis by high-performance liquid chromatography coupled with mass spectrometry techniques. Optimal conditions to obtain the best total extraction yield were determined as follows: mortar grinding to a fine powder, two successive extractions, solid:liquid ratio (2:60) and 700 rpm stirring. Qualitative analysis of the metabolite profiling of these nine species extracted with the optimised method was corroborated using MS and MS/MS approaches. Nine main compounds were identified: 1 β-orcinol, 2 orsellinic acid, 3 putative choline sulphate, 4 roccellic acid, 5 montagnetol, 6 lecanoric acid, 7 erythrin, 8 lepraric acid and 9 acetylportentol, and several other compounds were reported. Identification was performed using the m/z ratio, fragmentation pathway and/or after isolation by NMR analysis. The variation of the metabolite profile in differently organised parts of two Roccella species suggests a specific role of major compounds in developmental stages of this symbiotic association. Metabolic profiles represent specific chemical species and depend on the extraction conditions, the kind of the photobiont partner and the in situ localisation of major compounds. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Metabolite

    MedlinePlus

    A metabolite is any substance produced during metabolism (digestion or other bodily chemical processes). The term metabolite may also refer to the product that remains after a drug is broken down (metabolized) by the body.

  3. Combining DI-ESI–MS and NMR datasets for metabolic profiling

    PubMed Central

    Marshall, Darrell D.; Lei, Shulei; Worley, Bradley; Huang, Yuting; Garcia-Garcia, Aracely; Franco, Rodrigo; Dodds, Eric D.; Powers, Robert

    2014-01-01

    Metabolomics datasets are commonly acquired by either mass spectrometry (MS) or nuclear magnetic resonance spectroscopy (NMR), despite their fundamental complementarity. In fact, combining MS and NMR datasets greatly improves the coverage of the metabolome and enhances the accuracy of metabolite identification, providing a detailed and high-throughput analysis of metabolic changes due to disease, drug treatment, or a variety of other environmental stimuli. Ideally, a single metabolomics sample would be simultaneously used for both MS and NMR analyses, minimizing the potential for variability between the two datasets. This necessitates the optimization of sample preparation, data collection and data handling protocols to effectively integrate direct-infusion MS data with one-dimensional (1D) 1H NMR spectra. To achieve this goal, we report for the first time the optimization of (i) metabolomics sample preparation for dual analysis by NMR and MS, (ii) high throughput, positive-ion direct infusion electrospray ionization mass spectrometry (DI-ESI-MS) for the analysis of complex metabolite mixtures, and (iii) data handling protocols to simultaneously analyze DI-ESI-MS and 1D 1H NMR spectral data using multiblock bilinear factorizations, namely multiblock principal component analysis (MB-PCA) and multiblock partial least squares (MB-PLS). Finally, we demonstrate the combined use of backscaled loadings, accurate mass measurements and tandem MS experiments to identify metabolites significantly contributing to class separation in MB-PLS-DA scores. We show that integration of NMR and DI-ESI-MS datasets yields a substantial improvement in the analysis of neurotoxin involvement in dopaminergic cell death. PMID:25774104

  4. Concurrent profiling of polar metabolites and lipids in human plasma using HILIC-FTMS

    NASA Astrophysics Data System (ADS)

    Cai, Xiaoming; Li, Ruibin

    2016-11-01

    Blood plasma is the most popularly used sample matrix for metabolite profiling studies, which aim to achieve global metabolite profiling and biomarker discovery. However, most of the current studies on plasma metabolite profiling focused on either the polar metabolites or lipids. In this study, a comprehensive analysis approach based on HILIC-FTMS was developed to concurrently examine polar metabolites and lipids. The HILIC-FTMS method was developed using mixed standards of polar metabolites and lipids, the separation efficiency of which is better in HILIC mode than in C5 and C18 reversed phase (RP) chromatography. This method exhibits good reproducibility in retention times (CVs < 3.43%) and high mass accuracy (<3.5 ppm). In addition, we found MeOH/ACN/Acetone (1:1:1, v/v/v) as extraction cocktail could achieve desirable gathering of demanded extracts from plasma samples. We further integrated the MeOH/ACN/Acetone extraction with the HILIC-FTMS method for metabolite profiling and smoking-related biomarker discovery in human plasma samples. Heavy smokers could be successfully distinguished from non smokers by univariate and multivariate statistical analysis of the profiling data, and 62 biomarkers for cigarette smoke were found. These results indicate that our concurrent analysis approach could be potentially used for clinical biomarker discovery, metabolite-based diagnosis, etc.

  5. Concurrent profiling of polar metabolites and lipids in human plasma using HILIC-FTMS.

    PubMed

    Cai, Xiaoming; Li, Ruibin

    2016-11-07

    Blood plasma is the most popularly used sample matrix for metabolite profiling studies, which aim to achieve global metabolite profiling and biomarker discovery. However, most of the current studies on plasma metabolite profiling focused on either the polar metabolites or lipids. In this study, a comprehensive analysis approach based on HILIC-FTMS was developed to concurrently examine polar metabolites and lipids. The HILIC-FTMS method was developed using mixed standards of polar metabolites and lipids, the separation efficiency of which is better in HILIC mode than in C5 and C18 reversed phase (RP) chromatography. This method exhibits good reproducibility in retention times (CVs < 3.43%) and high mass accuracy (<3.5 ppm). In addition, we found MeOH/ACN/Acetone (1:1:1, v/v/v) as extraction cocktail could achieve desirable gathering of demanded extracts from plasma samples. We further integrated the MeOH/ACN/Acetone extraction with the HILIC-FTMS method for metabolite profiling and smoking-related biomarker discovery in human plasma samples. Heavy smokers could be successfully distinguished from non smokers by univariate and multivariate statistical analysis of the profiling data, and 62 biomarkers for cigarette smoke were found. These results indicate that our concurrent analysis approach could be potentially used for clinical biomarker discovery, metabolite-based diagnosis, etc.

  6. Concurrent profiling of polar metabolites and lipids in human plasma using HILIC-FTMS

    PubMed Central

    Cai, Xiaoming; Li, Ruibin

    2016-01-01

    Blood plasma is the most popularly used sample matrix for metabolite profiling studies, which aim to achieve global metabolite profiling and biomarker discovery. However, most of the current studies on plasma metabolite profiling focused on either the polar metabolites or lipids. In this study, a comprehensive analysis approach based on HILIC-FTMS was developed to concurrently examine polar metabolites and lipids. The HILIC-FTMS method was developed using mixed standards of polar metabolites and lipids, the separation efficiency of which is better in HILIC mode than in C5 and C18 reversed phase (RP) chromatography. This method exhibits good reproducibility in retention times (CVs < 3.43%) and high mass accuracy (<3.5 ppm). In addition, we found MeOH/ACN/Acetone (1:1:1, v/v/v) as extraction cocktail could achieve desirable gathering of demanded extracts from plasma samples. We further integrated the MeOH/ACN/Acetone extraction with the HILIC-FTMS method for metabolite profiling and smoking-related biomarker discovery in human plasma samples. Heavy smokers could be successfully distinguished from non smokers by univariate and multivariate statistical analysis of the profiling data, and 62 biomarkers for cigarette smoke were found. These results indicate that our concurrent analysis approach could be potentially used for clinical biomarker discovery, metabolite-based diagnosis, etc. PMID:27819279

  7. Improved Quantum Chemical NMR Chemical Shift Prediction of Metabolites in Aqueous Solution toward the Validation of Unknowns.

    PubMed

    Hoffmann, Felix; Li, Da-Wei; Sebastiani, Daniel; Brüschweiler, Rafael

    2017-04-27

    A quantum-chemistry based protocol, termed MOSS-DFT, is presented for the prediction of (13)C and (1)H NMR chemical shifts of a wide range of organic molecules in aqueous solution, including metabolites. Molecular motif-specific linear scaling parameters are reported for five different density functional theory (DFT) methods (B97-2/pcS-1, B97-2/pcS-2, B97-2/pcS-3, B3LYP/pcS-2, and BLYP/pcS-2), which were applied to a large set of 176 metabolite molecules. The chemical shift root-mean-square deviations (RMSD) for the best method, B97-2/pcS-3, are 1.93 and 0.154 ppm for (13)C and (1)H chemical shifts, respectively. Excellent results have been obtained for chemical shifts of methyl and aromatic (13)C and (1)H that are not directly bonded to a heteroatom (O, N, S, or P) with RMSD values of 1.15/0.079 and 1.31/0.118 ppm, respectively. This study not only demonstrates how NMR chemical shift in aqueous environment can be improved over the commonly used global linear scaling approach, but also allows for motif-specific error estimates, which are useful for an improved chemical shift-based verification of metabolite candidates of metabolomics samples containing unknown components.

  8. Pharmacokinetic and pharmacodynamic profile of bendamustine and its metabolites.

    PubMed

    Darwish, Mona; Bond, Mary; Hellriegel, Edward; Robertson, Philmore; Chovan, James P

    2015-06-01

    Bendamustine is a unique alkylating agent indicated for the treatment of chronic lymphocytic leukemia and rituximab-refractory, indolent B cell non-Hodgkin's lymphoma. Despite the extensive experience with bendamustine, its pharmacokinetic profile has only recently been described. This overview summarizes the pharmacokinetics, pharmacokinetic/pharmacodynamic relationships, and drug-drug interactions of bendamustine in adult and pediatric patients with hematologic malignancies. A literature search and data on file (including a human mass balance study, pharmacokinetic population analyses in adult and pediatric patients, and modeling analyses) were evaluated for inclusion. Bendamustine concentrations peak at end of intravenous infusion (~1 h). Subsequent elimination is triphasic, with the intermediate t 1/2 (~40 min) as the effective t 1/2 since the final phase represents <1 % of the area under the curve. Bendamustine is rapidly hydrolyzed to monohydroxy-bendamustine and dihydroxy-bendamustine, which have little or no activity. Cytochrome P450 (CYP) 1A2 oxidation yields the active metabolites γ-hydroxybendamustine and N-desmethyl-bendamustine, at low concentrations, which contribute minimally to cytotoxicity. Minor involvement of CYP1A2 in bendamustine elimination suggests a low likelihood of drug-drug interactions with CYP1A2 inhibitors. Systemic exposure to bendamustine 120 mg/m(2) is comparable between adult and pediatric patients; age, race, and sex have been shown to have no significant effect on systemic exposure in either population. The effect of hepatic/renal impairment on bendamustine pharmacokinetics remains to be elucidated. Higher bendamustine concentrations may be associated with increased probability of nausea or infection. No clear exposure-efficacy response relationship has been observed. Altogether, the findings support dosing based on body surface area for most patient populations.

  9. Antistaphylococcal activity and metabolite profiling of manuka honey (Leptospermum scoparium L.) after in vitro simulated digestion.

    PubMed

    Mannina, Luisa; Sobolev, Anatoly P; Coppo, Erika; Di Lorenzo, Arianna; Nabavi, Seyed Mohammad; Marchese, Anna; Daglia, Maria

    2016-03-01

    The antistaphylococcal activity against methicillin-susceptible and -resistant Staphylococcus aureus and the metabolite profiling of manuka honey (MH) were investigated before and after in vitro simulated gastric (GD) and gastroduodenal (GDD) digestions. Undigested manuka honey showed antibacterial activity against all the tested strains, the GD sample showed no activity against S. aureus, and the GDD honey showed an antistaphylococcal activity, which was slightly reduced in comparison with the undigested sample. To explain these results, methylglyoxal (MGO), to which most of the antibacterial activity of MH is ascribed, was subjected to in vitro simulated GD and GDD. After digestion, MGO showed antibacterial activity at concentrations definitively higher than those registered in digested MH samples. These results showed that the antistaphylococcal activity registered after digestion cannot be ascribed to MGO. Thus metabolite analysis, carried out using an explorative untargeted NMR-based approach and a targeted RP-HPLC-PAD-ESI-MSn analysis focused on bio-active substances, was used to highlight the chemical modifications occurring from digestion. The results showed that (1) the level of MGO decreases and (2) the content of aromatic compounds, such as leptosin and methyl syringate, markers of manuka honey, was stable under gastric and gastroduodenal conditions, whereas (3) the levels of acetic and lactic acids increase in particular after gastroduodenal digestion, being 1.5 and 2.8 times higher in GDD-MH than in UND-MH, respectively. Overall, the results obtained from chemical analysis provide at least a partial explanation of the registered antibacterial activity observed after gastroduodenal digestion.

  10. Metabolic Profiling and Classification of Propolis Samples from Southern Brazil: An NMR-Based Platform Coupled with Machine Learning.

    PubMed

    Maraschin, Marcelo; Somensi-Zeggio, Amélia; Oliveira, Simone K; Kuhnen, Shirley; Tomazzoli, Maíra M; Raguzzoni, Josiane C; Zeri, Ana C M; Carreira, Rafael; Correia, Sara; Costa, Christopher; Rocha, Miguel

    2016-01-22

    The chemical composition of propolis is affected by environmental factors and harvest season, making it difficult to standardize its extracts for medicinal usage. By detecting a typical chemical profile associated with propolis from a specific production region or season, certain types of propolis may be used to obtain a specific pharmacological activity. In this study, propolis from three agroecological regions (plain, plateau, and highlands) from southern Brazil, collected over the four seasons of 2010, were investigated through a novel NMR-based metabolomics data analysis workflow. Chemometrics and machine learning algorithms (PLS-DA and RF), including methods to estimate variable importance in classification, were used in this study. The machine learning and feature selection methods permitted construction of models for propolis sample classification with high accuracy (>75%, reaching ∼90% in the best case), better discriminating samples regarding their collection seasons comparatively to the harvest regions. PLS-DA and RF allowed the identification of biomarkers for sample discrimination, expanding the set of discriminating features and adding relevant information for the identification of the class-determining metabolites. The NMR-based metabolomics analytical platform, coupled to bioinformatic tools, allowed characterization and classification of Brazilian propolis samples regarding the metabolite signature of important compounds, i.e., chemical fingerprint, harvest seasons, and production regions.

  11. 1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite—Malylglutamate

    PubMed Central

    2017-01-01

    Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance. PMID:28753027

  12. Pathway Activity Profiling (PAPi): from the metabolite profile to the metabolic pathway activity.

    PubMed

    Aggio, Raphael B M; Ruggiero, Katya; Villas-Bôas, Silas Granato

    2010-12-01

    Metabolomics is one of the most recent omics-technologies and uses robust analytical techniques to screen low molecular mass metabolites in biological samples. It has evolved very quickly during the last decade. However, metabolomics datasets are considered highly complex when used to relate metabolite levels to metabolic pathway activity. Despite recent developments in bioinformatics, which have improved the quality of metabolomics data, there is still no straightforward method capable of correlating metabolite level to the activity of different metabolic pathways operating within the cells. Thus, this kind of analysis still depends on extremely laborious and time-consuming processes. Here, we present a new algorithm Pathway Activity Profiling (PAPi) with which we are able to compare metabolic pathway activities from metabolite profiles. The applicability and potential of PAPi was demonstrated using a previously published data from the yeast Saccharomyces cerevisiae. PAPi was able to support the biological interpretations of the previously published observations and, in addition, generated new hypotheses in a straightforward manner. However, PAPi is time consuming to perform manually. Thus, we also present here a new R-software package (PAPi) which implements the PAPi algorithm and facilitates its usage to quickly compare metabolic pathways activities between different experimental conditions. Using the identified metabolites and their respective abundances as input, the PAPi package calculates pathways' Activity Scores, which represents the potential metabolic pathways activities and allows their comparison between conditions. PAPi also performs principal components analysis and analysis of variance or t-test to investigate differences in activity level between experimental conditions. In addition, PAPi generates comparative graphs highlighting up- and down-regulated pathway activity. These datasets are available in http://www.4shared

  13. Characterization of an Nmr Homolog That Modulates GATA Factor-Mediated Nitrogen Metabolite Repression in Cryptococcus neoformans

    PubMed Central

    Lee, I. Russel; Lim, Jonathan W. C.; Ormerod, Kate L.; Morrow, Carl A.; Fraser, James A.

    2012-01-01

    Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis. PMID:22470421

  14. Genetic engineering and metabolite profiling for overproduction of polyhydroxybutyrate in cyanobacteria.

    PubMed

    Hondo, Sayaka; Takahashi, Masatoshi; Osanai, Takashi; Matsuda, Mami; Hasunuma, Tomohisa; Tazuke, Akio; Nakahira, Yoichi; Chohnan, Shigeru; Hasegawa, Morifumi; Asayama, Munehiko

    2015-11-01

    Genetic engineering and metabolite profiling for the overproduction of polyhydroxybutyrate (PHB), which is a carbon material in biodegradable plastics, were examined in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Transconjugants harboring cyanobacterial expression vectors that carried the pha genes for PHB biosynthesis were constructed. The overproduction of PHB by the engineering cells was confirmed through microscopic observations using Nile red, transmission electron microscopy (TEM), or nuclear magnetic resonance (NMR). We successfully recovered PHB from transconjugants prepared from nitrogen-depleted medium without sugar supplementation in which PHB reached approximately 7% (w/w) of the dry cell weight, showing a value of 12-fold higher productivity in the transconjugant than that in the control strain. We also measured the intracellular levels of acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA (3HB-CoA), which are intermediate products for PHB. The results obtained indicated that these products were absent or at markedly low levels when cells were subjected to the steady-state growth phase of cultivation under nitrogen depletion for the overproduction of bioplastics. Based on these results, efficient factors were discussed for the overproduction of PHB in recombinant cyanobacteria.

  15. Characterizing Exposures of Fish to Wastewater Treatment Plant Effluent: An Integrated Metabolite and Lipid Profiling Approach

    EPA Science Inventory

    Metabolite and lipid profiling are well established techniques for studying chemical-induced alterations to normal biological function in numerous organisms. These techniques have been used successfully to identify biomarkers of chemical exposure, screen for chemical potency, or ...

  16. Characterizing Exposures of Fish to Wastewater Treatment Plant Effluent: An Integrated Metabolite and Lipid Profiling Approach

    EPA Science Inventory

    Metabolite and lipid profiling are well established techniques for studying chemical-induced alterations to normal biological function in numerous organisms. These techniques have been used successfully to identify biomarkers of chemical exposure, screen for chemical potency, or ...

  17. Metabolic Profiling and Antioxidant Assay of Metabolites from Three Radish Cultivars (Raphanus sativus).

    PubMed

    Park, Chang Ha; Baskar, Thanislas Bastin; Park, Soo-Yun; Kim, Sun-Ju; Valan Arasu, Mariadhas; Al-Dhabi, Naif Abdullah; Kim, Jae Kwang; Park, Sang Un

    2016-01-28

    A total of 13 anthocyanins and 33 metabolites; including organic acids, phenolic acids, amino acids, organic compounds, sugar acids, sugar alcohols, and sugars, were profiled in three radish cultivars by using high-performance liquid chromatography (HPLC) and gas chromatography time-of-flight mass spectrometry (GC-TOFMS)-based metabolite profiling. Total phenolics and flavonoids and their in vitro antioxidant activities were assessed. Pelargonidins were found to be the major anthocyanin in the cultivars studied. The cultivar Man Tang Hong showed the highest level of anthocyanins (1.89 ± 0.07 mg/g), phenolics (0.0664 ± 0.0033 mg/g) and flavonoids (0.0096 ± 0.0004 mg/g). Here; the variation of secondary metabolites in the radishes is described, as well as their association with primary metabolites. The low-molecular-weight hydrophilic metabolite profiles were subjected to principal component analysis (PCA), hierarchical clustering analysis (HCA), Pearson's correlation analysis. PCA fully distinguished the three radish cultivars tested. The polar metabolites were strongly correlated between metabolites that participate in the TCA cycle. The chemometrics results revealed that TCA cycle intermediates and free phenolic acids as well as anthocyanins were higher in the cultivar Man Tang Hong than in the others. Furthermore; superoxide radical scavenging activities and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging were investigated to elucidate the antioxidant activity of secondary metabolites in the cultivars. Man Tang Hong showed the highest superoxide radical scavenging activity (68.87%) at 1000 μg/mL, and DPPH activity (20.78%), followed by Seo Ho and then Hong Feng No. 1. The results demonstrate that GC-TOFMS-based metabolite profiling, integrated with chemometrics, is an applicable method for distinguishing phenotypic variation and determining biochemical reactions connecting primary and secondary metabolism. Therefore; this study might provide

  18. Application of an integrated LC-UV-MS-NMR platform to the identification of secondary metabolites from cell cultures: benzophenanthridine alkaloids from elicited Eschscholzia californica (california poppy) cell cultures().

    PubMed

    Gathungu, Rose M; Oldham, John T; Bird, Susan S; Lee-Parsons, Carolyn W T; Vouros, Paul; Kautz, Roger

    2012-01-01

    Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. Cultures can be optimized for high yields of desired metabolites using rapid profiling assays such as HPLC. We describe an approach to establishing a rapid assay for profiling cell culture expression systems using a novel microscale LC-UV-MS-NMR platform, designed to acquire both MS and NMR each at their optimal sensitivity, by using nanosplitter MS from 4 mm analytical HPLC columns, and offline microdroplet NMR. The approach is demonstrated in the analysis of elicited Eschscholzia californica cell cultures induced with purified yeast extract to produce benzophenanthridine alkaloids. Preliminary HPLC-UV provides an overview of the changes in the production of alkaloids with time after elicitation. At the time point corresponding to the production of the most alkaloids, the integrated LC-MS-microcoil NMR platform is used for structural identification of extracted alkaloids. Eight benzophenanthridine alkaloids were identified at the sub-microgram level. This paper demonstrates the utility of the nanosplitter LC-MS/microdroplet NMR platform when establishing cell culture expression systems.

  19. 1H NMR-based metabolic fingerprinting of urine metabolites after consumption of lingonberries (Vaccinium vitis-idaea) with a high-fat meal.

    PubMed

    Lehtonen, Henna-Maria; Lindstedt, Anni; Järvinen, Riikka; Sinkkonen, Jari; Graça, Gonçalo; Viitanen, Matti; Kallio, Heikki; Gil, Ana M

    2013-06-01

    The use of NMR metabolomics in clinical trials is growing; however, reports of postprandial experiments in humans are scarce. The present study investigated whether consumption of lingonberries as a supplement to an oil-rich meal modifies the postprandial fingerprints of human urine. Urine samples were analysed by (1)H NMR, and untargeted multivariate analysis was applied to the data for comprehensive fingerprinting. A clear separation of postprandial lingonberry meal samples was revealed. To evaluate statistical differences, a targeted approach was applied for the informative spectral areas. Significantly (p<0.05) increased levels of polyphenol metabolites, hippuric acid and 4-hydroxyhippuric acid, and decreased creatinine and dimethylamine levels were the major explanations for the grouping of the postprandial samples after the different meals. Thus, inclusion of polyphenol-rich lingonberry powder in a rapeseed oil-rich meal modifies the metabolic profile of urine which may be used to reveal both consumption of berries and health-promoting changes in the common metabolism.

  20. Urinary metabolite profiling identifies novel colonic metabolites and conjugates of phenolics in healthy volunteers.

    PubMed

    Pimpão, Rui C; Dew, Tristan; Figueira, Maria E; McDougall, Gordon J; Stewart, Derek; Ferreira, Ricardo B; Santos, Claudia N; Williamson, Gary

    2014-07-01

    The colonic metabolism of dietary flavonoids, phenolic acids and their phenolic metabolites is complex and many metabolites and conjugates have not yet been unambiguously identified in humans. Urine samples from nine healthy human volunteers obtained after the ingestion of a puree of five (poly)phenol-rich berry fruits were analysed using LC-Orbitrap MS to provide a preliminary indication of possible metabolites based on exact mass. In most cases, the identity of compounds was confirmed using standards produced either chemically or enzymically followed by analysis using LC-triple quadrupole MS. Sulphated, glucuronidated and methylated forms of catechol, pyrogallol and protocatechuic acid mostly appeared in urine after 8 h, suggesting colonic metabolism. Gallic acid and (-)-epicatechin conjugates appeared mainly before 4 h, indicative of absorption from the small intestine. Conjugates of ferulic, caffeic, and vanillic acid appeared at intermediate times. We have positively identified metabolites and conjugates, some novel, in the urine of healthy volunteers after intake of multiple phenolics from a mixed puree from berry fruits, with each being excreted at specific and signature times. Some of these compounds could potentially be used as biomarkers of fruit intake. The possible biological activities of these colonic metabolites require further assessment. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Application of comprehensive NMR-based analysis strategy in annotation, isolation and structure elucidation of low molecular weight metabolites of Ricinus communis seeds.

    PubMed

    Vučković, Ivan; Rapinoja, Marja-Leena; Vaismaa, Matti; Vanninen, Paula; Koskela, Harri

    2016-01-01

    Powder-like extract of Ricinus communis seeds contain a toxic protein, ricin, which has a history of military, criminal and terroristic use. As the detection of ricin in this "terrorist powder" is difficult and time-consuming, related low mass metabolites have been suggested to be useful for screening as biomarkers of ricin. To apply a comprehensive NMR-based analysis strategy for annotation, isolation and structure elucidation of low molecular weight plant metabolites of Ricinus communis seeds. The seed extract was prepared with a well-known acetone extraction approach. The common metabolites were annotated from seed extract dissolved in acidic solution using (1)H NMR spectroscopy with spectrum library comparison and standard addition, whereas unconfirmed metabolites were identified using multi-step off-line HPLC-DAD-NMR approach. In addition to the common plant metabolites, two previously unreported compounds, 1,3-digalactoinositol and ricinyl-alanine, were identified with support of MS analyses. The applied comprehensive NMR-based analysis strategy provided identification of the prominent low molecular weight metabolites with high confidence. Copyright © 2015 John Wiley & Sons, Ltd.

  2. 1H NMR Metabolic Fingerprinting to Probe Temporal Postharvest Changes on Qualitative Attributes and Phytochemical Profile of Sweet Cherry Fruit

    PubMed Central

    Goulas, Vlasios; Minas, Ioannis S.; Kourdoulas, Panayiotis M.; Lazaridou, Athina; Molassiotis, Athanassios N.; Gerothanassis, Ioannis P.; Manganaris, George A.

    2015-01-01

    Sweet cherry fruits (Prunus avium cvs. ‘Canada Giant’, ‘Ferrovia’) were harvested at commercial maturity stage and analyzed at harvest and after maintenance at room temperature (storage at ∼20°C, shelf life) for 1, 2, 4, 6, and 8 days, respectively. Fruit were initially analyzed for respiration rate, qualitative attributes and textural properties: ‘Canada Giant’ fruit were characterized by higher weight losses and stem browning index, being more intense over the late stages of shelf life period; meanwhile ‘Ferrovia’ possessed appreciably better performance even after extended shelf life period. A gradual decrease of respiration rate was monitored in both cultivars, culminated after 8 days at 20°C. The sweet cherry fruit nutraceutical profile was monitored using an array of instrumental techniques (spectrophotometric assays, HPLC, 1H-NMR). Fruit antioxidant capacity was enhanced with the progress of shelf life period, concomitant with the increased levels of total anthocyanin and of phenolic compounds. ‘Ferrovia’ fruit presented higher contents of neochlorogenic acid and p-coumaroylquinic acid throughout the shelf life period. We further developed an 1H-NMR method that allows the study of primary and secondary metabolites in a single running, without previous separation and isolation procedures. Diagnostic peaks were located in the aliphatic region for sugars and organic acids, in the aromatic region for phenolic compounds and at 8.2–8.6 ppm for anthocyanins. This NMR-based methodology provides a unifying tool for quantitative and qualitative characterization of metabolite changes of sweet cherry fruits; it is also expected to be further exploited for monitoring temporal changes in other fleshy fruits. PMID:26617616

  3. Different profiles of quercetin metabolites in rat plasma: comparison of two administration methods.

    PubMed

    Kawai, Yoshichika; Saito, Satomi; Nishikawa, Tomomi; Ishisaka, Akari; Murota, Kaeko; Terao, Junji

    2009-03-23

    The bioavailability of polyphenols in human and rodents has been discussed regarding their biological activity. We found different metabolite profiles of quercetin in rat plasma between two administration procedures. A single intragastric administration (50 mg/kg) resulted in the appearance of a variety of metabolites in the plasma, whereas only a major fraction was detected by free access (1% quercetin). The methylated/non-methylated metabolites ratio was much higher in the free access group. Mass spectrometric analyses showed that the fraction from free access contained highly conjugated quercetin metabolites such as sulfo-glucuronides of quercetin and methylquercetin. The metabolite profile of human plasma after an intake of onion was similar to that with intragastric administration in rats. In vitro oxidation of human low-density lipoprotein showed that methylation of the catechol moiety of quercetin significantly attenuated the antioxidative activity. These results might provide information about the bioavailability of quercetin when conducting animal experiments.

  4. Digital NMR profiles as building blocks: assembling ¹H fingerprints of steviol glycosides.

    PubMed

    Napolitano, José G; Simmler, Charlotte; McAlpine, James B; Lankin, David C; Chen, Shao-Nong; Pauli, Guido F

    2015-04-24

    This report describes a fragment-based approach to the examination of congeneric organic compounds by NMR spectroscopy. The method combines the classic interpretation of 1D- and 2D-NMR data sets with contemporary computer-assisted NMR analysis. Characteristic NMR profiles of key structural motifs were generated by (1)H iterative full spin analysis and then joined together as building blocks to recreate the (1)H NMR spectra of increasingly complex molecules. To illustrate the methodology described, a comprehensive analysis of steviol (1), seven steviol glycosides (2-8) and two structurally related isosteviol compounds (9, 10) was carried out. The study also assessed the potential impact of this method on relevant aspects of natural product research including structural verification, chemical dereplication, and mixture analysis.

  5. Digital NMR Profiles as Building Blocks: Assembling 1H Fingerprints of Steviol Glycosides

    PubMed Central

    Napolitano, José G.; Simmler, Charlotte; McAlpine, James B.; Lankin, David C.; Chen, Shao-Nong; Pauli, Guido F.

    2015-01-01

    This report describes a fragment-based approach to the examination of congeneric organic compounds by NMR spectroscopy. The method combines the classic interpretation of 1D- and 2D-NMR data sets with contemporary computer-assisted NMR analysis. Characteristic NMR profiles of key structural motifs were generated by 1H iterative full spin analysis and then joined together as building blocks to recreate the 1H NMR spectra of increasingly complex molecules. To illustrate the methodology described, a comprehensive analysis of steviol (1), seven steviol glycosides (2–8) and two structurally related isosteviol compounds (9, 10) was carried out. The study also assessed the potential impact of this method on relevant aspects of natural product research including structural verification, chemical dereplication, and mixture analysis. PMID:25714117

  6. A Carbonyl Capture Approach for Profiling Oxidized Metabolites in Cell Extracts

    PubMed Central

    Mattingly, Stephanie J.; Xu, Tao; Nantz, Michael H.; Higashi, Richard M.; Fan, Teresa W.-M.

    2012-01-01

    Fourier-transform ion-cyclotron resonance mass spectrometry (FT-ICR-MS) detection of oxidized cellular metabolites is described using isotopologic, carbonyl-selective derivatizing agents that integrate aminooxy functionality for carbonyl capture, quaternary nitrogen for electrospray enhancement, and a hydrophobic domain for sample cleanup. These modular structural features enable rapid, sensitive analysis of complex mixtures of metabolite-derivatives by FT-ICR-MS via continuous nanoelectrospray infusion. Specifically, this approach can be used to globally assess levels of low abundance and labile aldehyde and ketone metabolites quantitatively and in high throughput manner. These metabolites are often key and unique indicators of various biochemical pathways and their perturbations. Analysis of lung adenocarcinoma A549 cells established a profile of carbonyl metabolites spanning multiple structural classes. We also demonstrate a procedure for metabolite quantification using pyruvate as a model analyte. PMID:23175637

  7. Profiling of Disease-Related Metabolites in Grapevine Internode Tissues Infected with Agrobacterium vitis

    PubMed Central

    Jung, Sung-Min; Hur, Youn-Young; Preece, John E.; Fiehn, Oliver; Kim, Young-Ho

    2016-01-01

    Green shoot cuttings of 10 different grapevine species were inoculated with Agrobacterium vitis to find disease-related metabolites in the grapevine. Crown galls formed 60 days after inoculation varied in gall severity (GS) evaluated by gall incidence (GI) and gall diameter (GD), which were classified into three response types as RR (low GI and small GD), SR (high GI and small GD), and SS (high GI and large GD), corresponding to resistant, moderately resistant, and susceptible responses, respectively. In this, 4, 4, and 2 Vitis species were classified into RR, SR, and SS, respectively. Gas chromatography mass spectrometry (GC-MS) analysis of the grapevine stem metabolites with A. vitis infection showed 134 metabolites in various compound classes critically occurred, which were differentially clustered with the response types by the principal component analysis. Multivariate analysis of the metabolite profile revealed that 11 metabolites increased significantly in relation to the response types, mostly at post-inoculation stages, more prevalently (8 metabolites) at two days after inoculation than other stages, and more related to SS (7 metabolites) than RR (3 metabolites) or SR (one metabolite). This suggests most of the disease-related metabolites may be rarely pre-existing but mostly induced by pathogen infection largely for facilitating gall development except stilbene compound resveratrol, a phytoalexin that may be involved in the resistance response. All of these aspects may be used for the selection of resistant grapevine cultivars and their rootstocks for the control of the crown gall disease of the grapevine. PMID:27904455

  8. Profiling of Disease-Related Metabolites in Grapevine Internode Tissues Infected with Agrobacterium vitis.

    PubMed

    Jung, Sung-Min; Hur, Youn-Young; Preece, John E; Fiehn, Oliver; Kim, Young-Ho

    2016-12-01

    Green shoot cuttings of 10 different grapevine species were inoculated with Agrobacterium vitis to find disease-related metabolites in the grapevine. Crown galls formed 60 days after inoculation varied in gall severity (GS) evaluated by gall incidence (GI) and gall diameter (GD), which were classified into three response types as RR (low GI and small GD), SR (high GI and small GD), and SS (high GI and large GD), corresponding to resistant, moderately resistant, and susceptible responses, respectively. In this, 4, 4, and 2 Vitis species were classified into RR, SR, and SS, respectively. Gas chromatography mass spectrometry (GC-MS) analysis of the grapevine stem metabolites with A. vitis infection showed 134 metabolites in various compound classes critically occurred, which were differentially clustered with the response types by the principal component analysis. Multivariate analysis of the metabolite profile revealed that 11 metabolites increased significantly in relation to the response types, mostly at post-inoculation stages, more prevalently (8 metabolites) at two days after inoculation than other stages, and more related to SS (7 metabolites) than RR (3 metabolites) or SR (one metabolite). This suggests most of the disease-related metabolites may be rarely pre-existing but mostly induced by pathogen infection largely for facilitating gall development except stilbene compound resveratrol, a phytoalexin that may be involved in the resistance response. All of these aspects may be used for the selection of resistant grapevine cultivars and their rootstocks for the control of the crown gall disease of the grapevine.

  9. 1H-NMR metabolic profiling of cerebrospinal fluid in patients with complex regional pain syndrome-related dystonia.

    PubMed

    Meissner, Axel; van der Plas, Anton A; van Dasselaar, Nick T; Deelder, André M; van Hilten, Jacobus J; Mayboroda, Oleg A

    2014-01-01

    In complex regional pain syndrome (CRPS)-related dystonia, compelling evidence points to the involvement of the central nervous system, but the underpinning pathobiology is still unclear. Thus, to enable a hypothesis-free, unbiased view of the problem and to obtain new insight into the pathobiology of dystonia in CRPS, we applied an exploratory metabolomics analysis of cerebrospinal fluid (CSF) of patients with CRPS-related dystonia. (1)H-NMR spectroscopy in combination with multivariate modeling were used to investigate metabolic profiles of a total of 105 CSF samples collected from patients with CRPS-related dystonia and controls. We found a significantly different metabolic profile of CSF in CRPS patients compared to controls. The differences were already reflected in the first two principal components of the principal component analysis model, which is an indication that the variance associated with CRPS is stronger than variance caused by such classical confounders as gender, age, or individual differences. A supervised analysis generated a strong model pinpointing the most important metabolites contributed to the metabolic signature of patients with CRPS-related dystonia. From the set of identified discriminators, the most relevant metabolites were 2-keto-isovalerate, glucose, glutamine, and lactate, which all showed increased concentrations, and urea, which showed decreased concentration in CRPS subjects. Our findings point at a catabolic state in chronic CRPS patients with dystonia that is likely associated with inflammation.

  10. Metabolite profiling in retinoblastoma identifies novel clinicopathological subgroups

    PubMed Central

    Kohe, Sarah; Brundler, Marie-Anne; Jenkinson, Helen; Parulekar, Manoj; Wilson, Martin; Peet, Andrew C; McConville, Carmel M

    2015-01-01

    Background: Tumour classification, based on histopathology or molecular pathology, is of value to predict tumour behaviour and to select appropriate treatment. In retinoblastoma, pathology information is not available at diagnosis and only exists for enucleated tumours. Alternative methods of tumour classification, using noninvasive techniques such as magnetic resonance spectroscopy, are urgently required to guide treatment decisions at the time of diagnosis. Methods: High-resolution magic-angle spinning magnetic resonance spectroscopy (HR-MAS MRS) was undertaken on enucleated retinoblastomas. Principal component analysis and cluster analysis of the HR-MAS MRS data was used to identify tumour subgroups. Individual metabolite concentrations were determined and were correlated with histopathological risk factors for each group. Results: Multivariate analysis identified three metabolic subgroups of retinoblastoma, with the most discriminatory metabolites being taurine, hypotaurine, total-choline and creatine. Metabolite concentrations correlated with specific histopathological features: taurine was correlated with differentiation, total-choline and phosphocholine with retrolaminar optic nerve invasion, and total lipids with necrosis. Conclusions: We have demonstrated that a metabolite-based classification of retinoblastoma can be obtained using ex vivo magnetic resonance spectroscopy, and that the subgroups identified correlate with histopathological features. This result justifies future studies to validate the clinical relevance of these subgroups and highlights the potential of in vivo MRS as a noninvasive diagnostic tool for retinoblastoma patient stratification. PMID:26348444

  11. 1H High Resolution Magic-Angle Coil Spinning (HR-MACS) - NMR Metabolic Profiling of whole Saccharomyces cervisiae cells: A Demonstrative Study

    NASA Astrophysics Data System (ADS)

    Wong, Alan; Boutin, Celine; Aguiar, Pedro

    2014-06-01

    The low sensitivity of Nuclear Magnetic Resonance (NMR) is its prime shortcoming compared to other analytical methods for metabolomic studies. It relies on large sample volume (30-50 µl for HR-MAS) for rich metabolic profiling, hindering high-throughput screening especially when the sample requires a labor-intensive preparation or is a sacred specimen. This is indeed the case for some living organisms. This study evaluates a 1H HR-MAS approach for metabolic profiling of small volume (250 nl) whole bacterial cells, Saccharomyces cervisiae, using an emerging micro-NMR technology: high-resolution magic-angle coil spinning (HR-MACS). As a demonstrative study for whole cells, we perform two independent metabolomics studies identifying the significant metabolites associated with osmotic stress and aging.

  12. Gender- and Age-Associated Differences in Serum Metabolite Profiles among Japanese Populations.

    PubMed

    Saito, Kosuke; Maekawa, Keiko; Kinchen, Jason M; Tanaka, Rieko; Kumagai, Yuji; Saito, Yoshiro

    2016-01-01

    Serum metabolites can reflect the diffusion/export of biochemicals from various organs. They can serve as biomarkers related to diseases and therapeutic efficacy/toxicity. While studies in Caucasians suggested that subject gender and age can affect circulating metabolite profiles, the Japanese population has not been surveyed. Our objective was to delineate gender- and age-associated differences in serum metabolite profiles among Japanese populations. Using a mass spectrometry-based global metabolomics approach, 516 endogenous metabolites were detected in sera from Japanese individuals. The principal component analysis identified gender as the primary component, followed by age, suggesting that these two criteria were key contributors to variations in the dataset. Gender-associated differences were observed in 31 and 25% of metabolites in the young (age 25-35) and old (ages 55-65) populations, respectively, in redox homeostasis, and in steroid and purine nucleotide metabolism pathways. Age-associated differences were observed in 24 and 23% of metabolites in men and women, respectively. No pathway was commonly highlighted. Thus, gender and age impact on metabolite profiles in the Japanese population. Our results provide useful information to explore biomarkers for clinical applications in the Japanese population and to assess the applicability of known biomarkers identified in other populations to the Japanese population.

  13. 1HNMR-Based Discriminatory Analysis of Eurycoma longifolia from Different Locations and Establishing a Profile for Primary Metabolites Identification and Quassinoids Quantification.

    PubMed

    Ebrahimi, Forough; Ibrahim, Baharudin; Teh, Chin Hoe; Murugaiyah, Vikneswaran; Lam, Chan Kit

    2017-01-01

    Quassinoids, the major secondary metabolites of Eurycoma longifolia roots, improve male fertility. Hence, it is crucial to investigate their quantitative level in E. longifolia extracts. A profile was established to identify the primary metabolites and major quassinoids, and quantify quassinoids using external calibration curves. Furthermore, the metabolic discrimination of E. longifolia roots from different regions was investigated. The (1)H-NMR spectra of the quassinoids, eurycomanone, eurycomanol, 13,21-dihydroeurycomanone, and eurycomanol-2-O-β-D-glycopyranoside were obtained. The (1)H-NMR profiles of E. longifolia root aqueous extracts from Perak (n = 30) were obtained and used to identify primary metabolites and the quassinoids. Selangor, Kedah, Terengganu (n = 5 for each), and Perak samples were checked for metabolic discrimination. Hotelling's T(2) plot was used to check for outliers. Orthogonal partial least-squares discriminant analysis was run to reveal the discriminatory metabolites. Perak samples contained formic, succinic, methylsuccinic, fumaric, lactic, acetic and syringic acids as well as choline, alanine, phenylalanine, tyrosine, α-glucose, eurycomanone, eurycomanol, 13,21-dihydroeurycomanone, and eurycomanol-2-O-β-D-glycopyranoside. The extracts from other locations contained the same metabolites. The limit of quantification values were 1.96 (eurycomanone), 15.62 (eurycomanol), 3.91 (13,21-dihydroeurycomanone), and 31.25 (eurycomanol-2-O-β-D-glycopyranoside) ppm. The Hotelling's T(2) plot revealed no outlier. The orthogonal partial least-squares discriminant analysis model showed that choline, eurycomanol, eurycomanol-2-O-β-D-glycopyranoside, and lactic and succinic acid levels were different among regions. Terengganu and Perak samples contained higher amounts of eurycomanol and eurycomanol-2-O-β-D-glycopyranoside, respectively. The current approach efficiently detected E. longifolia root metabolites, quantified the quassinoids, and

  14. Stable isotope coded derivatizing reagents as internal standards in metabolite profiling.

    PubMed

    Bruheim, Per; Kvitvang, Hans Fredrik Nyvold; Villas-Boas, Silas G

    2013-06-28

    Gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometric (MS) detection have become the two main techniques for the analysis of metabolite pools (i.e. Metabolomics). These technologies are especially suited for Metabolite Profiling analysis of various metabolite groups due to high separation capabilities of the chromatographs and high sensitivity of the mass analysers. The trend in quantitative Metabolite Profiling is to add more metabolites and metabolite groups in a single method. This should not be done by compromising the analytical precision. Mass spectrometric detection comes with certain limitations, especially in the quantitative aspects as standards are needed for conversion of ion abundance to concentration and ionization efficiencies are directly dependent on eluent conditions. This calls for novel strategies to counteract all variables that can influence the quantitative precision. Usually, internal standards are used to correct any technical variation. For quantitation of single or just a few analytes this can be executed with spiking isotopically labeled standards. However, for more comprehensive analytical tasks, e.g. profiling tens or hundreds of analytes simultaneously, this strategy becomes expensive and in many cases isotopically labeled standards are not available. An alternative is to introduce a derivatizing step where the sample is derivatized with naturally labeled reagent, while a standard solution is separately derivatized with isotopically labeled reagent and spiked into the sample solution prior to analysis. This strategy, named isotope coded derivatization - ICD, is attractive in the emerging field of quantitative Metabolite Profiling where current protocols can easily comprise over hundred metabolites. This review provides an overview of isotopically labeled derivatizing reagents that have been developed for important metabolite groups with the aim to improve analytical performance and precision.

  15. Multidimensional High-Resolution Magic Angle Spinning and Solution-State NMR Characterization of 13C-labeled Plant Metabolites and Lignocellulose

    PubMed Central

    Mori, Tetsuya; Tsuboi, Yuuri; Ishida, Nobuhiro; Nishikubo, Nobuyuki; Demura, Taku; Kikuchi, Jun

    2015-01-01

    Lignocellulose, which includes mainly cellulose, hemicellulose, and lignin, is a potential resource for the production of chemicals and for other applications. For effective production of materials derived from biomass, it is important to characterize the metabolites and polymeric components of the biomass. Nuclear magnetic resonance (NMR) spectroscopy has been used to identify biomass components; however, the NMR spectra of metabolites and lignocellulose components are ambiguously assigned in many cases due to overlapping chemical shift peaks. Using our 13C-labeling technique in higher plants such as poplar samples, we demonstrated that overlapping peaks could be resolved by three-dimensional NMR experiments to more accurately assign chemical shifts compared with two-dimensional NMR measurements. Metabolites of the 13C-poplar were measured by high-resolution magic angle spinning NMR spectroscopy, which allows sample analysis without solvent extraction, while lignocellulose components of the 13C-poplar dissolved in dimethylsulfoxide/pyridine solvent were analyzed by solution-state NMR techniques. Using these methods, we were able to unambiguously assign chemical shifts of small and macromolecular components in 13C-poplar samples. Furthermore, using samples of less than 5 mg, we could differentiate between two kinds of genes that were overexpressed in poplar samples, which produced clearly modified plant cell wall components. PMID:26143886

  16. Comparative metabolite profiling of the insecticide thiamethoxam in plant and cell suspension culture of tomato.

    PubMed

    Karmakar, Rajib; Bhattacharya, Ramcharan; Kulshrestha, Gita

    2009-07-22

    The metabolism of thiamethoxam [(EZ)-3-(2-chloro-1,3-thiazol-5-yl-methyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene (nitro) amine] was investigated in whole plant, callus, and heterotrophic cell suspension culture of aseptically and field grown tomato (Lycopersicon esculentum Mill.) plants. The structure of the metabolites was elucidated by chromatographic (HPLC) and spectroscopic (IR, NMR, and MS) methods. Thiamethoxam metabolism proceeded by the formation of a urea derivative, a nitroso product, and nitro guanidine. Both urea and nitro guanidine metabolites further degraded in plants, and a mechanism has been proposed. In the plant, organ-specific differences in thiamethoxam metabolism were observed. Only one metabolite was formed in whole plant against four in callus and eight metabolites in cell suspension culture under aseptic conditions. Out of six metabolites of thiamethoxam in tomato fruits in field conditions, five were similar to those formed in the cell suspension culture. In the cell suspension culture, thiamethoxam degraded to maximum metabolites within 72 h, whereas in plants, such extensive conversion could only be observed after 10 days.

  17. Steroid receptor profiling of vinclozolin and its primary metabolites

    SciTech Connect

    Molina-Molina, Jose-Manuel; Hillenweck, Anne; Jouanin, Isabelle; Zalko, Daniel; Cravedi, Jean-Pierre; Fernandez, Mariana-Fatima; Pillon, Arnaud; Nicolas, Jean-Claude; Olea, Nicolas; Balaguer, Patrick . E-mail: balaguer@montp.inserm.fr

    2006-10-01

    Several pesticides and fungicides commonly used to control agricultural and indoor pests are highly suspected to display endocrine-disrupting effects in animals and humans. Endocrine disruption is mainly caused by the interference of chemicals at the level of steroid receptors: it is now well known that many of these chemicals can display estrogenic effects and/or anti-androgenic effects, but much less is known about the interaction of these compounds with other steroid receptors. Vinclozolin, a dicarboximide fungicide, like its primary metabolites 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1), and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), is known to bind androgen receptor (AR). Although vinclozolin and its metabolites were characterized as anti-androgens, relatively little is known about their effects on the function of the progesterone (PR), glucocorticoid (GR), mineralocorticoid (MR) or estrogen receptors (ER{alpha} and ER{beta}). Objectives of the study were to determine the ability of vinclozolin and its two primary metabolites to activate AR, PR, GR, MR and ER. For this purpose, we used reporter cell lines bearing luciferase gene under the control of wild type or chimeric Gal4 fusion AR, PR, GR, MR or ERs. We confirmed that all three were antagonists for AR, whereas only M2 was found a partial agonist. Interestingly, M2 was also a PR, GR and MR antagonist (MR >> PR > GR) while vinclozolin was an MR and PR antagonist. Vinclozolin, M1 and M2 were agonists for both ERs with a lower affinity for ER{beta}. Although the potencies of the fungicide and its metabolites are low when compared to natural ligands, their ability to act via more than one mechanism and the potential for additive or synergistic effect must be taken into consideration in the risk assessment process.

  18. Identification of endogenous metabolites in human sperm cells using proton nuclear magnetic resonance ((1) H-NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS).

    PubMed

    Paiva, C; Amaral, A; Rodriguez, M; Canyellas, N; Correig, X; Ballescà, J L; Ramalho-Santos, J; Oliva, R

    2015-05-01

    The objective of this study was to contribute to the first comprehensive metabolomic characterization of the human sperm cell through the application of two untargeted platforms based on proton nuclear magnetic resonance ((1) H-NMR) spectroscopy and gas chromatography coupled to mass spectrometry (GC-MS). Using these two complementary strategies, we were able to identify a total of 69 metabolites, of which 42 were identified using NMR, 27 using GC-MS and 4 by both techniques. The identity of some of these metabolites was further confirmed by two-dimensional (1) H-(1) H homonuclear correlation spectroscopy (COSY) and (1) H-(13) C heteronuclear single-quantum correlation (HSQC) spectroscopy. Most of the metabolites identified are reported here for the first time in mature human spermatozoa. The relationship between the metabolites identified and the previously reported sperm proteome was also explored. Interestingly, overrepresented pathways included not only the metabolism of carbohydrates, but also of lipids and lipoproteins. Of note, a large number of the metabolites identified belonged to the amino acids, peptides and analogues super class. The identification of this initial set of metabolites represents an important first step to further study their function in male gamete physiology and to explore potential reasons for dysfunction in future studies. We also demonstrate that the application of NMR and MS provides complementary results, thus constituting a promising strategy towards the completion of the human sperm cell metabolome.

  19. Metabolic profiling identification of metabolites formed in Mediterranean mussels (Mytilus galloprovincialis) after diclofenac exposure.

    PubMed

    Bonnefille, Bénilde; Arpin-Pont, Lauren; Gomez, Elena; Fenet, Hélène; Courant, Frédérique

    2017-04-01

    Despite the growing concern on the presence of pharmaceutically active compounds in the environment, few studies have been conducted on their metabolism in marine organisms. In this study, a non-targeted strategy based on the generation of chemical profiles generated by liquid chromatography combined with high resolution mass spectrometry was used to highlight metabolite production by the Mediterranean mussel (Mytilus galloprovincialis) after diclofenac exposure. This method allowed revealing the production of 13 metabolites in mussel tissues. Three of them were phase I metabolites, including 4'-hydroxy-diclofenac and 5-hydroxy-diclofenac. The remaining 10 were phase II metabolites, including sulfate and amino acids conjugates. Among all of the metabolites highlighted, 5 were reported for the first time in an aquatic organism exposed to diclofenac.

  20. Biomarkers are used to predict quantitative metabolite concentration profiles in human red blood cells

    PubMed Central

    Palsson, Bernhard O.

    2017-01-01

    Deep-coverage metabolomic profiling has revealed a well-defined development of metabolic decay in human red blood cells (RBCs) under cold storage conditions. A set of extracellular biomarkers has been recently identified that reliably defines the qualitative state of the metabolic network throughout this metabolic decay process. Here, we extend the utility of these biomarkers by using them to quantitatively predict the concentrations of other metabolites in the red blood cell. We are able to accurately predict the concentration profile of 84 of the 91 (92%) measured metabolites (p < 0.05) in RBC metabolism using only measurements of these five biomarkers. The median of prediction errors (symmetric mean absolute percent error) across all metabolites was 13%. The ability to predict numerous metabolite concentrations from a simple set of biomarkers offers the potential for the development of a powerful workflow that could be used to evaluate the metabolic state of a biological system using a minimal set of measurements. PMID:28264007

  1. Comparative metabolic profiling reveals secondary metabolites correlated with soybean salt tolerance.

    PubMed

    Wu, Wei; Zhang, Qing; Zhu, Yanming; Lam, Hon-Ming; Cai, Zongwei; Guo, Dianjing

    2008-12-10

    High-performance liquid chromatography-ultraviolet-electrospray ionization mass spectrometry (HPLC-UV-ESI-MS) and HPLC-ESI-MS(n) analysis methods were used for metabolic profiling and simultaneous identification of isoflavonoids and saponins in soybean seeds. Comparative targeted metabolic profiling revealed marked differences in the metabolite composition between salt-sensitive and salt-tolerant soybean varieties. Principle component analysis clearly demonstrated that it is possible to use secondary metabolites, for example, isoflavones and saponins, to discriminate between closely related soybean genotypes. Genistin and group B saponins were identified as the key secondary metabolites correlated with salt tolerance. These individual metabolites may provide additional insight into the salt tolerance and adaptation of plants.

  2. Temperament Type Specific Metabolite Profiles of the Prefrontal Cortex and Serum in Cattle

    PubMed Central

    Brand, Bodo; Hadlich, Frieder; Brandt, Bettina; Schauer, Nicolas; Graunke, Katharina L.; Langbein, Jan; Repsilber, Dirk; Ponsuksili, Siriluk; Schwerin, Manfred

    2015-01-01

    In the past decade the number of studies investigating temperament in farm animals has increased greatly because temperament has been shown not only to affect handling but also reproduction, health and economically important production traits. However, molecular pathways underlying temperament and molecular pathways linking temperament to production traits, health and reproduction have yet to be studied in full detail. Here we report the results of metabolite profiling of the prefrontal cortex and serum of cattle with distinct temperament types that were performed to further explore their molecular divergence in the response to the slaughter procedure and to identify new targets for further research of cattle temperament. By performing an untargeted comprehensive metabolite profiling, 627 and 1097 metabolite features comprising 235 and 328 metabolites could be detected in the prefrontal cortex and serum, respectively. In total, 54 prefrontal cortex and 51 serum metabolite features were indicated to have a high relevance in the classification of temperament types by a sparse partial least square discriminant analysis. A clear discrimination between fearful/neophobic-alert, interested-stressed, subdued/uninterested-calm and outgoing/neophilic-alert temperament types could be observed based on the abundance of the identified relevant prefrontal cortex and serum metabolites. Metabolites with high relevance in the classification of temperament types revealed that the main differences between temperament types in the response to the slaughter procedure were related to the abundance of glycerophospholipids, fatty acyls and sterol lipids. Differences in the abundance of metabolites related to C21 steroid metabolism and oxidative stress indicated that the differences in the metabolite profiles of the four extreme temperament types could be the result of a temperament type specific regulation of molecular pathways that are known to be involved in the stress and fear response

  3. Temperament type specific metabolite profiles of the prefrontal cortex and serum in cattle.

    PubMed

    Brand, Bodo; Hadlich, Frieder; Brandt, Bettina; Schauer, Nicolas; Graunke, Katharina L; Langbein, Jan; Repsilber, Dirk; Ponsuksili, Siriluk; Schwerin, Manfred

    2015-01-01

    In the past decade the number of studies investigating temperament in farm animals has increased greatly because temperament has been shown not only to affect handling but also reproduction, health and economically important production traits. However, molecular pathways underlying temperament and molecular pathways linking temperament to production traits, health and reproduction have yet to be studied in full detail. Here we report the results of metabolite profiling of the prefrontal cortex and serum of cattle with distinct temperament types that were performed to further explore their molecular divergence in the response to the slaughter procedure and to identify new targets for further research of cattle temperament. By performing an untargeted comprehensive metabolite profiling, 627 and 1097 metabolite features comprising 235 and 328 metabolites could be detected in the prefrontal cortex and serum, respectively. In total, 54 prefrontal cortex and 51 serum metabolite features were indicated to have a high relevance in the classification of temperament types by a sparse partial least square discriminant analysis. A clear discrimination between fearful/neophobic-alert, interested-stressed, subdued/uninterested-calm and outgoing/neophilic-alert temperament types could be observed based on the abundance of the identified relevant prefrontal cortex and serum metabolites. Metabolites with high relevance in the classification of temperament types revealed that the main differences between temperament types in the response to the slaughter procedure were related to the abundance of glycerophospholipids, fatty acyls and sterol lipids. Differences in the abundance of metabolites related to C21 steroid metabolism and oxidative stress indicated that the differences in the metabolite profiles of the four extreme temperament types could be the result of a temperament type specific regulation of molecular pathways that are known to be involved in the stress and fear response.

  4. High-resolution mass spectrometry elucidates metabonate (false metabolite) formation from alkylamine drugs during in vitro metabolite profiling.

    PubMed

    Barbara, Joanna E; Kazmi, Faraz; Muranjan, Seema; Toren, Paul C; Parkinson, Andrew

    2012-10-01

    In vitro metabolite profiling and characterization experiments are widely employed in early drug development to support safety studies. Samples from incubations of investigational drugs with liver microsomes or hepatocytes are commonly analyzed by liquid chromatography/mass spectrometry for detection and structural elucidation of metabolites. Advanced mass spectrometers with accurate mass capabilities are becoming increasingly popular for characterization of drugs and metabolites, spurring changes in the routine workflows applied. In the present study, using a generic full-scan high-resolution data acquisition approach with a time-of-flight mass spectrometer combined with postacquisition data mining, we detected and characterized metabonates (false metabolites) in microsomal incubations of several alkylamine drugs. If a targeted approach to mass spectrometric detection (without full-scan acquisition and appropriate data mining) were employed, the metabonates may not have been detected, hence their formation underappreciated. In the absence of accurate mass data, the metabonate formation would have been incorrectly characterized because the detected metabonates manifested as direct cyanide-trapped conjugates or as cyanide-trapped metabolites formed from the parent drugs by the addition of 14 Da, the mass shift commonly associated with oxidation to yield a carbonyl. This study demonstrates that high-resolution mass spectrometry and the associated workflow is very useful for the detection and characterization of unpredicted sample components and that accurate mass data were critical to assignment of the correct metabonate structures. In addition, for drugs containing an alkylamine moiety, the results suggest that multiple negative controls and chemical trapping agents may be necessary to correctly interpret the results of in vitro experiments.

  5. NMR-Derived Models of Amidopyrine and its Metabolites Complexed to Rabbit Cytochrome P450 2B4 Reveal a Structural Mechanism of Sequential N-Dealkylation†

    PubMed Central

    Roberts, Arthur G.; Sjögren, Sara E. A.; Fomina, Nadezda; Vu, Kathy T.; Almutairi, Adah; Halpert, James R.

    2011-01-01

    To understand the molecular basis of sequential N-dealkylation by cytochrome P450 2B enzymes, the binding of amidopyrine (AP) as well as the metabolites of this reaction, desmethylamidopyrine (DMAP) and aminoantipyrine (AAP), were studied using the X-ray crystal structure of rabbit P450 2B4 and two nuclear magnetic resonance (NMR) techniques: saturation transfer difference (STD) spectroscopy and longitudinal (T1) relaxation NMR. STD NMR of AP and its metabolites bound to P450 2B4 were similar, suggesting that they occupy similar niches within the enzyme’s active site. The model-dependent relaxation rates (RM) determined from T1 relaxation NMR of AP and DMAP suggest that the N-linked methyl is closest to the heme. To determine the orientation(s) of AP and its metabolites within the P450 2B4 active site, distances calculated from the relaxation rates were used to constrain the metabolites to the X-ray crystal structure of P450 2B4. Simulated annealing of the complex revealed that the metabolites do indeed occupy similar hydrophobic pockets within the active site, while the N-linked methyl(s) are free to rotate between two binding modes. From these bound structures, a model of N-demethylation was developed, where the N-linked methyl functional groups rotate between catalytic and non-catalytic positions. This study is the first to provide a structural model of a drug and its metabolites complexed to a cytochrome P450 based on NMR and to provide a structural mechanism for how a drug can undergo sequential oxidations without unbinding. The rotation of the amide functional group might represent a common structural mechanism for N-dealkylation reactions for other drugs such as the local anesthetic lidocaine. PMID:21375273

  6. Obesity-related metabolite profiles of black women spanning the epidemiologic transition.

    PubMed

    Dugas, Lara R; Chorell, Elin; Plange-Rhule, Jacob; Lambert, Estelle V; Cao, Guichan; Cooper, Richard S; Layden, Brian T; Scholten, Denise; Olsson, Tommy; Luke, Amy; Goedecke, Julia H

    2016-03-01

    In developed countries, specific metabolites have been associated with obesity and metabolic diseases, e.g. type 2 diabetes. It is unknown whether a similar profile persists across populations of African-origin, at increased risk for obesity and related diseases. In a cross-sectional study of normal-weight and obese black women (33.3 ± 6.3 years) from the US (N = 69, 65 % obese), South Africa (SA, N = 97, 49 % obese) and Ghana (N = 82, 33 % obese) serum metabolite profiles were characterized via gas chromatography-time of flight/mass spectrometry. In US and SA women, BMI correlated with branched-chain and aromatic amino acids, as well as dopamine and aminoadipic acid. The relationship between BMI and lipid metabolites differed by site; BMI correlated positively with palmitoleic acid (16:1) in the US; negatively with stearic acid (18:0) in SA, and positively with arachidonic acid (20:4) in Ghana. BMI was also positively associated with sugar-related metabolites in the US; i.e. uric acid, and mannitol, and with glucosamine, glucoronic acid and mannitol in SA. While we identified a common amino acid metabolite profile associated with obesity in black women from the US and SA, we also found site-specific obesity-related metabolites suggesting that the local environment is a key moderator of obesity.

  7. Profiling methods to identify cold-regulated primary metabolites using gas chromatography coupled to mass spectrometry.

    PubMed

    Dethloff, Frederik; Erban, Alexander; Orf, Isabel; Alpers, Jessica; Fehrle, Ines; Beine-Golovchuk, Olga; Schmidt, Stefanie; Schwachtje, Jens; Kopka, Joachim

    2014-01-01

    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 single leaves from plants that were exposed to temperature stress experiments. The described method is modular. The modules include a rapid sampling and metabolic inactivation protocol for samples in a wide size range, a sample extraction procedure, a chemical derivatization step that is required to make the metabolite fraction amenable to gas chromatographic analysis, a routine GC-MS method, and finally the 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 method has two applications: (1) the rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification of cold-regulated metabolites by exact quantification using a GC-MS protocol with extended internal and external standardization.

  8. NMR-Based Metabolic Profiling of Field-Grown Leaves from Sugar Beet Plants Harbouring Different Levels of Resistance to Cercospora Leaf Spot Disease

    PubMed Central

    Sekiyama, Yasuyo; Okazaki, Kazuyuki; Kikuchi, Jun; Ikeda, Seishi

    2017-01-01

    Cercospora leaf spot (CLS) is one of the most serious leaf diseases for sugar beet (Beta vulgaris L.) worldwide. The breeding of sugar beet cultivars with both high CLS resistance and high yield is a major challenge for breeders. In this study, we report the nuclear magnetic resonance (NMR)-based metabolic profiling of field-grown leaves for a subset of sugar beet genotypes harbouring different levels of CLS resistance. Leaves were collected from 12 sugar beet genotypes at four time points: seedling, early growth, root enlargement, and disease development stages. 1H-NMR spectra of foliar metabolites soluble in a deuterium-oxide (D2O)-based buffer were acquired and subjected to multivariate analyses. A principal component analysis (PCA) of the NMR data from the sugar beet leaves shows clear differences among the growth stages. At the later time points, the sugar and glycine betaine contents were increased, whereas the choline content was decreased. The relationship between the foliar metabolite profiles and resistance level to CLS was examined by combining partial least squares projection to latent structure (PLS) or orthogonal PLS (OPLS) analysis and univariate analyses. It was difficult to build a robust model for predicting precisely the disease severity indices (DSIs) of each genotype; however, GABA and Gln differentiated susceptible genotypes (genotypes with weak resistance) from resistant genotypes (genotypes with resistance greater than a moderate level) before inoculation tests. The results suggested that breeders might exclude susceptible genotypes from breeding programs based on foliar metabolites profiled without inoculation tests, which require an enormous amount of time and effort. PMID:28134762

  9. Metabolite profiling of (14)C-omacetaxine mepesuccinate in plasma and excreta of cancer patients.

    PubMed

    Nijenhuis, Cynthia M; Lucas, Luc; Rosing, Hilde; Robertson, Philmore; Hellriegel, Edward T; Schellens, Jan H M; Beijnen, And Jos H

    2016-12-01

    Omacetaxine mepesuccinate (hereafter referred to as omacetaxine) is a protein translation inhibitor approved by the US Food and Drug Administration for adult patients with chronic myeloid leukemia with resistance and/or intolerance to two or more tyrosine kinase inhibitors. The objective was to investigate the metabolite profile of omacetaxine in plasma, urine and faeces samples collected up to 72 h after a single 1.25-mg/m(2) subcutaneous dose of (14)C-omacetaxine in cancer patients. High-performance liquid chromatography mass spectrometry (MS) (high resolution) in combination with off-line radioactivity detection was used for metabolite identification. In total, six metabolites of omacetaxine were detected. The reactions represented were mepesuccinate ester hydrolysis, methyl ester hydrolysis, pyrocatechol conversion from the 1,3-dioxole ring. Unchanged omacetaxine was the most prominent omacetaxine-related compound in plasma. In urine, unchanged omacetaxine was also dominant, together with 4'-DMHHT. In feces very little unchanged omacetaxine was found and the pyrocatechol metabolite of omacetaxine, M534 and 4'-desmethyl homoharringtonine (4'-DMHHT) was the most abundant metabolites. Omacetaxine was extensively metabolized, with subsequent renal and hepatic elimination of the metabolites. The low levels of the metabolites found in plasma indicate that the metabolites are unlikely to contribute materially to the efficacy and/or toxicity of omacetaxine.

  10. Tissue targeted metabonomics: metabolic profiling by microdialysis sampling and microcoil NMR.

    PubMed

    Price, Kristin E; Vandaveer, Shannon S; Lunte, Craig E; Larive, Cynthia K

    2005-08-10

    The concentration of low molecular weight compounds in tissues can yield valuable information about the metabolic state of an organism. Studies of changes in the metabolic state or metabonomics can reflect disease pathways, drug action, or toxicity. This research aims to develop a new approach, tissue targeted metabonomics. Microdialysis sampling and microcoil NMR analysis are employed to compare basal and ischemic metabolic states of various tissues (blood, brain, and heart) of Sprague-Dawley rats. Microdialysis sampling is localized, making the metabolic profile tissue specific. Coupling to NMR analysis is highly advantageous, because a complete metabolic profile is obtained in a single spectrum. However, small sample volumes and low analyte concentrations make analysis of microdialysis samples challenging. Microcoil NMR uses low sample volumes and has improved mass sensitivity, relative to standard 5 mm probes. The coupling of these techniques is a potentially powerful tool for metabonomics analysis.

  11. Evaluation of saffron (Crocus sativus L.) adulteration with plant adulterants by (1)H NMR metabolite fingerprinting.

    PubMed

    Petrakis, Eleftherios A; Cagliani, Laura R; Polissiou, Moschos G; Consonni, Roberto

    2015-04-15

    In the present work, a preliminary study for the detection of adulterated saffron and the identification of the adulterant used by means of (1)H NMR and chemometrics is reported. Authentic Greek saffron and four typical plant-derived materials utilised as bulking agents in saffron, i.e., Crocus sativus stamens, safflower, turmeric, and gardenia were investigated. A two-step approach, relied on the application of both OPLS-DA and O2PLS-DA models to the (1)H NMR data, was adopted to perform authentication and prediction of authentic and adulterated saffron. Taking into account the deficiency of established methodologies to detect saffron adulteration with plant adulterants, the method developed resulted reliable in assessing the type of adulteration and could be viable for dealing with extensive saffron frauds at a minimum level of 20% (w/w).

  12. Changes in the charged metabolite and sugar profiles of pasteurized and unpasteurized Japanese sake with storage.

    PubMed

    Sugimoto, Masahiro; Kaneko, Miku; Onuma, Hiromi; Sakaguchi, Yasuko; Mori, Masayo; Abe, Shinobu; Soga, Tomoyoshi; Tomita, Masaru

    2012-03-14

    Japanese sake (rice wine) is commonly heat treated (pasteurized) to maintain its quality. In this study, temporal changes in the metabolite profiles of pasteurized and unpasteurized sake were investigated during storage. Metabolomic analyses were conducted for eight sets of pasteurized and unpasteurized sake obtained from single process batches stored at 8 or 20 °C for 0, 1, 2, or 4 months. Capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry were used to obtain charged metabolite and sugar profiles, respectively. The total amino acid concentration decreased with storage, and the decrease was faster in pasteurized sake than in unpasteurized. The organic acid concentrations were relatively constant in both types of sake. Peptide and glucose concentrations increased and polysaccharide concentrations decreased in unpasteurized sake, while they were relatively constant in pasteurized sake. Rather than stabilizing the sake metabolite profile during storage, pasteurization results in characteristic changes compared to unpasteurized sake.

  13. Garlic sprouting is associated with increased antioxidant activity and concomitant changes in the metabolite profile.

    PubMed

    Zakarova, Alexandra; Seo, Ji Yeon; Kim, Hyang Yeon; Kim, Jeong Hwan; Shin, Jung-Hye; Cho, Kye Man; Lee, Choong Hwan; Kim, Jong-Sang

    2014-02-26

    Although garlic (Allium sativum) has been extensively studied for its health benefits, sprouted garlic has received little attention. We hypothesized that sprouting garlic would stimulate the production of various phytochemicals that improve health. Ethanolic extracts from garlic sprouted for different periods had variable antioxidant activities when assessed with in vitro assays, including the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay and the oxygen radical absorbance capacity assay. Extracts from garlic sprouted for 5 days had the highest antioxidant activity, whereas extracts from raw garlic had relatively low antioxidant activity. Furthermore, sprouting changed the metabolite profile of garlic: the metabolite profile of garlic sprouted for 5-6 days was distinct from the metabolite profile of garlic sprouted for 0-4 days, which is consistent with the finding that garlic sprouted for 5 days had the highest antioxidant activity. Therefore, sprouting may be a useful way to improve the antioxidant potential of garlic.

  14. Transcript and metabolite profiling in cell cultures of 18 plant species that produce benzylisoquinoline alkaloids.

    PubMed

    Farrow, Scott C; Hagel, Jillian M; Facchini, Peter J

    2012-05-01

    Benzylisoquinoline alkaloids (BIAs) are a large and diverse group of ~2500 specialized metabolites found predominantly in plants of the order Ranunculales. Research focused on BIA metabolism in a restricted number of plant species has identified many enzymes and cognate genes involved in the biosynthesis of compounds such as morphine, sanguinarine and berberine. However, the formation of most BIAs remains uncharacterized at the molecular biochemical level. Herein a compendium of sequence- and metabolite-profiling resources from 18 species of BIA-accumulating cell cultures was established, representing four related plant families. Our integrated approach consisted of the construction of EST libraries each containing approximately 3500 unigenes per species for a total of 58,787 unigenes. The EST libraries were manually triaged using known BIA-biosynthetic genes as queries to identify putative homologs with similar or potentially different functions. Sequence resources were analyzed in the context of the targeted metabolite profiles obtained for each cell culture using electrospray-ionization and collision-induced dissociation mass spectrometry. Fragmentation analysis was used for the identification or structural characterization coupled with the relative quantification of 72 BIAs, which establishes a key resource for future work on alkaloid biosynthesis. The metabolite profile obtained for each species provides a rational basis for the prediction of enzyme function in BIA metabolism. The metabolic frameworks assembled through the integration of transcript and metabolite profiles allow a comparison of BIA metabolism across several plant species and families. Taken together, these data represent an important tool for the discovery of BIA biosynthetic genes.

  15. [Determination of the profiles of secondary metabolites characteristic of Alternaria strains isolated from tomato].

    PubMed

    Benavidez Rozo, Martha Elizabeth; Patriarca, Andrea; Cabrera, Gabriela; Fernández Pinto, Virginia E

    2014-01-01

    Many Alternaria species have been studied for their ability to produce bioactive secondary metabolites, such as tentoxin (TEN), some of which have toxic properties. The main food contaminant toxins are tenuazonic acid, alternariol (AOH), alternariol monomethyl ether (AME), altenuene, and altertoxins i, ii and iii. To determine the profiles of secondary metabolites characteristic of Alternaria strains isolated from tomato for their chemotaxonomic classification. The profiles of secondary metabolites were determined by HPLC MS. The Alternaria isolates obtained from spoiled tomatoes belong, according to their morphological characteristics, to the species groups Alternaria alternata, Alternaria tenuissima and Alternaria arborescens, with A. tenuissima being the most frequent. The most frequent profiles of secondary metabolites belonging to the species groups A. alternata (AOH, AME, TEN), A. tenuissima (AOH, AME, TEN, tenuazonic acid) and A. arborescens (AOH, AME, TEN, tenuazonic acid) were determined, with some isolates of the latter being able to synthesize AAL toxins. Secondary metabolite profiles are a useful tool for the differentiation of small spored Alternaria isolates not easily identifiable by their morphological characteristics. Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

  16. COVALENT BINDING OF REDUCED METABOLITES OF [15N3] TNT TO SOIL ORGANIC MATTER DURING A BIOREMEDIATION PROCESS ANALYZED BY 15N NMR SPECTROSCOPY. (R826646)

    EPA Science Inventory

    Evidence is presented for the covalent binding of
    biologically reduced metabolites of 2,4,6-15N3-trinitrotoluene
    (TNT) to different soil fractions (humic acids, fulvic
    acids, and humin) using liquid 15N NMR spectroscopy. A
    silylation p...

  17. COVALENT BINDING OF REDUCED METABOLITES OF [15N3] TNT TO SOIL ORGANIC MATTER DURING A BIOREMEDIATION PROCESS ANALYZED BY 15N NMR SPECTROSCOPY. (R826646)

    EPA Science Inventory

    Evidence is presented for the covalent binding of
    biologically reduced metabolites of 2,4,6-15N3-trinitrotoluene
    (TNT) to different soil fractions (humic acids, fulvic
    acids, and humin) using liquid 15N NMR spectroscopy. A
    silylation p...

  18. 1H-NMR and 13C-NMR lipid profiles of human renal tissues.

    PubMed

    Tugnoli, V; Bottura, G; Fini, G; Reggiani, A; Tinti, A; Trinchero, A; Tosi, M R

    2003-01-01

    Lipids from human renal tissues are studied by means of (1)H- and (13)C-NMR spectroscopy. The total lipid fractions obtained from healthy kidneys, malignant renal cell carcinomas, and benign oncocytomas are characterized and analyzed to elucidate the main differences between the functional and neoplastic tissues. In all cases the lipid components are well identified. The healthy kidney is characterized by high amounts of triglycerides and the presence of cholesterol in its free form. On the contrary, renal cell carcinomas contain high amounts of cholesterol that are almost completely esterified as oleate, suggesting an intracellular localization of the cholesteryl esters synthesis. Cholesteryl esters are considered markers of renal cell carcinomas, thus supporting recent theories that these compounds play a leading role in cell proliferation. Oncocytomas are particularly rich in phosphatidylcholine and, analogous to the healthy kidney, are completely lacking in cholesteryl esters. Healthy kidneys and oncocytomas appear to have other similarities if compared with renal cell carcinomas: a very high fatty acyl/cholesterol ratio, the presence of dolichols, and a higher grade of unsaturation. The (13)C data suggest a new method for the direct evaluation of the saturated/unsaturated fatty acyl ratio.

  19. Profiling of carotenoids in tomato juice by one- and two-dimensional NMR.

    PubMed

    Tiziani, Stefano; Schwartz, Steven J; Vodovotz, Yael

    2006-08-09

    Epidemiological data have shown a link between dietary intake of tomatoes and tomato products (rich in carotenoids) and a decreased risk of chronic diseases. The carotenoid profile in tomato products depends on tomato variety as well as the thermal conditions used in processing. The final carotenoid profile may affect the bioaccessibility and bioavailability of these biomolecules. Therefore, nondestructive, reliable methods are needed to characterize the structural and stereochemical variation of carotenoids. CDCl(3) rapid extraction was used to extract carotenoids from tomato juice as an alternative rapid procedure that minimizes solvents and time consumption prior to NMR analysis. The profile of these biomolecules was characterized by application of high-resolution multidimensional NMR techniques using a cryogenic probe. The combination of homonuclear and heteronuclear two-dimensional NMR techniques served to identify (all-E)-, (5Z)-, (9Z)-, and (13Z)-lycopene isomers and other carotenoids such as (all-E)-beta-carotene and (15Z)-phytoene dissolved in the extracted lipid mixture. The use of one-dimensional NMR enabled the rapid identification of lycopene isomers, thereby minimizing further isomerization of (all-E)-lycopene as compared to HPLC data. On the basis of the assignments accomplished, the carotenoid profile of typical tomato juice was successfully determined with minimal purification procedures.

  20. Temperature dependence of (1)H NMR chemical shifts and its influence on estimated metabolite concentrations.

    PubMed

    Wermter, Felizitas C; Mitschke, Nico; Bock, Christian; Dreher, Wolfgang

    2017-07-06

    Temperature dependent chemical shifts of important brain metabolites measured by localised (1)H MRS were investigated to test how the use of incorrect prior knowledge on chemical shifts impairs the quantification of metabolite concentrations. Phantom measurements on solutions containing 11 metabolites were performed on a 7 T scanner between 1 and 43 °C. The temperature dependence of the chemical shift differences was fitted by a linear model. Spectra were simulated for different temperatures and analysed by the AQSES program (jMRUI 5.2) using model functions with chemical shift values for 37 °C. Large differences in the temperature dependence of the chemical shift differences were determined with a maximum slope of about ±7.5 × 10(-4) ppm/K. For 32-40 °C, only minor quantification errors resulted from using incorrect chemical shifts, with the exception of Cr and PCr. For 1-10 °C considerable quantification errors occurred if the temperature dependence of the chemical shifts was neglected. If (1)H MRS measurements are not performed at 37 °C, for which the published chemical shift values have been determined, the temperature dependence of chemical shifts should be considered to avoid systematic quantification errors, particularly for measurements on animal models at lower temperatures.

  1. Metabolite Variation in Lean and Obese Streptozotocin (STZ)-Induced Diabetic Rats via (1)H NMR-Based Metabolomics Approach.

    PubMed

    Abu Bakar Sajak, Azliana; Mediani, Ahmed; Maulidiani; Ismail, Amin; Abas, Faridah

    2016-12-19

    Diabetes mellitus (DM) is considered as a complex metabolic disease because it affects the metabolism of glucose and other metabolites. Although many diabetes studies have been conducted in animal models throughout the years, the pathogenesis of this disease, especially between lean diabetes (ND + STZ) and obese diabetes (OB + STZ), is still not fully understood. In this study, the urine from ND + STZ, OB + STZ, lean/control (ND), and OB + STZ rats were collected and compared by using (1)H NMR metabolomics. The results from multivariate data analysis (MVDA) showed that the diabetic groups (ND + STZ and OB + STZ) have similarities and dissimilarities for a certain level of metabolites. Differences between ND + STZ and OB + STZ were particularly noticeable in the synthesis of ketone bodies, branched-chain amino acid (BCAA), and sensitivity towards the oral T2DM diabetes drug metformin. This finding suggests that the ND + STZ group was more similar to the T1DM model and OB + STZ to the T2DM model. In addition, we also managed to identify several pathways and metabolism aspects shared by obese (OB) and OB + STZ. The results from this study are useful in developing drug target-based research as they can increase understanding regarding the cause and effect of DM.

  2. Separation of small metabolites and lipids in spectra from biopsies by diffusion-weighted HR-MAS NMR: a feasibility study.

    PubMed

    Diserens, G; Vermathen, M; Precht, C; Broskey, N T; Boesch, C; Amati, F; Dufour, J-F; Vermathen, P

    2015-01-07

    High Resolution Magic Angle Spinning (HR-MAS) NMR allows metabolic characterization of biopsies. HR-MAS spectra from tissues of most organs show strong lipid contributions that are overlapping metabolite regions, which hamper metabolite estimation. Metabolite quantification and analysis would benefit from a separation of lipids and small metabolites. Generally, a relaxation filter is used to reduce lipid contributions. However, the strong relaxation filter required to eliminate most of the lipids also reduces the signals for small metabolites. The aim of our study was therefore to investigate different diffusion editing techniques in order to employ diffusion differences for separating lipid and small metabolite contributions in the spectra from different organs for unbiased metabonomic analysis. Thus, 1D and 2D diffusion measurements were performed, and pure lipid spectra that were obtained at strong diffusion weighting (DW) were subtracted from those obtained at low DW, which include both small metabolites and lipids. This subtraction yielded almost lipid free small metabolite spectra from muscle tissue. Further improved separation was obtained by combining a 1D diffusion sequence with a T2-filter, with the subtraction method eliminating residual lipids from the spectra. Similar results obtained for biopsies of different organs suggest that this method is applicable in various tissue types. The elimination of lipids from HR-MAS spectra and the resulting less biased assessment of small metabolites have potential to remove ambiguities in the interpretation of metabonomic results. This is demonstrated in a reproducibility study on biopsies from human muscle.

  3. Metabolite Profiling of Italian Tomato Landraces with Different Fruit Types

    PubMed Central

    Baldina, Svetlana; Picarella, Maurizio E.; Troise, Antonio D.; Pucci, Anna; Ruggieri, Valentino; Ferracane, Rosalia; Barone, Amalia; Fogliano, Vincenzo; Mazzucato, Andrea

    2016-01-01

    Increased interest toward traditional tomato varieties is fueled by the need to rescue desirable organoleptic traits and to improve the quality of fresh and processed tomatoes in the market. In addition, the phenotypic and genetic variation preserved in tomato landraces represents a means to understand the genetic basis of traits related to health and organoleptic aspects and improve them in modern varieties. To establish a framework for this approach, we studied the content of several metabolites in a panel of Italian tomato landraces categorized into three broad fruit type classes (flattened/ribbed, pear/oxheart, round/elongate). Three modern hybrids, corresponding to the three fruit shape typologies, were included as reference. Red ripe fruits were morphologically characterized and biochemically analyzed for their content in glycoalkaloids, phenols, amino acids, and Amadori products. The round/elongate types showed a higher content in glycoalkaloids, whereas flattened types had higher levels of phenolic compounds. Flattened tomatoes were also rich in total amino acids and in particular in glutamic acid. Multivariate analysis of amino acid content clearly separated the three classes of fruit types. Making allowance of the very low number of genotypes, phenotype-marker relationships were analyzed after retrieving single nucleotide polymorphisms (SNPs) among the landraces available in the literature. Sixty-six markers were significantly associated with the studied traits. The positions of several of these SNPs showed correspondence with already described genomic regions and QTLs supporting the reliability of the association. Overall the data indicated that significant changes in quality-related metabolites occur depending on the genetic background in traditional tomato germplasm, frequently according to specific fruit shape categories. Such a variability is suitable to harness association mapping for metabolic quality traits using this germplasm as an experimental

  4. Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.

    PubMed

    García-Cañaveras, Juan Carlos; López, Silvia; Castell, José Vicente; Donato, M Teresa; Lahoz, Agustín

    2016-02-01

    MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.

  5. Metabolite profiles of the biocontrol yeast Pichia anomala J121 grown under oxygen limitation.

    PubMed

    Fredlund, E; Broberg, A; Boysen, M E; Kenne, L; Schnürer, J

    2004-04-01

    The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (A), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (A), but continued to be produced in (B). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (B). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (B) than in (A), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.

  6. Phenolic and heterocyclic metabolite profiles of the grapevine pathogen Eutypa lata.

    PubMed

    Mahoney, Noreen; Lardner, Richard; Molyneux, Russell J; Scott, Eileen S; Smith, Leverett R; Schoch, Thomas K

    2003-09-01

    The ascomycete Eutypa lata is the causative agent of eutypa dieback in grapevines, a serious economic problem in major wine grape producing areas. In order to develop a predictive, non-destructive assay for early detection of fungal infection, the phenolic metabolite profiles of 11 strains of E. lata grown on four different artificial growth media were analyzed by HPLC and their variability compared with growth on Cabernet Sauvignon grapevine wood and wood extracts. Six compounds were generally produced in significant amounts, namely eutypinol, eulatachromene, and eutypine and its benzofuran cyclization product, together with siccayne and eulatinol. The two most widely distributed and abundant metabolites were eutypinol and eulatachromene, which were present in 8 of the strains grown on grapewood aqueous extract fortified with sucrose. Metabolite production on grapevine extract was greatly enhanced relative to the artificial media, indicating that this native substrate provides optimal conditions and a more representative profile of the metabolites produced in the natural disease state. The primary metabolites were tested in a grapeleaf disc bioassay to establish their relative toxicity. Neither eutypinol nor siccayne were phytotoxic; eulatachromene, eulatinol, eutypine, and the benzofuran exhibited necrotic effects in the bioassay. The results indicate that eutypa dieback may be caused by several E. lata metabolites rather than a single compound.

  7. Detection of hepatotoxicity potential with metabolite profiling (metabolomics) of rat plasma.

    PubMed

    Mattes, W; Davis, K; Fabian, E; Greenhaw, J; Herold, M; Looser, R; Mellert, W; Groeters, S; Marxfeld, H; Moeller, N; Montoya-Parra, G; Prokoudine, A; van Ravenzwaay, B; Strauss, V; Walk, T; Kamp, H

    2014-11-04

    While conventional parameters used to detect hepatotoxicity in drug safety assessment studies are generally informative, the need remains for parameters that can detect the potential for hepatotoxicity at lower doses and/or at earlier time points. Previous work has shown that metabolite profiling (metabonomics/metabolomics) can detect signals of potential hepatotoxicity in rats treated with doxorubicin at doses that do not elicit hepatotoxicity as monitored with conventional parameters. The current study extended this observation to the question of whether such signals could be detected in rats treated with compounds that can elicit hepatotoxicity in humans (i.e., drug-induced liver injury, DILI) but have not been reported to do so in rats. Nine compounds were selected on the basis of their known DILI potential, with six other compounds chosen as negative for DILI potential. A database of rat plasma metabolite profiles, MetaMap(®)Tox (developed by metanomics GmbH and BASF SE) was used for both metabolite profiles and mode of action (MoA) metabolite signatures for a number of known toxicities. Eight of the nine compounds with DILI potential elicited metabolite profiles that matched with MoA patterns of various rat liver toxicities, including cholestasis, oxidative stress, acetaminophen-type toxicity and peroxisome proliferation. By contrast, only one of the six non-DILI compounds showed a weak match with rat liver toxicity. These results suggest that metabolite profiling may indeed have promise to detect signals of hepatotoxicity in rats treated with compounds having DILI potential. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Plasma-metabolite-biomarkers for the therapeutic response in depressed patients by the traditional Chinese medicine formula Xiaoyaosan: A (1)H NMR-based metabolomics approach.

    PubMed

    Liu, Cai-Chun; Wu, Yan-Fei; Feng, Guang-Ming; Gao, Xiao-Xia; Zhou, Yu-Zhi; Hou, Wen-Jing; Qin, Xue-Mei; Du, Guan-Hua; Tian, Jun-Sheng

    2015-10-01

    Depression is one of the most prevalent and serious mental disorders. Xiaoyaosan, a well-known Chinese prescription, has been widely used for the treatment of depression in China. Both clinical studies and animal experiments indicate that Xiaoyaosan has an obvious antidepressant activity. Additionally, a large number of candidate biomarkers have emerged that can be used for early disease detection and for monitoring ongoing treatment response to therapy because of their correlations with the characteristics of the disease. However, there have been few reports on biomarkers that measure the treatment response to the clinical use of Xiaoyaosan using a metabolomics approach. The current study is aimed at discovering biomarkers and biochemical pathways to facilitate the diagnosis of depression and the efficient evaluation of Xiaoyaosan using plasma metabolomics profiles based on (1)H NMR. Sixteen depressed patients diagnosed by standard methods (HAMD and CGI-SI) and sixteen healthy volunteers were recruited. (1)H NMR-based metabolomics techniques and multivariate statistical methods were used to analyze the plasma metabolites of the depressed patients before and after treatment and to compare them with healthy controls. The plasma levels of trimethylamine oxide, glutamine and lactate in depressed patients increased significantly (p≤0.05) compared with healthy controls, whereas the levels of phenylalanine, valine, alanine, glycine, leucine, citrate, choline, lipids and glucose decreased significantly (p≤0.05). Additionally, alanine, choline, trimethylamine oxide, glutamine, lactate and glucose were returned to normal levels after Xiaoyaosan treatment. These statistically significant perturbations are involved in energy metabolism, amino acid metabolism and gut microbiota metabolism. Additional experimentation with gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) is required to confirm our findings. Application of these

  9. Kidney Tissue Targeted Metabolic Profiling of Unilateral Ureteral Obstruction Rats by NMR

    PubMed Central

    Li, Zhenyu; Li, Aiping; Gao, Jining; Li, Hong; Qin, Xuemei

    2016-01-01

    Renal interstitial fibrosis is a common pathological process in the progression of kidney disease. A nuclear magnetic resonance (NMR) based metabolomic approach was used to analyze the kidney tissues of rats with renal interstitial fibrosis (RIF), induced by unilateral ureteral obstruction (UUO). The combination of a variety of statistical methods were used to screen out 14 significantly changed potential metabolites, which are related with multiple biochemical processes including amino acid metabolism, adenine metabolism, energy metabolism, osmolyte change and induced oxidative stress. The exploration of the contralateral kidneys enhanced the understanding of the disease, which was also supported by serum biochemistry and kidney histopathology results. In addition, the pathological parameters (clinical chemistry, histological and immunohistochemistry results) were correlated with the significantly changed differential metabolites related with RIF. This study showed that targeted tissue metabolomic analysis can be used as a useful tool to understand the mechanism of the disease and provide a novel insight in the pathogenesis of RIF. PMID:27695416

  10. Consumption of pasteurized human lysozyme transgenic goats’ milk alters serum metabolite profile in young pigs

    PubMed Central

    Brundige, Dottie R.; Maga, Elizabeth A.; Klasing, Kirk C.

    2009-01-01

    Nutrition, bacterial composition of the gastrointestinal tract, and general health status can all influence the metabolic profile of an organism. We previously demonstrated that feeding pasteurized transgenic goats’ milk expressing human lysozyme (hLZ) can positively impact intestinal morphology and modulate intestinal microbiota composition in young pigs. The objective of this study was to further examine the effect of consuming hLZ-containing milk on young pigs by profiling serum metabolites. Pigs were placed into two groups and fed a diet of solid food and either control (non-transgenic) goats’ milk or milk from hLZ-transgenic goats for 6 weeks. Serum samples were collected at the end of the feeding period and global metabolite profiling was performed. For a total of 225 metabolites (160 known, 65 unknown) semi-quantitative data was obtained. Levels of 18 known and 4 unknown metabolites differed significantly between the two groups with the direction of change in 13 of the 18 known metabolites being almost entirely congruent with improved health status, particularly in terms of the gastrointestinal tract health and immune response, with the effects of the other five being neutral or unknown. These results further support our hypothesis that consumption of hLZ-containing milk is beneficial to health. PMID:19847666

  11. Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatography.

    PubMed

    Callahan, Damien L; De Souza, David; Bacic, Antony; Roessner, Ute

    2009-07-01

    Highly polar metabolites, such as sugars and most amino acids are not retained by conventional RP LC columns. Without sufficient retention low concentration compounds are not detected due ion suppression and structural isomers are not resolved. In contrast, hydrophilic interaction chromatography (HILIC) and aqueous normal phase chromatography (ANP) retain compounds based on their hydrophilicity and therefore provides a means of separating highly polar compounds. Here, an ANP method based on the diamond hydride stationary phase is presented for profiling biological small molecules by LC. A rapid separation system based upon a fast gradient that delivers reproducible chromatography is presented. Approximately 1000 compounds were reproducibly detected in human urine samples and clear differences between these samples were identified. This chromatography was also applied to xylem fluid from soyabean (Glycine max) plants to which 400 compounds were detected. This method greatly increases the metabolite coverage over RP-only metabolite profiling in biological samples. We show that both forms of chromatography are necessary for untargeted comprehensive metabolite profiling and that the diamond hydride stationary phase provides a good option for polar metabolite analysis.

  12. Comparative metabolite profiling of Solanum tuberosum against six wild Solanum species with Colorado potato beetle resistance.

    PubMed

    Tai, Helen H; Worrall, Kraig; Pelletier, Yvan; De Koeyer, David; Calhoun, Larry A

    2014-09-10

    The Colorado potato beetle Leptinotarsa decemlineata (Say) (CPB) is a coleopteran herbivore that feeds on the foliage on Solanum species, in particular, potato. Six resistant wild Solanum species were identified, and two of these species had low levels of glycoalkaloids. Comparative analysis of the untargeted metabolite profiles of the foliage using UPLC-qTOF-MS was done to find metabolites shared between the wild species but not with Solanum tuberosum (L.) to identify resistance-related metabolites. It was found that only S. tuberosum produced the triose glycoalkaloids solanine and chaconine. Instead, the six wild species produced glycoalkaloids that shared in common tetrose sugar side chains. Additionally, there were non-glycoalkaloid metabolites associated with resistance including hydroxycoumarin and a phenylpropanoid, which were produced in all wild species but not in S. tuberosum.

  13. Semiautomated Alignment of High-Throughput Metabolite Profiles with Chemometric Tools

    PubMed Central

    Xiao, Zi-dan; Liang, Yi-zeng; Chau, Foo-tim; Chan, Hoi-yan

    2017-01-01

    The rapid increase in the use of metabolite profiling/fingerprinting techniques to resolve complicated issues in metabolomics has stimulated demand for data processing techniques, such as alignment, to extract detailed information. In this study, a new and automated method was developed to correct the retention time shift of high-dimensional and high-throughput data sets. Information from the target chromatographic profiles was used to determine the standard profile as a reference for alignment. A novel, piecewise data partition strategy was applied for the determination of the target components in the standard profile as markers for alignment. An automated target search (ATS) method was proposed to find the exact retention times of the selected targets in other profiles for alignment. The linear interpolation technique (LIT) was employed to align the profiles prior to pattern recognition, comprehensive comparison analysis, and other data processing steps. In total, 94 metabolite profiles of ginseng were studied, including the most volatile secondary metabolites. The method used in this article could be an essential step in the extraction of information from high-throughput data acquired in the study of systems biology, metabolomics, and biomarker discovery. PMID:28168083

  14. Leveraging non-targeted metabolite profiling via statistical genomics

    USDA-ARS?s Scientific Manuscript database

    One of the challenges of systems biology is to integrate multiple sources of data in order to build a cohesive view of the system of study. Here we describe the mass spectrometry based profiling of maize kernels, a model system for genomic studies and a cornerstone of the agroeconomy. Using a networ...

  15. Ameliorating effects of Mango (Mangifera indica L.) fruit on plasma ethanol level in a mouse model assessed with H-NMR based metabolic profiling.

    PubMed

    Kim, So-Hyun; K Cho, Somi; Min, Tae-Sun; Kim, Yujin; Yang, Seung-Ok; Kim, Hee-Su; Hyun, Sun-Hee; Kim, Hana; Kim, Young-Suk; Choi, Hyung-Kyoon

    2011-05-01

    The ameliorating effects of Mango (Mangifera indica L.) flesh and peel samples on plasma ethanol level were investigated using a mouse model. Mango fruit samples remarkably decreased mouse plasma ethanol levels and increased the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase. The (1)H-NMR-based metabolomic technique was employed to investigate the differences in metabolic profiles of mango fruits, and mouse plasma samples fed with mango fruit samples. The partial least squares-discriminate analysis of (1)H-NMR spectral data of mouse plasma demonstrated that there were clear separations among plasma samples from mice fed with buffer, mango flesh and peel. A loading plot demonstrated that metabolites from mango fruit, such as fructose and aspartate, might stimulate alcohol degradation enzymes. This study suggests that mango flesh and peel could be used as resources for functional foods intended to decrease plasma ethanol level after ethanol uptake.

  16. The metabolic profile of lemon juice by proton HR-MAS NMR: the case of the PGI Interdonato Lemon of Messina.

    PubMed

    Cicero, Nicola; Corsaro, Carmelo; Salvo, Andrea; Vasi, Sebastiano; Giofré, Salvatore V; Ferrantelli, Vincenzo; Di Stefano, Vita; Mallamace, Domenico; Dugo, Giacomo

    2015-01-01

    We have studied by means of High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) the metabolic profile of the famous Sicilian lemon known as 'Interdonato Lemon of Messina PGI'. The PGI Interdonato Lemon of Messina possesses high organoleptic and healthy properties and is recognised as one of the most nutrient fruits. In particular, some of its constituents are actively studied for their chemo-preventive and therapeutic properties. In this paper, we have determined by means of HR-MAS NMR spectroscopy the molar concentration of the main metabolites constituent the juice of PGI Interdonato Lemon of Messina in comparison with that of the not-PGI Interdonato Lemon of Turkey. Our aim is to develop an analytical technique, in order to determine a metabolic fingerprint able to reveal commercial frauds in national and international markets.

  17. Ameliorating effects of Mango (Mangifera indica L.) fruit on plasma ethanol level in a mouse model assessed with 1H-NMR based metabolic profiling

    PubMed Central

    Kim, So-Hyun; K. Cho, Somi; Min, Tae-Sun; Kim, Yujin; Yang, Seung-Ok; Kim, Hee-Su; Hyun, Sun-Hee; Kim, Hana; Kim, Young-Suk; Choi, Hyung-Kyoon

    2011-01-01

    The ameliorating effects of Mango (Mangifera indica L.) flesh and peel samples on plasma ethanol level were investigated using a mouse model. Mango fruit samples remarkably decreased mouse plasma ethanol levels and increased the activities of alcohol dehydrogenase and acetaldehyde dehydrogenase. The 1H-NMR-based metabolomic technique was employed to investigate the differences in metabolic profiles of mango fruits, and mouse plasma samples fed with mango fruit samples. The partial least squares-discriminate analysis of 1H-NMR spectral data of mouse plasma demonstrated that there were clear separations among plasma samples from mice fed with buffer, mango flesh and peel. A loading plot demonstrated that metabolites from mango fruit, such as fructose and aspartate, might stimulate alcohol degradation enzymes. This study suggests that mango flesh and peel could be used as resources for functional foods intended to decrease plasma ethanol level after ethanol uptake. PMID:21562641

  18. Classification of terverticillate penicillia based on profiles of mycotoxins and other secondary metabolites.

    PubMed Central

    Frisvad, J C; Filtenborg, O

    1983-01-01

    Strains of available terverticillate penicillium species and varieties were analyzed for profiles of known mycotoxins and other secondary metabolites produced on Czapek yeast autolysate agar (intracellular metabolites) and yeast extract-sucrose agar (extracellular metabolites) by using simple thin-layer chromatography screening techniques. These strains (2,473 in all) could be classified into 29 groups based on profiles of secondary metabolites. Most of these profiles of secondary metabolites were distinct, containing several biosynthetically different mycotoxins and unknown metabolites characterized by distinct colors and retardation factors on thin-layer chromatography plates. Some species (P. italicum and P. atramentosum) only produced one or two metabolites by the simple screening methods. The 29 groups based on profiles of secondary metabolites were known species or subgroups thereof. These species and subgroups were independently identifiable by using morphological and physiological criteria. The species accepted, the number of isolates in each species investigated, and the mycotoxins they produced were: P. atramentosum, 4; P. aurantiogriseum, 510 (group I: penicillic acid and S-toxin and group II: penicillic acid, penitrem A [low frequency], terrestric acid [low frequency], viomellein, and xanthomegnin); P. brevicompactum, 81 (brevianamid A and mycophenolic acid); P. camembertii group I, 38, and group II, 114 (cyclopiazonic acid); P. chrysogenum, 87 (penicillin, roquefortine C, and PR-toxin); P. claviforme, 4 (patulin and roquefortine C); P. clavigerum, 4 (penitrem A); P. concentricum group I, 10 (griseofulvin and roquefortine C), and group II, 3 (patulin and roquefortine C); P. crustosum, 123 (penitrem A, roquefortine C, and terrestric acid); P. echinulatum, 13; P. expansum, 91 (citrinin, patulin, and roquefortine C); P. granulatum, 6 (patulin, penitrem A, and roquefortine C [traces]); P. griseofulvum, 21 (cyclopiazonic acid, griseofulvin, patulin, and

  19. (1)H-(13)C NMR-Based Profiling of Biotechnological Starch Utilization.

    PubMed

    Sundekilde, Ulrik K; Meier, Sebastian

    2016-10-04

    Starch is used in food- and nonfood applications as a renewable and degradable source of carbon and energy. Insight into the chemical detail of starch degradation remains challenging as the starch constituents amylose and amylopectin are homopolymers. We show that considerable molecular detail of starch fragmentation can be obtained from multivariate analysis of spectral features in optimized (1)H-(13)C NMR spectroscopy of starch fragments to identify relevant features that distinguish processes in starch utilization. As a case study, we compare the profiles of starch fragments in commercial beer samples. Spectroscopic profiles of homooligomeric starch fragments can be excellent indicators of process conditions. In addition, differences in the structure and composition of starch fragments have predictive value for downstream process output such as ethanol production from starch. Thus, high-resolution (1)H-(13)C NMR spectroscopic profiles of homooligomeric fragment mixtures in conjunction with chemometric methods provide a useful addition to the analytical chemistry toolbox of biotechnological starch utilization.

  20. Investigating correlations in the altered metabolic profiles of obese and diabetic subjects in a South Indian Asian population using an NMR-based metabolomic approach.

    PubMed

    Gogna, Navdeep; Krishna, Murahari; Oommen, Anup Mammen; Dorai, Kavita

    2015-02-01

    It is well known that obesity/high body mass index (BMI) plays a key role in the evolution of insulin resistance and type-2 diabetes mellitus (T2DM). However, the exact mechanism underlying its contribution is still not fully understood. This work focuses on an NMR-based metabolomic investigation of the serum profiles of diabetic, obese South Indian Asian subjects. (1)H 1D and 2D NMR experiments were performed to profile the altered metabolic patterns of obese diabetic subjects and multivariate statistical methods were used to identify metabolites that contributed significantly to the differences in the samples of four different subject groups: diabetic and non-diabetic with low and high BMIs. Our analysis revealed that the T2DM-high BMI group has higher concentrations of saturated fatty acids, certain amino acids (leucine, isoleucine, lysine, proline, threonine, valine, glutamine, phenylalanine, histidine), lactic acid, 3-hydroxybutyric acid, choline, 3,7-dimethyluric acid, pantothenic acid, myoinositol, sorbitol, glycerol, and glucose, as compared to the non-diabetic-low BMI (control) group. Of these 19 identified significant metabolites, the levels of saturated fatty acids, lactate, valine, isoleucine, and phenylalanine are also higher in obese non-diabetic subjects as compared to control subjects, implying that this set of metabolites could be identified as potential biomarkers for the onset of diabetes in subjects with a high BMI. Our work validates the utility of NMR-based metabolomics in conjunction with multivariate statistical analysis to provide insights into the underlying metabolic pathways that are perturbed in diabetic subjects with a high BMI.

  1. Anionic metabolite profiling by capillary electrophoresis-mass spectrometry using a noncovalent polymeric coating. Orange juice and wine as case studies.

    PubMed

    Acunha, Tanize; Simó, Carolina; Ibáñez, Clara; Gallardo, Alberto; Cifuentes, Alejandro

    2016-01-08

    In several metabolomic studies, it has already been demonstrated that capillary electrophoresis hyphenated to mass spectrometry (CE-MS) can detect an important group of highly polar and ionized metabolites that are overseen by techniques such as NMR, LC-MS and GC-MS, providing complementary information. In this work, we present a strategy for anionic metabolite profiling by CE-MS using a cationic capillary coating. The polymer, abbreviated as PTH, is composed of a poly-(N,N,N',N'-tetraethyldiethylenetriamine, N-(2-hydroxypropyl) methacrylamide, TEDETAMA-co-HPMA (50:50) copolymer. A CE-MS method based on PTH-coating was optimized for the analysis of a group of 16 standard anionic metabolites. Separation was achieved within 12min, with high separation efficiency (up to 92,000 theoretical plates per meter), and good repeatability, namely, relative standard deviation values for migration times and peak areas were below 0.2 and 2.1%, respectively. The optimized method allowed the detection of 87 metabolites in orange juice and 142 metabolites in red wine, demonstrating the good possibilities of this strategy for metabolomic applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt.

    PubMed

    Settachaimongkon, Sarn; Nout, M J Robert; Antunes Fernandes, Elsa C; Hettinga, Kasper A; Vervoort, Jacques M; van Hooijdonk, Toon C M; Zwietering, Marcel H; Smid, Eddy J; van Valenberg, Hein J F

    2014-05-02

    Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Host plant secondary metabolite profiling shows a complex, strain-dependent response of maize to plant growth-promoting rhizobacteria of the genus Azospirillum.

    PubMed

    Walker, Vincent; Bertrand, Cédric; Bellvert, Floriant; Moënne-Loccoz, Yvan; Bally, René; Comte, Gilles

    2011-01-01

    Most Azospirillum plant growth-promoting rhizobacteria (PGPR) benefit plant growth through source effects related to free nitrogen fixation and/or phytohormone production, but little is known about their potential effects on plant physiology. These effects were assessed by comparing the early impacts of three Azospirillum inoculant strains on secondary metabolite profiles of two different maize (Zea mays) cultivars. After 10d of growth in nonsterile soil, maize methanolic extracts were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) and secondary metabolites identified by liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). Seed inoculation resulted in increased shoot biomass (and also root biomass with one strain) of hybrid PR37Y15 but had no stimulatory effect on hybrid DK315. In parallel, Azospirillum inoculation led to major qualitative and quantitative modifications of the contents of secondary metabolites, especially benzoxazinoids, in the maize plants. These modifications depended on the PGPR strain×plant cultivar combination. Thus, Azospirillum inoculation resulted in early, strain-dependent modifications in the biosynthetic pathways of benzoxazine derivatives in maize in compatible interactions. This is the first study documenting a PGPR effect on plant secondary metabolite profiles, and suggests the establishment of complex interactions between Azospirillum PGPR and maize.

  4. Metabolite identification and profiling in drug design: current practice and future directions.

    PubMed

    Zhang, Zhoupeng; Zhu, Mingshe; Tang, Wei

    2009-01-01

    Drug metabolism and pharmacokinetics (DMPK) represents a critical component in support of drug discovery and development. This is because the therapeutic efficacy of a drug is dependent on its exposure which in turn is dictated in part by metabolic stability of the molecule. In addition, drug metabolism may lead to the formation of metabolites that can either be pharmacologically active or elicit adverse effect. On this basis, metabolite identification and profiling have become a routine exercise during lead optimization and subsequent development processes. The current communication provides an overview on the account of metabolite identification and profiling in support of drug design with an additional emphasis on the commonly used analytical techniques. The discussion is supported by case studies. Future directions are discussed in the context of newer platforms of technology and bioanalytical approaches enabling better operation efficiency in pharmaceutical research.

  5. Nontargeted metabolite profiles and sensory properties of strawberry cultivars grown both organically and conventionally.

    PubMed

    Kårlund, Anna; Hanhineva, Kati; Lehtonen, Marko; Karjalainen, Reijo O; Sandell, Mari

    2015-01-28

    Strawberry (Fragaria × ananassa Duch.) contains many secondary metabolites potentially beneficial for human health, and several of these compounds contribute to strawberry sensory properties, as well. In this study, three strawberry cultivars grown both conventionally and organically were subjected to nontargeted metabolite profiling analysis with LC-qTOF-ESI-MS and to descriptive sensory evaluation by a trained panel. Combined metabolome and sensory data (PLS model) revealed that 79% variation in the metabolome explained 88% variation in the sensory profiles. Flavonoids and condensed and hydrolyzable tannins determined the orosensory properties, and fatty acids contributed to the odor attributes of strawberry. Overall, the results indicated that the chemical composition and sensory quality of strawberries grown in different cultivation systems vary mostly according to cultivar. Organic farming practices may enhance the accumulation of some plant metabolites in specific strawberry genotypes. Careful cultivar selection is a key factor for the improvement of nutritional quality and marketing value of organic strawberries.

  6. Human Ozone (O3) Exposure Alters Serum Profile of Lipid Metabolites

    EPA Science Inventory

    HUMAN OZONE (O3) EXPOSURE ALTERS SERUM PROFILE OF LIPID METABOLITES Miller, D B.1; Kodavanti, U P.2 Karoly, E D.3; Cascio W.E2, Ghio, A J. 21. UNC-Chapel Hill, Chapel Hill, N.C., United States. 2. NHEERL, U.S. EPA, RTP, N.C., United States. 3. METABOLON INC., Durham, N.C., United...

  7. Human Ozone (O3) Exposure Alters Serum Profile of Lipid Metabolites

    EPA Science Inventory

    HUMAN OZONE (O3) EXPOSURE ALTERS SERUM PROFILE OF LIPID METABOLITES Miller, D B.1; Kodavanti, U P.2 Karoly, E D.3; Cascio W.E2, Ghio, A J. 21. UNC-Chapel Hill, Chapel Hill, N.C., United States. 2. NHEERL, U.S. EPA, RTP, N.C., United States. 3. METABOLON INC., Durham, N.C., United...

  8. Metabolite profiling of maize kernels--genetic modification versus environmental influence.

    PubMed

    Frank, Thomas; Röhlig, Richard M; Davies, Howard V; Barros, Eugenia; Engel, Karl-Heinz

    2012-03-28

    A metabolite profiling approach based on gas chromatography-mass spectrometry (GC-MS) was applied to investigate the metabolite profiles of genetically modified (GM) Bt-maize (DKC78-15B, TXP 138F) and Roundup Ready-maize (DKC78-35R). For the comparative investigation of the impact of genetic modification versus environmental influence on the metabolite profiles, GM maize was grown together with the non-GM near-isogenic comparators under different environmental conditions, including several growing locations and seasons in Germany and South Africa. Analyses of variance (ANOVA) revealed significant differences between GM and non-GM maize grown in Germany and South Africa. For the factor genotype, 4 and 3%, respectively, of the total number of peaks detected by GC-MS showed statistically significant differences (p < 0.01) in peak heights as compared to the respective isogenic lines. However, ANOVA for the factor environment (growing location, season) revealed higher numbers of significant differences (p < 0.01) between the GM and the non-GM maize grown in Germany (42%) and South Africa (10%), respectively. This indicates that the majority of differences observed are related to natural variability rather than to the genetic modifications. In addition, multivariate data assessment by means of principal component analysis revealed that environmental factors, that is, growing locations and seasons, were dominant parameters driving the variability of the maize metabolite profiles.

  9. Prediction of Clinically Relevant Safety Signals of Nephrotoxicity through Plasma Metabolite Profiling

    PubMed Central

    Mattes, W. B.; Kamp, H. G.; Fabian, E.; Herold, M.; Krennrich, G.; Looser, R.; Mellert, W.; Prokoudine, A.; Strauss, V.; van Ravenzwaay, B.; Walk, T.; Naraoka, H.; Omura, K.; Schuppe-Koistinen, I.; Nadanaciva, S.; Bush, E. D.; Moeller, N.; Ruiz-Noppinger, P.; Piccoli, S. P.

    2013-01-01

    Addressing safety concerns such as drug-induced kidney injury (DIKI) early in the drug pharmaceutical development process ensures both patient safety and efficient clinical development. We describe a unique adjunct to standard safety assessment wherein the metabolite profile of treated animals is compared with the MetaMap Tox metabolomics database in order to predict the potential for a wide variety of adverse events, including DIKI. To examine this approach, a study of five compounds (phenytoin, cyclosporin A, doxorubicin, captopril, and lisinopril) was initiated by the Technology Evaluation Consortium under the auspices of the Drug Safety Executive Council (DSEC). The metabolite profiles for rats treated with these compounds matched established reference patterns in the MetaMap Tox metabolomics database indicative of each compound's well-described clinical toxicities. For example, the DIKI associated with cyclosporine A and doxorubicin was correctly predicted by metabolite profiling, while no evidence for DIKI was found for phenytoin, consistent with its clinical picture. In some cases the clinical toxicity (hepatotoxicity), not generally seen in animal studies, was detected with MetaMap Tox. Thus metabolite profiling coupled with the MetaMap Tox metabolomics database offers a unique and powerful approach for augmenting safety assessment and avoiding clinical adverse events such as DIKI. PMID:23762827

  10. Metabolite Profile Changes in Xylem Sap and Leaf Extracts of Strategy I Plants in Response to Iron Deficiency and Resupply

    PubMed Central

    Rellán-Álvarez, Rubén; El-Jendoubi, Hamdi; Wohlgemuth, Gert; Abadía, Anunciación; Fiehn, Oliver; Abadía, Javier; Álvarez-Fernández, Ana

    2011-01-01

    The metabolite profile changes induced by Fe deficiency in leaves and xylem sap of several Strategy I plant species have been characterized. We have confirmed that Fe deficiency causes consistent changes both in the xylem sap and leaf metabolite profiles. The main changes in the xylem sap metabolite profile in response to Fe deficiency include consistent decreases in amino acids, N-related metabolites and carbohydrates, and increases in TCA cycle metabolites. In tomato, Fe resupply causes a transitory flush of xylem sap carboxylates, but within 1 day the metabolite profile of the xylem sap from Fe-deficient plants becomes similar to that of Fe-sufficient controls. The main changes in the metabolite profile of leaf extracts in response to Fe deficiency include consistent increases in amino acids and N-related metabolites, carbohydrates and TCA cycle metabolites. In leaves, selected pairs of amino acids and TCA cycle metabolites show high correlations, with the sign depending of the Fe status. These data suggest that in low photosynthesis, C-starved Fe-deficient plants anaplerotic reactions involving amino acids can be crucial for short-term survival. PMID:22645546

  11. Effects of fungicides on galanthamine and metabolite profiles in Narcissus bulbs.

    PubMed

    Lubbe, Andrea; Verpoorte, Robert; Choi, Young Hae

    2012-09-01

    Large-scale plant cultivation usually involves the use of pesticides. Apart from eliminating the target organism, the external chemicals may affect the metabolism of the crop plant. This may have implications for plants cultivated for specific medicinal compounds. In this study the effects of diverse fungicides on the metabolism of Narcissus pseudonarcissus cv. Carlton bulbs were investigated. N. pseudonarcissus cv. Carlton is being cultivated for the extraction of the alkaloid galanthamine. Fungicides typically used in Narcissus cultivation were applied in a field experiment. The aim was to determine whether fungicide applications changed the concentration of galanthamine in the bulbs. (1)H NMR spectroscopy allowed quantitative analysis of galanthamine and other metabolites in bulb extracts. Multivariate data analysis revealed changes in bulb metabolite patterns caused by fungicides. Bulbs treated before planting generally had higher levels of alkaloids, while foliar field applications caused lower alkaloid levels but altered carbohydrate metabolism. Within these groups, certain fungicide treatments caused changes in specific metabolites. This study shows that the fungicides used in Narcissus cultivation can cause a change in the metabolome still detectable in the bulbs after harvest. The standard cultivation practices in terms of fungicide treatment were found suitable for the production of N. pseudonarcissus cv. Carlton as raw material for galanthamine extraction. In the cultivation of medicinal plants for secondary metabolites the potential effect of pesticides and other agrochemicals should be taken into account. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  12. Reproductive features and faecal progesterone metabolite profile in female ferrets.

    PubMed

    Proháczik, A; Kulcsár, M; Glávits, R; Driancourt, M A; Huszenicza, G Y

    2011-02-01

    Elevated post-partum progesterone metabolite (P(4) -met) levels have been recently postulated to occur in lactating lynxes. The aims of this study were to monitor reproductive features in female ferrets, changes in the faecal P(4) -met concentrations throughout the breeding season and ovarian activity in post-partum lactating and non-lactating (NL) female ferrets. Our results indicate that coinciding with the results described in the lynx, elevated faecal P4-met concentrations occur in lactating ferrets, furthermore, that the duration of elevated secretion of P(4) seems to be dependent on the duration of lactation (P4-met at delivery, n=47: <500 ng/g; 5-7 days after delivery, during lactation, n=47: ≥ 500-800 ng/g; in females weaned at delivery, n=4: baseline levels). Three days after ovariohysterectomy of lactating females, P(4) -met concentrations decreased to baseline levels. In lactating females, the ovarian stroma is more active than that in NL ones implicating that the ovary is at least in part responsible for the elevated P4-met concentrations. Ovaries of lactating females contained many luteinized cells either as luteinized granulose cells in the wall of late pre-antral/early antral follicles or as corpus luteum (CL)-like structures. Early resumption of the entire ovarian activity (developed follicles and oestrus) occurs in NL post-partum females, while final follicular development is blocked (follicles stalls at antral stage) in the lactating ones (however, occasionally lactational oestrus may occur). We suppose that the elevated faecal P4-met during lactation together with suckling and other hormonal effects may contribute to prevention of early returning to oestrus in nursing female ferrets. © 2010 Blackwell Verlag GmbH.

  13. Analysis of immediate changes of water-soluble metabolites in alkali-burned rabbit cornea, aqueous humor and lens by high-resolution 1H-NMR spectroscopy.

    PubMed

    Risa, Oystein; Saether, Oddbjørn; Midelfart, Anna; Krane, Jostein; Cejková, Jitka

    2002-01-01

    To investigate immediate changes in water-soluble metabolites of ocular tissue in alkali-burned eyes by using high-resolution 1H-NMR spectroscopy. Adult New Zealand rabbit eyes were burned with 1 M NaOH for 1 min. Normal eyes were used as control. Samples from aqueous humor and perchloric acid extracts of the cornea and lens were analyzed on a NMR spectrometer operating at 500 MHz for protons. Metabolites were quantified by comparing peak area with an added internal standard, TSP (3'-trimethylsilylpropinate-2,2,3,3-d4). Alkali burn of corneal surface causes immediate changes in concentration of many water-soluble metabolites in the anterior segment. Even as far away as the lens a significant increase in lactate was found. Cornea showed a significant increase in glucose and a significant decrease in hypo-taurine concentration. Most changes were observed in aqueous humor, with significant increases in succinate, creatine, scyllo- and myo-inositol and a significant decrease in citrate concentration. Furthermore, a small decrease in ascorbate concentration in aqueous humor was observed. The present study provides a valuable contribution to the knowledge of metabolic alterations in alkali-burned eyes. It shows that 1H-NMR spectroscopy is well suited for simultaneous qualitative and quantitative analysis of changes of metabolite concentrations in damaged tissues. This can help us to better evaluate and understand the biological alterations due to alkali burn.

  14. Fragment Assembly Approach Based on Graph/Network Theory with Quantum Chemistry Verifications for Assigning Multidimensional NMR Signals in Metabolite Mixtures.

    PubMed

    Ito, Kengo; Tsutsumi, Yu; Date, Yasuhiro; Kikuchi, Jun

    2016-04-15

    The abundant observation of chemical fragment information for molecular complexities is a major advantage of biological NMR analysis. Thus, the development of a novel technique for NMR signal assignment and metabolite identification may offer new possibilities for exploring molecular complexities. We propose a new signal assignment approach for metabolite mixtures by assembling H-H, H-C, C-C, and Q-C fragmental information obtained by multidimensional NMR, followed by the application of graph and network theory. High-speed experiments and complete automatic signal assignments were achieved for 12 combined mixtures of (13)C-labeled standards. Application to a (13)C-labeled seaweed extract showed 66 H-C, 60 H-H, 326 C-C, and 28 Q-C correlations, which were successfully assembled to 18 metabolites by the automatic assignment. The validity of automatic assignment was supported by quantum chemical calculations. This new approach can predict entire metabolite structures from peak networks of biological extracts.

  15. NMR-based metabolic profiling in healthy individuals overfed different types of fat: links to changes in liver fat accumulation and lean tissue mass

    PubMed Central

    Elmsjö, A; Rosqvist, F; Engskog, M K R; Haglöf, J; Kullberg, J; Iggman, D; Johansson, L; Ahlström, H; Arvidsson, T; Risérus, U; Pettersson, C

    2015-01-01

    Background: Overeating different dietary fatty acids influence the amount of liver fat stored during weight gain, however, the mechanisms responsible are unclear. We aimed to identify non-lipid metabolites that may differentiate between saturated (SFA) and polyunsaturated fatty acid (PUFA) overfeeding using a non-targeted metabolomic approach. We also investigated the possible relationships between plasma metabolites and body fat accumulation. Methods: In a randomized study (LIPOGAIN study), n=39 healthy individuals were overfed with muffins containing SFA or PUFA. Plasma samples were precipitated with cold acetonitrile and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Pattern recognition techniques were used to overview the data, identify variables contributing to group classification and to correlate metabolites with fat accumulation. Results: We previously reported that SFA causes a greater accumulation of liver fat, visceral fat and total body fat, whereas lean tissue levels increases less compared with PUFA, despite comparable weight gain. In this study, lactate and acetate were identified as important contributors to group classification between SFA and PUFA (P<0.05). Furthermore, the fat depots (total body fat, visceral adipose tissue and liver fat) and lean tissue correlated (P(corr)>0.5) all with two or more metabolites (for example, branched amino acids, alanine, acetate and lactate). The metabolite composition differed in a manner that may indicate higher insulin sensitivity after a diet with PUFA compared with SFA, but this needs to be confirmed in future studies. Conclusion: A non-lipid metabolic profiling approach only identified a few metabolites that differentiated between SFA and PUFA overfeeding. Whether these metabolite changes are involved in depot-specific fat storage and increased lean tissue mass during overeating needs further investigation. PMID:26479316

  16. NMR-based metabolic profiling in healthy individuals overfed different types of fat: links to changes in liver fat accumulation and lean tissue mass.

    PubMed

    Elmsjö, A; Rosqvist, F; Engskog, M K R; Haglöf, J; Kullberg, J; Iggman, D; Johansson, L; Ahlström, H; Arvidsson, T; Risérus, U; Pettersson, C

    2015-10-19

    Overeating different dietary fatty acids influence the amount of liver fat stored during weight gain, however, the mechanisms responsible are unclear. We aimed to identify non-lipid metabolites that may differentiate between saturated (SFA) and polyunsaturated fatty acid (PUFA) overfeeding using a non-targeted metabolomic approach. We also investigated the possible relationships between plasma metabolites and body fat accumulation. In a randomized study (LIPOGAIN study), n=39 healthy individuals were overfed with muffins containing SFA or PUFA. Plasma samples were precipitated with cold acetonitrile and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Pattern recognition techniques were used to overview the data, identify variables contributing to group classification and to correlate metabolites with fat accumulation. We previously reported that SFA causes a greater accumulation of liver fat, visceral fat and total body fat, whereas lean tissue levels increases less compared with PUFA, despite comparable weight gain. In this study, lactate and acetate were identified as important contributors to group classification between SFA and PUFA (P<0.05). Furthermore, the fat depots (total body fat, visceral adipose tissue and liver fat) and lean tissue correlated (P(corr)>0.5) all with two or more metabolites (for example, branched amino acids, alanine, acetate and lactate). The metabolite composition differed in a manner that may indicate higher insulin sensitivity after a diet with PUFA compared with SFA, but this needs to be confirmed in future studies. A non-lipid metabolic profiling approach only identified a few metabolites that differentiated between SFA and PUFA overfeeding. Whether these metabolite changes are involved in depot-specific fat storage and increased lean tissue mass during overeating needs further investigation.

  17. (1)H NMR-based metabolic profiling of liver in chronic unpredictable mild stress rats with genipin treatment.

    PubMed

    Chen, Jian-Li; Shi, Bi-Yun; Xiang, Huan; Hou, Wen-Jing; Qin, Xue-Mei; Tian, Jun-Sheng; Du, Guan-Hua

    2015-11-10

    Genipin, a hydrolyzed metabolite of geniposide extracted from the fruit of Gardenia jasminoides Ellis, has shown promise in alleviating depressive symptoms, however, the antidepressant mechanism of genipin remains unclear and incomprehensive. In this study, the metabolic profiles of aqueous and lipophilic extracts in liver of the chronic unpredictable mild stress (CUMS)-induced rat with genipin treatment were investigated using proton nuclear magnetic resonance ((1)H NMR) spectroscopy coupled with multivariate data analysis. Significant differences in the metabolic profiles of rats in the CUMS model group (MS) and the control group (NS) were observed with metabolic effects including decreasing in choline, glycerol and glycogen, increasing in lactate, alanine and succinate, and a disordered lipid metabolism, while the moderate dose (50mg/kg) of genipin could significantly regulate the concentrations of glycerol, lactate, alanine, succinate and the lipid to their normal levels. These biomakers were involved in metabolism pathways such as glycolysis/gluconeogensis, tricarboxylic acid (TCA) cycle and lipid metabolism, which may be helpful for understanding of antidepressant mechanism of genipin. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Metabolite profile of cerebrospinal fluid in patients with spina bifida: a proton magnetic resonance spectroscopy study.

    PubMed

    Pal, Kamalesh; Sharma, Uma; Gupta, D K; Pratap, Akshay; Jagannathan, N R

    2005-02-01

    The present study was carried out to assess the metabolic differences between cerebrospinal fluid samples of patients with spina bifida and age-matched control individuals. To study the metabolite profile of cerebrospinal fluid of patients with spina bifida using proton magnetic resonance spectroscopy, compare the levels of metabolites with controls, establish correlation of underlying neuronal dysfunction with metabolic changes in patients with spina bifida, and evaluate the potential use of this technique as an additional tool for diagnostic assessment. Combination of embryopathy, stretching, ischemia, compression, and trauma is responsible for cord dysfunction in spina bifida. Changes in neuronal metabolism leads to changes in the local milieu of cerebrospinal fluid in the cord. Change in metabolite profile of cerebrospinal fluid in spina bifida in terms of increase in products of anaerobic metabolism, nerve membrane integrity, and nerve ischemia has not yet been studied. Cerebrospinal fluid obtained from patients and control individuals were characterized using various one- and two-dimensional proton magnetic resonance spectroscopy techniques. Concentration of various metabolites was calculated using the area under the nuclear magnetic resonance peak. Statistically significantly higher levels of lactate, choline, glycerophosphocholine, acetate, and alanine in the cerebrospinal fluid of patients with spina bifida was observed compared with control individuals. Significantly higher levels of metabolites were observed in patients with spina bifida, representing a state of nerve ischemia, anaerobic metabolism, and disruption of neuronal membrane.

  19. The effects of GA and ABA treatments on metabolite profile of germinating barley.

    PubMed

    Huang, Yuqing; Cai, Shengguan; Ye, Lingzhen; Hu, Hongliang; Li, Chengdao; Zhang, Guoping

    2016-02-01

    Sugar degradation during grain germination is important for malt quality. In malting industry, gibberellin (GA) is frequently used for improvement of malting quality. In this study, the changes of metabolite profiles and starch-degrading enzymes during grain germination, and as affected by GA and abscisic acid (ABA) were investigated using two wild barley accessions XZ72 and XZ95. Totally fifty-two metabolites with known structures were detected and the change of metabolite during germination was time- and genotype dependent. Sugars and amino acids were the most dramatically changed compounds. Addition of GA enhanced the activities of starch-degrading enzymes, and increased most metabolites, especially sugars and amino acids, whereas ABA had the opposite effect. The effect varied with the barley accessions. The current study is the first attempt in investigating the effect of hormones on metabolite profiles in germinating barley grain, being helpful for identifying the factors affecting barley germination or malt quality. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Efficacy of Withania somnifera on seminal plasma metabolites of infertile males: a proton NMR study at 800 MHz.

    PubMed

    Gupta, Ashish; Mahdi, Abbas Ali; Shukla, Kamla Kant; Ahmad, Mohammad Kaleem; Bansal, Navneeta; Sankhwar, Pushplata; Sankhwar, Satya Narain

    2013-08-26

    Traditional Indian systems of medicine use roots of Withania somnifera for impotence, infertility treatment, stress, and the aging process. Although Withania somnifera improves semen quality by regulating reproductive hormone levels and oxidative stress, the molecular mechanism is not clear. Our study uses high-resolution Nuclear Magnetic Resonance (NMR) spectroscopy to explore the scientific basis to reveal the pre- and post-treatment efficacy of Withania somnifera on seminal plasma of infertile men-which remains unexplored to date. A total of 180 infertile male patients were administered Withania somnifera root powder at the rate of 5 g/d for a 3-month period. The study included age-matched, healthy men as a control (n=50) group. Proton NMR spectroscopy was used to measure lactate, alanine, glutamate, glutamine, citrate, lysine, choline, glycerophosphocholine (GPC), glycine, tyrosine, histidine, phenylalanine, and uridine in all seminal plasma samples. To appraise infertility levels, we also measured sperm concentration, motility, lipid peroxide, and hormonal perturbation. Withania somnifera therapy repairs the disturbed concentrations of lactate, alanine, citrate, GPC, histidine, and phenylalanine in seminal plasma and recovers the quality of semen of post-treated compared to pre-treated infertile men. Serum biochemistry was also improved over post-therapy in infertile men. Our findings reveal that Withania somnifera not only reboots enzymatic activity of metabolic pathways and energy metabolism but also invigorates the harmonic balance of seminal plasma metabolites and reproductive hormones in infertile men. The results suggest that Withania somnifera may be used as an empirical therapy for clinical management and treatment of infertility. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  1. Marking of metabolites in the diagnostics of metabolic diseases and in the investigation of xenobiotics metabolism using NMR spectroscopy.

    PubMed

    Krawczyk, Hanna

    2016-10-25

    There are currently no sound estimates of the number of children born with a serious congenital disorder attributable to genetic or environmental causes (World Health Organization) but there is a supposed number of babies born with birth defects per year: in the world approximately 7.9 million children (6% of births). There is conducted population-based screening by the individual countries. The specialised methods are used when it is not possible to diagnose disease in screening. In recent years in the diagnostics of these disorders the methods of Magnetic Resonance Spectroscopy of the brain (in vivo(1)H-MRS) and high resolution NMR spectroscopy gain in importance. The manuscript focused on developing the method of marking the metabolic diseases markers of various origins using NMR spectroscopy (including synthesis of markers). Considering the disorders occurring among children, according to Hoffman, Zschocke, Nyhan, there are three following groups of inherited metabolic diseases: disorders of intermediary metabolism, disorders of the biosynthesis and breakdown of complex molecules and neurotransmitter defects and related disorders. The presented investigation is focused on: a study of selected compounds that cause disorders of intermediary metabolism, a study of compounds that cause disorders of the biosynthesis and breakdown of complex molecules and a study of compounds that cause neurotransmitter defects and related disorders. In the subsequent chapter of manuscript there are presented the results of investigation concerning the metabolism of xenobiotics that could potentially be used in therapy of inherited metabolic diseases, basing on stilbene derivatives. In the last chapter there are presented the results of experiments with creatinine- the metabolite produced in muscles. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Metabolite Profiling of Root Exudates of Common Bean under Phosphorus Deficiency

    PubMed Central

    Tawaraya, Keitaro; Horie, Ryota; Saito, Saki; Wagatsuma, Tadao; Saito, Kazuki; Oikawa, Akira

    2014-01-01

    Root exudates improve the nutrient acquisition of plants and affect rhizosphere microbial communities. The plant nutrient status affects the composition of root exudates. The purpose of this study was to examine common bean (Phaseolus vulgaris L.) root exudates under phosphorus (P) deficiency using a metabolite profiling technique. Common bean plants were grown in a culture solution at P concentrations of 0 (P0), 1 (P1) and 8 (P8) mg P L−1 for 1, 10 and 20 days after transplanting (DAT). Root exudates were collected, and their metabolites were determined by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF MS). The shoot P concentration and dry weight of common bean plants grown at P0 were lower than those grown at P8. One hundred and fifty-nine, 203 and 212 metabolites were identified in the root exudates, and 16% (26/159), 13% (26/203) and 9% (20/212) of metabolites showed a P0/P8 ratio higher than 2.0 at 1, 10 and 20 DAT, respectively. The relative peak areas of several metabolites, including organic acids and amino acids, in root exudates were higher at P0 than at P8. These results suggest that more than 10% of primary and secondary metabolites are induced to exude from roots of common bean by P deficiency. PMID:25032978

  3. Metabolite profiling of wheat grains (Triticum aestivum L.) from organic and conventional agriculture.

    PubMed

    Zörb, Christian; Langenkämper, Georg; Betsche, Thomas; Niehaus, Karsten; Barsch, Aiko

    2006-10-18

    In some European community countries up to 8% of the agricultural area is managed organically. The aim was to obtain a metabolite profile for wheat (Triticum aestivum L.) grains grown under comparable organic and conventional conditions. These conditions cannot be found in plant material originating from different farms or from products purchased in supermarkets. Wheat grains from a long-term biodynamic, bioorganic, and conventional farming system from the harvest 2003 from Switzerland were analyzed. The presented data show that using a high throughput GC-MS technique, it was possible to determine relative levels of a set of 52 different metabolites including amino acids, organic acids, sugars, sugar alcohols, sugar phosphates, and nucleotides from wheat grains. Within the metabolites from all field trials, there was at the most a 50% reduction comparing highest and lowest mean values. The statistical analysis of the data shows that the metabolite status of the wheat grain from organic and mineralic farming did not differ in concentrations of 44 metabolites. This result indicates no impact or a small impact of the different farming systems. In consequence, we did not detect extreme differences in metabolite composition and quality of wheat grains.

  4. Antihepatotoxic Effect and Metabolite Profiling of Panicum turgidum Extract via UPLC-qTOF-MS

    PubMed Central

    Farag, Mohamed A.; El Fishawy, Ahlam M.; El-Toumy, Sayed A.; Amer, Khadiga F.; Mansour, Ahmed M.; Taha, Hala E.

    2016-01-01

    Background: Panicum turgidum, desert grass, has not reported any detailed phytochemical or biological study as yet Objective: To establish P. turgidum secondary metabolite profile and to assess its antihepatotoxic effect Materials and Methods: Ultra-performance liquid chromatography (UPLC) coupled to quadrupole high-resolution time of flight mass spectrometry (qTOF-MS) was used for large-scale secondary metabolites profiling in P. turgidum extract, alongside assessing median lethal dose (LD50) and hepatoprotective effect against carbon tetrachloride (CCl4) intoxication Results: A total of 39 metabolites were identified with flavonoids as the major class present as O/C-glycosides of luteolin, apigenin, isorhamnetin and naringenin, most of which are first time to be reported in Panicum sp. Antihepatotoxic effect of P. turgidum crude extract was revealed via improving several biochemical marker levels and mitigation against oxidative stress in the serum and liver tissues, compared with CCl4 intoxicated group and further confirmed by histopathological examination. Conclusion: This study reveals that P. turgidum, enriched in C-flavonoids, presents a novel source of safe antihepatotoxic agents and further demonstrates the efficacy of UPLC-MS metabolomics in the field of natural products drug discovery. SUMMARY UPLC coupled to qTOF-MS was used for large scale secondary metabolites profiling in P. turgidum.A total of 39 metabolites were identified with flavonoids amounting as the major metabolite class.Anti-hepatotoxic effect of P. turgidum extract was revealed via several biochemical markers and histopathological examination.This study reveals that P. turgidum, enriched in C-flavonoids, present a novel source of antihepatotoxic agents. Abbreviations used: UPLC: Ultra-performance liquid chromatography (UPLC), LD50: median lethal dose, MDA: malondialdehyde, GSH: glutathione reductase, CAT: catalase, SOD: superoxide dismutase, ALT: alanine aminotransferase, AST: aspartate

  5. Investigating the drought-stress response of hybrid poplar genotypes by metabolite profiling.

    PubMed

    Barchet, Genoa L H; Dauwe, Rebecca; Guy, Robert D; Schroeder, William R; Soolanayakanahally, Raju Y; Campbell, Malcolm M; Mansfield, Shawn D

    2014-11-01

    Drought stress is perhaps the most commonly encountered abiotic stress plants experience in the natural environment, and it is one of the most important factors limiting plant productivity. Here, we employed untargeted metabolite profiling to examine four drought-stressed hybrid poplar (Populus spp.) genotypes for their metabolite content, using gas chromatography coupled to mass spectrometry. The primary objective of these analyses was to characterize the metabolite profile of poplar trees to assess relative drought resistance and to investigate the underlying biochemical mechanisms employed by the genotypes to combat drought. Metabolite profiling identified key metabolites that increased or decreased in relative abundance upon exposure to drought stress. Overall, amino acids, the antioxidant phenolic compounds catechin and kaempferol, as well as the osmolytes raffinose and galactinol exhibited increased abundance under drought stress, whereas metabolites involved in photorespiration, redox regulation and carbon fixation showed decreased abundance under drought stress. One clone in particular, Okanese, displayed unique responses to the imposed drought conditions. This clone was found to have higher leaf water potential, but lower growth rate relative to the other clones tested. Okanese also had lower accumulation of osmolytes such as raffinose, galactinol and proline, but higher overall levels of antioxidants such as catechin and dehydroascorbic acid. As such, it was proposed that osmotic adjustment as a mechanism for drought avoidance in this clone is not as well developed in comparison with the other clones investigated in this study, and that a possible alternative mechanism for the enhanced drought avoidance displayed by Okanese may be due to differential allocation of resources or better retention of water.

  6. Metabolic Profiling of Cells in Response to Drug Treatment using (1)H High-resolution Magic Angle Spinning (HR-MAS) NMR Spectroscopy.

    PubMed

    Vermathen, Martina; Diserens, Gaëlle; Vermathen, Peter; Furrer, Julien

    2017-03-29

    High-resolution magic angle spinning (HR-MAS) is an NMR technique that provides access to well resolved liquid-like 1H NMR spectra of semi-solid samples. Therefore, 1H HR-MAS NMR spectroscopy has become an important tool for the direct analysis of biological samples such as tissues and cells in a mostly non-destructive way. Here, we focus on the application of HR-MAS NMR combined with multivariate statistical methods used for metabolic profiling of cells and in particular for the study of cellular metabolic responses to drug exposure. The principles of HR-MAS and the metabolomic approach are briefly described. As an example, a study on the metabolic response of different cell types towards treatment with a highly cytotoxic hexacationic ruthenium metallaprism as potential anti-cancer drug is presented. Specific metabolites and metabolic pathways are suggested to be associated with the cellular response. The study demonstrates the potential of HR-MAS metabolomics applied to cells for addressing the intracellular processes involved in the treatment with organometallic drugs.

  7. In Vitro Metabolite Profiling of ADB-FUBINACA, A New Synthetic Cannabinoid.

    PubMed

    Carlier, Jeremy; Diao, Xingxing; Wohlfarth, Ariane; Scheidweiler, Karl; Huestis, Marilyn A

    2016-11-08

    Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption. N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (ADB-FUBINACA) is an emerging synthetic cannabinoid whose toxicological and metabolic data are currently unavailable. We aimed to determine optimal markers for identifying ADB-FUBINACA intake. Metabolic stability was evaluated with human liver microsome incubations. Metabolites were identified after 1 and 3 h incubation with pooled human hepatocytes, liquid chromatography- high resolution mass spectrometry in positive-ion mode (5600+ TripleTOF®, Sciex) and several data mining approaches (MetabolitePilot™, Sciex). Metabolite separation was achieved on an Ultra Biphenyl column (Restek®); full-scan TOF-MS and information-dependent acquisition MS/MS data were acquired. ADB-FUBINACA microsomal half-life was 39.7 min, with a predicted hepatic clearance of 9.0 mL/min/kg and a 0.5 extraction ratio (intermediate-clearance drug). Twenty-three metabolites were identified. Major metabolic pathways were alkyl and indazole hydroxylation, terminal amide hydrolysis, subsequent glucuronide conjugations, and dehydrogenation. We recommend ADB-FUBINACA hydroxyalkyl, hydroxydehydroalkyl and hydroxylindazole metabolites as ADB-FUBINACA intake markers. N-dealkylated metabolites are not specific ADB-FUBINACA metabolites and should not be used as definitive markers of consumption. This is the first ADB-FUBINACA in vitro metabolism study; in vivo experiments enabling pharmacokinetic and pharmacodynamics studies or urine from authentic clinical/forensic cases are needed to confirm our results. .

  8. Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling.

    PubMed

    Huber, Kilian V M; Olek, Karin M; Müller, André C; Tan, Chris Soon Heng; Bennett, Keiryn L; Colinge, Jacques; Superti-Furga, Giulio

    2015-11-01

    Thermal stabilization of proteins after ligand binding provides an efficient means to assess the binding of small molecules to proteins. We show here that in combination with quantitative mass spectrometry, the approach allows for the systematic survey of protein engagement by cellular metabolites and drugs. We profiled the targets of the drugs methotrexate and (S)-crizotinib and the metabolite 2'3'-cGAMP in intact cells and identified the 2'3'-cGAMP cognate transmembrane receptor STING, involved in immune signaling.

  9. Ultrasensitive Profiling of Metabolites Using Tyramine-Functionalized Graphene Quantum Dots.

    PubMed

    Li, Nan; Than, Aung; Wang, Xuewan; Xu, Shaohai; Sun, Lei; Duan, Hongwei; Xu, Chenjie; Chen, Peng

    2016-03-22

    Graphene quantum dots (GQDs) are emerging fluorescence reporters attractive for optical sensing, owing to their high photostability, highly tunable photoluminescence, molecular size, atomically thin structure, biocompatibility, and ease of functionalization. Herein, we present a fluorometric sensing platform based on tyramine-functionalized GQDs, which is able to detect a spectrum of metabolites with high sensitivity and specificity. Furthermore, multiparametric blood analysis (glucose, cholesterol, L-lactate, and xanthine) is demonstrated. This convenient metabolite profiling technique could be instrumental for diagnosis, study, and management of metabolic disorders and associated diseases, such as diabetes, obesity, lactic acidosis, gout, and hypertension.

  10. Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent Metabolic Fingerprints in Perfusion Culture.

    PubMed

    Karst, Daniel J; Steinhoff, Robert F; Kopp, Marie R G; Serra, Elisa; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

    2016-12-20

    Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 10(6) cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 10(6) cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016.

  11. (13)C-NMR-Based Metabolomic Profiling of Typical Asian Soy Sauces.

    PubMed

    Kamal, Ghulam Mustafa; Yuan, Bin; Hussain, Abdullah Ijaz; Wang, Jie; Jiang, Bin; Zhang, Xu; Liu, Maili

    2016-09-02

    It has been a strong consumer interest to choose high quality food products with clear information about their origin and composition. In the present study, a total of 22 Asian soy sauce samples have been analyzed in terms of (13)C-NMR spectroscopy. Spectral data were analyzed by multivariate statistical methods in order to find out the important metabolites causing the discrimination among typical soy sauces from different Asian regions. It was found that significantly higher concentrations of glutamate in Chinese red cooking (CR) soy sauce may be the result of the manual addition of monosodium glutamate (MSG) in the final soy sauce product. Whereas lower concentrations of amino acids, like leucine, isoleucine and valine, observed in CR indicate the different fermentation period used in production of CR soy sauce, on the other hand, the concentration of some fermentation cycle metabolites, such as acetate and sucrose, can be divided into two groups. The concentrations of these fermentation cycle metabolites were lower in CR and Singapore Kikkoman (SK), whereas much higher in Japanese shoyu (JS) and Taiwan (China) light (TL), which depict the influence of climatic conditions. Therefore, the results of our study directly indicate the influences of traditional ways of fermentation, climatic conditions and the selection of raw materials and can be helpful for consumers to choose their desired soy sauce products, as well as for researchers in further authentication studies about soy sauce.

  12. Actions of mammalian insulin on a Neurospora variant: effects on intracellular metabolite levels as monitored by P-31 NMR spectroscopy

    SciTech Connect

    Greenfield, N.J.; McKenzie, M.A.; Jordan, F.; Takahashi, M.; Lenard, J.

    1986-05-01

    Fourier transform P-31 NMR spectroscopy (81 MHz) was used to investigate the biochemical nature of insulin action upon the cell wall-deficient slime mutant of Neurospora crassa. Spectra of oxygenated, living cells (ca.10/sup 9//ml.) in late logarithmic-early stationary phase of growth were accumulated for approximately 20 min. (350-450 pulses). Pronounced differences were seen in the metabolite levels of cells cultured for 18-21 hours in the presence of insulin (100 nM) as compared to cells cultured in its absence. Differences in the insulin-grown cells included higher levels of sugar phosphates, inorganic (cytoplasmic) phosphate, NAD+/NADH and UDP-glucose (UDPG) compared to control cells, in which UDP-N-acetylglucosamine (UDPNAG) was the prominent sugar nucleotide. When 100 mM glucose was administered with insulin immediately prior to measurement, short term effects were seen. There were significant increases of sugar phosphates, inorganic phosphate, NAD+/NADH, phosphodiesters and UDPG relative to the case of glucose addition alone. These results are wholly consistent with the known influence of insulin upon mammalian metabolism: stimulation of glucose uptake, phosphorylation and oxidation, phosphatide synthesis and Pi uptake.

  13. Metabolite signatures in hydrophilic extracts of mouse lungs exposed to cigarette smoke revealed by 1H NMR metabolomics investigation

    DOE PAGES

    Hu, Jian Z.; Wang, Xuan; Feng, Ju; ...

    2015-05-12

    Herein, 1H-NMR metabolomics are carried out to evaluate the changes of metabolites in lungs of mice exposed to cigarette smoke. It is found that the concentrations of adenosine derivatives (i.e. ATP, ADP and AMP), inosine and uridine are significantly fluctuated in the lungs of mice exposed to cigarette smoke compared with those of controls regardless the mouse is obese or regular weight. The decreased ATP, ADP, AMP and elevated inosine predict that the deaminases in charge of adenosine derivatives to inosine derivatives conversion are altered in lungs of mice exposed to cigarette smoke. Transcriptional analysis reveals that the concentrations ofmore » adenosine monophosphate deaminase and adenosine deaminase are different in the lungs of mice exposed to cigarette smoke, confirming the prediction from metabolomics studies. We also found, for the first time, that the ratio of glycerophosphocholine (GPC) to phosphocholine (PC) is significantly increased in the lungs of obese mice compared with regular weight mice. The ratio of GPC/PC is further elevated in the lungs of obese group by cigarette smoke exposure. Since GPC/PC ratio is a known biomarker for cancer, these results may suggest that obese group is more susceptible to lung cancer when exposed to cigarette smoke.« less

  14. A proton NMR study of the effect of a new intravasal injectable male contraceptive RISUG on seminal plasma metabolites.

    PubMed

    Sharma, U; Chaudhury, K; Jagannathan, N R; Guha, S K

    2001-09-01

    Nuclear magnetic resonance (NMR) spectroscopy was used to quantify citrate, glucose, lactate, glycerophosphorylcholine and choline in seminal plasma from subjects injected with a new male contraceptive RISUG, a copolymer of styrene maleic anhydride dissolved in dimethyl sulphoxide, and in seminal plasma from normal ejaculates. No significant difference in the concentration of citrate was observed between the groups, indicating that the prostate is not affected by the contraceptive. The concentrations of glucose, lactate, glycerophosphorylcholine and choline were significantly lower (P < 0.01) in subjects injected with RISUG compared with controls. In addition, metabolite ratios such as choline:citrate, citrate:lactate, choline:lactate and glycerophosphorylcholine:choline were calculated. Citrate:lactate and glycerophosphorylcholine:choline ratios were significantly lower in RISUG-injected subjects than in controls (P < 0.01), thereby indicating the occurrence of partial obstructive azoospermia. The most important finding of the present study was that the intervention of RISUG in the vas deferens even for a period as long as 8 years is absolutely safe and does not lead to prostatic diseases.

  15. Quantitative metabolomic profiling of serum, plasma, and urine by (1)H NMR spectroscopy discriminates between patients with inflammatory bowel disease and healthy individuals.

    PubMed

    Schicho, Rudolf; Shaykhutdinov, Rustem; Ngo, Jennifer; Nazyrova, Alsu; Schneider, Christopher; Panaccione, Remo; Kaplan, Gilaad G; Vogel, Hans J; Storr, Martin

    2012-06-01

    Serologic biomarkers for inflammatory bowel disease (IBD) have yielded variable differentiating ability. Quantitative analysis of a large number of metabolites is a promising method to detect IBD biomarkers. Human subjects with active Crohn's disease (CD) and active ulcerative colitis (UC) were identified, and serum, plasma, and urine specimens were obtained. We characterized 44 serum, 37 plasma, and 71 urine metabolites by use of (1)H NMR spectroscopy and "targeted analysis" to differentiate between diseased and non-diseased individuals, as well as between the CD and UC cohorts. We used multiblock principal component analysis and hierarchical OPLS-DA for comparing several blocks derived from the same "objects" (e.g., subject) to examine differences in metabolites. In serum and plasma of IBD patients, methanol, mannose, formate, 3-methyl-2-oxovalerate, and amino acids such as isoleucine were the metabolites most prominently increased, whereas in urine, maximal increases were observed for mannitol, allantoin, xylose, and carnitine. Both serum and plasma of UC and CD patients showed significant decreases in urea and citrate, whereas in urine, decreases were observed, among others, for betaine and hippurate. Quantitative metabolomic profiling of serum, plasma, and urine discriminates between healthy and IBD subjects. However, our results show that the metabolic differences between the CD and UC cohorts are less pronounced.

  16. Correlation between species-specific metabolite profiles and bioactivities of blueberries (Vaccinium spp.).

    PubMed

    Lee, Sarah; Jung, Eun Sung; Do, Seon-Gil; Jung, Ga-Young; Song, Gwanpil; Song, Jung-Min; Lee, Choong Hwan

    2014-03-05

    Metabolite profiling of three blueberry species (Vaccinium bracteatum Thunb., V. oldhamii Miquel., and V. corymbosum L.) was performed using gas chromatography-time-of-flight-mass spectrometry (GC-TOF-MS) and ultraperformance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS) combined multivariate analysis. Partial least-squares discriminant analysis clearly showed metabolic differences among species. GC-TOF-MS analysis revealed significant differences in amino acids, organic acids, fatty acids, sugars, and phenolic acids among the three blueberry species. UPLC-Q-TOF-MS analysis indicated that anthocyanins were the major metabolites distinguishing V. bracteatum from V. oldhamii. The contents of anthocyanins such as glycosides of cyanidin were high in V. bracteatum, while glycosides of delphinidin, petunidin, and malvidin were high in V. oldhamii. Antioxidant activities assessed using ABTS and DPPH assays showed the greatest activity in V. oldhamii and revealed the highest correlation with total phenolic, total flavonoid, and total anthocyanin contents and their metabolites.

  17. Differential Metabolite Profiles during Fruit Development in High-Yielding Oil Palm Mesocarp

    PubMed Central

    Teh, Huey Fang; Neoh, Bee Keat; Hong, May Ping Li; Low, Jaime Yoke Sum; Ng, Theresa Lee Mei; Ithnin, Nalisha; Thang, Yin Mee; Mohamed, Mohaimi; Chew, Fook Tim; Yusof, Hirzun Mohd.; Kulaveerasingam, Harikrishna; Appleton, David R.

    2013-01-01

    To better understand lipid biosynthesis in oil palm mesocarp, in particular the differences in gene regulation leading to and including de novo fatty acid biosynthesis, a multi-platform metabolomics technology was used to profile mesocarp metabolites during six critical stages of fruit development in comparatively high- and low-yielding oil palm populations. Significantly higher amino acid levels preceding lipid biosynthesis and nucleosides during lipid biosynthesis were observed in a higher yielding commercial palm population. Levels of metabolites involved in glycolysis revealed interesting divergence of flux towards glycerol-3-phosphate, while carbon utilization differences in the TCA cycle were proven by an increase in malic acid/citric acid ratio. Apart from insights into the regulation of enhanced lipid production in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programmes. PMID:23593468

  18. Metabolite Profiles in Various Plant Organs of Justicia gendarussa Burm.f. and Its in Vitro Cultures.

    PubMed

    Indrayoni, Putu; Purwanti, Diah Intan; Wongso, Suwidji; Prajogo, Bambang E W; Indrayanto, Gunawan

    2016-04-13

    Metabolite profiles of plant organs and their in vitro cultures of Justicia gendarussa have been studied by using Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight-Mass Spectrometry (UPLC-Qtof-MS). Samples of leaves, stems, roots, and shoot cultures showed similar patterns of metabolites, while samples of root cultures gave very different profiles. Concentrations of secondary metabolites in shoot cultures were relatively low compared to those in the leaves and roots of the plants. The results suggested that secondary metabolites in J. gendarussa were biosynthetized in the leaves, then transported to the roots.

  19. Metabolite Profiles in Various Plant Organs of Justicia gendarussa Burm.f. and Its in Vitro Cultures

    PubMed Central

    Indrayoni, Putu; Purwanti, Diah Intan; Wongso, Suwidji; Prajogo, Bambang E.W.; Indrayanto, Gunawan

    2016-01-01

    Metabolite profiles of plant organs and their in vitro cultures of Justicia gendarussa have been studied by using Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight-Mass Spectrometry (UPLC-Qtof-MS). Samples of leaves, stems, roots, and shoot cultures showed similar patterns of metabolites, while samples of root cultures gave very different profiles. Concentrations of secondary metabolites in shoot cultures were relatively low compared to those in the leaves and roots of the plants. The results suggested that secondary metabolites in J. gendarussa were biosynthetized in the leaves, then transported to the roots. PMID:28117321

  20. From Metabolomics to Fluxomics: A Computational Procedure to Translate Metabolite Profiles into Metabolic Fluxes

    PubMed Central

    Cortassa, Sonia; Caceres, Viviane; Bell, Lauren N.; O’Rourke, Brian; Paolocci, Nazareno; Aon, Miguel A.

    2015-01-01

    We describe a believed-novel procedure for translating metabolite profiles (metabolome) into the set of metabolic fluxes (fluxome) from which they originated. Methodologically, computational modeling is integrated with an analytical platform comprising linear optimization, continuation and dynamic analyses, and metabolic control. The procedure was tested with metabolite profiles obtained from ex vivo mice Langendorff-heart preparations perfused with glucose. The metabolic profiles were analyzed using a detailed kinetic model of the glucose catabolic pathways including glycolysis, pentose phosphate (PP), glycogenolysis, and polyols to translate the glucose metabolome of the heart into the fluxome. After optimization, the ability of the model to simulate the initial metabolite profile was confirmed, and metabolic fluxes as well as the structure of control and regulation of the glucose catabolic network could be calculated. We show that the step catalyzed by phosphofructokinase together with ATP demand and glycogenolysis exert the highest control on the glycolytic flux. The negative flux control exerted by phosphofructokinase on the PP and polyol pathways revealed that the extent of glycolytic flux directly affects flux redirection through these pathways, i.e., the higher the glycolytic flux the lower the PP and polyols. This believed-novel methodological approach represents a step forward that may help in designing therapeutic strategies targeted to diagnose, prevent, and treat metabolic diseases. PMID:25564863

  1. Ternary complexes between cationic GdIII chelates and anionic metabolites in aqueous solution: an NMR relaxometric study.

    PubMed

    Botta, Mauro; Aime, Silvio; Barge, Alessandro; Bobba, Gabriella; Dickins, Rachel S; Parker, David; Terreno, Enzo

    2003-05-09

    The (1)H and (17)O NMR relaxometric properties of two cationic complexes formed by Gd(III) with a macrocyclic heptadentate triamide ligand, L(1), and its Nmethylated analogue, L(2), have been investigated in aqueous media as a function of pH, temperature and magnetic field strength. The complexes possess two water molecules in their inner coordination sphere for which the rate of exchange has been found to be sensibly faster for the Nmethylated derivative and explained in terms of electronic effects (decrease of the charge density at the metal center) and perturbation of the network of hydrogen-bonded water molecules in the outer hydration sphere. The proton relaxivity shows a marked dependence from pH and decreases of about six units in the pH range 6.5 to 9.0. This has been accounted for by the displacement of the two water molecules by dissolved carbonate which acts as a chelating anion. The formation of ternary complexes with lactate, malonate, citrate, acetate, fluoride and hydrogenphosphate has been monitored by (1)H NMR relaxometric titrations at 20 MHz and pH 6.3 and the value of the affinity constant, K, and of the relaxivity of the adducts could be obtained. Lactate, malonate and citrate interact strongly with the complexes (log K > or =3.7) and coordinate in a bidendate mode by displacing both water molecules. Larger affinity constants have been measured for GdL(2). Acetate, fluoride and hydrogenphosphate form monoaqua ternary complexes which were investigated in detail with regard to their relaxometric properties. The NMR dispersion (NMRD) profiles indicate a large contribution to the relaxivity of the adducts from water molecules belonging to the second hydration shell of the complexes and hydrogen-bonded to the anion. A VT (17)O NMR study has shown a marked increase of the rate of water exchange upon binding which is explained by coordination of the anion in an equatorial site, thus leaving the water molecule in an apical position, more accessible for

  2. Spatio-Temporal Metabolite Profiling of the Barley Germination Process by MALDI MS Imaging

    PubMed Central

    Gorzolka, Karin; Kölling, Jan; Nattkemper, Tim W.; Niehaus, Karsten

    2016-01-01

    MALDI mass spectrometry imaging was performed to localize metabolites during the first seven days of the barley germination. Up to 100 mass signals were detected of which 85 signals were identified as 48 different metabolites with highly tissue-specific localizations. Oligosaccharides were observed in the endosperm and in parts of the developed embryo. Lipids in the endosperm co-localized in dependency on their fatty acid compositions with changes in the distributions of diacyl phosphatidylcholines during germination. 26 potentially antifungal hordatines were detected in the embryo with tissue-specific localizations of their glycosylated, hydroxylated, and O-methylated derivates. In order to reveal spatio-temporal patterns in local metabolite compositions, multiple MSI data sets from a time series were analyzed in one batch. This requires a new preprocessing strategy to achieve comparability between data sets as well as a new strategy for unsupervised clustering. The resulting spatial segmentation for each time point sample is visualized in an interactive cluster map and enables simultaneous interactive exploration of all time points. Using this new analysis approach and visualization tool germination-dependent developments of metabolite patterns with single MS position accuracy were discovered. This is the first study that presents metabolite profiling of a cereals’ germination process over time by MALDI MSI with the identification of a large number of peaks of agronomically and industrially important compounds such as oligosaccharides, lipids and antifungal agents. Their detailed localization as well as the MS cluster analyses for on-tissue metabolite profile mapping revealed important information for the understanding of the germination process, which is of high scientific interest. PMID:26938880

  3. Analysis of the urinary excretion of ifosfamide and its N-dechloroethylated metabolites in children using 31P-NMR spectroscopy.

    PubMed

    Misiura, Konrad; Zubowska, Małgorzata; Zielińska, Elzbieta

    2003-01-01

    Amounts of ifosfamide (CAS 3778-73-2) and its N-dechloroethylated metabolites excreted in the urine were measured using 31P-NMR spectroscopy in 26 cancer children treated with this drug. Strong inter-patient variation in levels of these compounds were found. These differences were independent from patients age, body surface area, and sex, the dose of the drug, suggesting genetic base of observed variations in ifosfamide metabolism.

  4. An optimized method for NMR-based plant seed metabolomic analysis with maximized polar metabolite extraction efficiency, signal-to-noise ratio, and chemical shift consistency.

    PubMed

    Wu, Xiangyu; Li, Ning; Li, Hongde; Tang, Huiru

    2014-04-07

    Plant metabolomic analysis has become an essential part of functional genomics and systems biology and requires effective extraction of both primary and secondary metabolites from plant cells. To establish an optimized extraction method for the NMR-based analysis, we used the seeds of mungbean (Vigna radiata cv. Elü no. 1) as a model and systematically investigated the dependence of the metabolite composition in plant extracts on various extraction parameters including cell-breaking methods, extraction solvents, number of extraction repeats, tissue-to-solvent ratio, and extract-to-buffer ratio (for final NMR analysis). We also compared two NMR approaches for quantitative metabolomic analysis from completely relaxed spectra directly and from partially relaxed spectra calculated with T1. By maximizing the extraction efficiency and signal-to-noise ratio but minimizing inter-sample chemical-shift variations and metabolite degradations, we established a parameter-optimized protocol for NMR-based plant seed metabolomic analysis. We concluded that aqueous methanol was the best extraction solvent with an optimal tissue-to-solvent ratio of about 1 : 10-1 : 15 (mg per μL). The combination of tissuelyser homogenization with ultrasonication was the choice of cell-breaking method with three repeated extractions being necessary. For NMR analysis, the optimal extract-to-solvent was around 5-8 mg mL(-1) and completely relaxed spectra were ideal for intrinsically quantitative metabolomic analysis although partially relaxed spectra were employable for comparative metabolomics. This optimized method will offer ensured data quality for high-throughput and reliable plant metabolomics studies.

  5. Covalent binding of reduced metabolites of [{sup 15}N{sub 3}]TNT to soil organic matter during a bioremediation process analyzed by {sup 15}N NMR spectroscopy

    SciTech Connect

    Achtnich, C.; Fernandes, E.; Bollag, J.M.; Knackmuss, H.J.; Lenke, H.

    1999-12-15

    Evidence is presented for the covalent binding of biologically reduced metabolites of 2,4,6-{sup 15}N{sub 3}-trinitrotoluene (TNT) to different soil fractions, using liquid {sup 15}N NMR spectroscopy. A silylation procedure was used to release soil organic matter from humin and whole soil for spectroscopic measurements. TNT-contaminated soil was spiked with 2,4,6-{sup 15}N{sub 3}-trinitrotoluene and {sup 14}C-ring labeled TNT, before treatment in a soil slurry reactor. During the anaerobic/aerobic incubation the amount of radioactivity detected in the fulvic and humic acid fractions did not change significantly whereas the radioactivity bound to humin increased to 71%. The {sup 15}N NMR spectra of the fulvic acid samples were dominated by a large peak that corresponded to aliphatic amines or ammonia. In the early stages of incubation, {sup 15}N NMR analysis of the humic acids indicated bound azoxy compounds. The signals arising from nitro and azoxy groups disappeared with further anaerobic treatment. At the end of incubation, the NMR shifts showed that nitrogen was covalently bound to humic acid as substituted amines and amides. The NMR spectra of the silylated humin suggest formation of azoxy compounds and imine linkages. Bound metabolites possessing nitro groups were also detected. Primary amines formed during the anaerobic incubation disappeared during the aerobic treatment. Simultaneously, the amount of amides and tertiary amines increased. Nitro and azoxy groups of bound molecules were still present in humin at the end of the incubation period. Formation of azoxy compounds from partially reduced TNT followed by binding and further reduction appears to be an important mechanism for the immobilization of metabolites of TNT to soil.

  6. Metabolic profiling of Klebsiella oxytoca: evaluation of methods for extraction of intracellular metabolites using UPLC/Q-TOF-MS.

    PubMed

    Park, Changhun; Yun, Seokhun; Lee, Sang Yup; Park, Kyungmoon; Lee, Jinwon

    2012-06-01

    The global pool of intracellular metabolites is a reflection of all the metabolic functions of an organism. In the absence of in situ methods capable of directly measuring metabolite pools, intracellular metabolite measurements need to be performed after an extraction procedure. In this study, we evaluated the optimization of technologies for generation of a global metabolomics profile for intracellular metabolites in Klebsiella oxytoca. Intracellular metabolites of K. oxytoca were extracted at the early stationary phase using six different common extraction procedures, including cold methanol, boiling ethanol, methanol/chloroform combinations, hot water, potassium hydroxide, and perchloric acid. The metabolites were subsequently collected for further analysis, and intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. During analysis, the stability of metabolites extracted using cold methanol was clearly higher than that obtained by other extraction methods. For the majority of metabolites, extracts generated in this manner exhibited the greatest recovery, with high reproducibility. Therefore, the use of cold ethanol was the best extraction method for attaining a metabolic profile. However, in another parallel extraction method, perchloric acid may also be required to maximize the range of metabolites recovered, particularly to extract glucose 1-phosphate and NADPH.

  7. Comparative Metabolite Profiling of Two Rice Genotypes with Contrasting Salt Stress Tolerance at the Seedling Stage

    PubMed Central

    Zhang, Fan; Deng, Jianli; Li, Zhikang; Fu, Binying

    2014-01-01

    Background Rice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level. Methods Gas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated. Results We characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na+ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment. Conclusions Metabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress. PMID:25265195

  8. Stability of targeted metabolite profiles of urine samples under different storage conditions.

    PubMed

    Rotter, Markus; Brandmaier, Stefan; Prehn, Cornelia; Adam, Jonathan; Rabstein, Sylvia; Gawrych, Katarzyna; Brüning, Thomas; Illig, Thomas; Lickert, Heiko; Adamski, Jerzy; Wang-Sattler, Rui

    2017-01-01

    Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS). We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics. Pooled, fasting urine samples were collected and stored at -80 °C (biobank standard), -20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ™ p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature. The concentrations of 63 investigated metabolites were stable at -20 and 4 °C for up to 24 h when compared to samples immediately stored at -80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E-04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature. Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.

  9. Quantitative 1H Nuclear Magnetic Resonance Metabolite Profiling as a Functional Genomics Platform to Investigate Alkaloid Biosynthesis in Opium Poppy1[W

    PubMed Central

    Hagel, Jillian M.; Weljie, Aalim M.; Vogel, Hans J.; Facchini, Peter J.

    2008-01-01

    Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The plant is widely cultivated as the only commercial source of the narcotic analgesics morphine and codeine. Variations in plant secondary metabolism as a result of genetic diversity are often associated with perturbations in other metabolic pathways. As part of a functional genomics platform, we used 1H nuclear magnetic resonance (NMR) metabolite profiling for the analysis of primary and secondary metabolism in opium poppy. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principle component analysis of the 1H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety and a high-thebaine, low-morphine cultivar. Distinction was also made between pharmaceutical-grade opium poppy cultivars and a condiment variety. Such phenotypic differences were not observed in root extracts. Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Quantification of 34 root and 21 latex metabolites, performed using Chenomx NMR Suite version 4.6, showed major differences in the accumulation of specific alkaloids in the latex of the low-alkaloid and high-thebaine, low-morphine varieties. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. Exceptions in the low-alkaloid cultivar included an increased accumulation of the alkaloid precursor tyramine and reduced levels of sucrose, some amino acids, and malate. Real-time polymerase chain reaction analysis of 42 genes involved in primary and secondary metabolism showed differential gene expression mainly associated with alkaloid biosynthesis. Reduced alkaloid levels in the condiment variety were associated with the

  10. Mass spectrometry-based metabolite profiling in the mouse liver following exposure to ultraviolet B radiation.

    PubMed

    Park, Hye Min; Shon, Jong Cheol; Lee, Mee Youn; Liu, Kwang-Hyeon; Kim, Jeong Kee; Lee, Sang Jun; Lee, Choong Hwan

    2014-01-01

    Although many studies have been performed on the effects of ultraviolet (UV) radiation on the skin, only a limited number of reports have investigated these effects on non-skin tissue. This study aimed to describe the metabolite changes in the liver of hairless mice following chronic exposure to UVB radiation. We did not observe significant macroscopic changes or alterations in hepatic cholesterol and triglyceride levels in the liver of UVB-irradiated mice, compared with those for normal mice. In this study, we detected hepatic metabolite changes by UVB exposure and identified several amino acids, fatty acids, nucleosides, carbohydrates, phospholipids, lysophospholipids, and taurine-conjugated cholic acids as candidate biomarkers in response to UVB radiation in the mouse liver by using various mass spectrometry (MS)-based metabolite profiling including ultra-performance liquid chromatography-quadrupole time-of-flight (TOF)-MS, gas chromatography-TOF-MS and nanomate LTQ-MS. Glutamine exhibited the most dramatic change with a 5-fold increase in quantity. The results from altering several types of metabolites suggest that chronic UVB irradiation may impact significantly on major hepatic metabolism processes, despite the fact that the liver is not directly exposed to UVB radiation. MS-based metabolomic approach for determining regulatory hepatic metabolites following UV irradiation will provide a better understanding of the relationship between internal organs and UV light.

  11. Multi-Omics Analysis Reveals a Correlation between the Host Phylogeny, Gut Microbiota and Metabolite Profiles in Cyprinid Fishes.

    PubMed

    Li, Tongtong; Long, Meng; Li, Huan; Gatesoupe, François-Joël; Zhang, Xujie; Zhang, Qianqian; Feng, Dongyue; Li, Aihua

    2017-01-01

    Gut microbiota play key roles in host nutrition and metabolism. However, little is known about the relationship between host genetics, gut microbiota and metabolic profiles. Here, we used high-throughput sequencing and gas chromatography/mass spectrometry approaches to characterize the microbiota composition and the metabolite profiles in the gut of five cyprinid fish species with three different feeding habits raised under identical husbandry conditions. Our results showed that host species and feeding habits significantly affect not only gut microbiota composition but also metabolite profiles (ANOSIM, p ≤ 0.05). Mantel test demonstrated that host phylogeny, gut microbiota, and metabolite profiles were significantly related to each other (p ≤ 0.05). Additionally, the carps with the same feeding habits had more similarity in gut microbiota composition and metabolite profiles. Various metabolites were correlated positively with bacterial taxa involved in food degradation. Our results shed new light on the microbiome and metabolite profiles in the gut content of cyprinid fishes, and highlighted the correlations between host genotype, fish gut microbiome and putative functions, and gut metabolite profiles.

  12. Multi-Omics Analysis Reveals a Correlation between the Host Phylogeny, Gut Microbiota and Metabolite Profiles in Cyprinid Fishes

    PubMed Central

    Li, Tongtong; Long, Meng; Li, Huan; Gatesoupe, François-Joël; Zhang, Xujie; Zhang, Qianqian; Feng, Dongyue; Li, Aihua

    2017-01-01

    Gut microbiota play key roles in host nutrition and metabolism. However, little is known about the relationship between host genetics, gut microbiota and metabolic profiles. Here, we used high-throughput sequencing and gas chromatography/mass spectrometry approaches to characterize the microbiota composition and the metabolite profiles in the gut of five cyprinid fish species with three different feeding habits raised under identical husbandry conditions. Our results showed that host species and feeding habits significantly affect not only gut microbiota composition but also metabolite profiles (ANOSIM, p ≤ 0.05). Mantel test demonstrated that host phylogeny, gut microbiota, and metabolite profiles were significantly related to each other (p ≤ 0.05). Additionally, the carps with the same feeding habits had more similarity in gut microbiota composition and metabolite profiles. Various metabolites were correlated positively with bacterial taxa involved in food degradation. Our results shed new light on the microbiome and metabolite profiles in the gut content of cyprinid fishes, and highlighted the correlations between host genotype, fish gut microbiome and putative functions, and gut metabolite profiles. PMID:28367147

  13. Associations between intensive diabetes therapy and NMR-determined lipoprotein subclass profiles in type 1 diabetes.

    PubMed

    Zhang, Ying; Jenkins, Alicia J; Basu, Arpita; Stoner, Julie A; Lopes-Virella, Maria F; Klein, Richard L; Lyons, Timothy J

    2016-02-01

    Our objective is to define differences in circulating lipoprotein subclasses between intensive versus conventional management of type 1 diabetes during the randomization phase of the Diabetes Control and Complications Trial (DCCT). NMR-determined lipoprotein subclass profiles (NMR-LSPs), which estimate molar subclass concentrations and mean particle diameters, were determined in 1,294 DCCT subjects after a median of 5 years (interquartile range: 4-6 years) of randomization to intensive or conventional diabetes management. In cross-sectional analyses, we compared standard lipids and NMR-LSPs between treatment groups. Standard total, LDL, and HDL cholesterol levels were similar between randomization groups, while triglyceride levels were lower in the intensively treated group. NMR-LSPs showed that intensive therapy was associated with larger LDL diameter (20.7 vs. 20.6 nm, P = 0.01) and lower levels of small LDL (median: 465 vs. 552 nmol/l, P = 0.007), total IDL/LDL (mean: 1,000 vs. 1,053 nmol/l, P = 0.01), and small HDL (mean: 17.3 vs. 18.6 μmol/l, P < 0.0001), the latter accounting for reduced total HDL (mean: 33.8 vs. 34.8 μmol/l, P = 0.01). In conclusion, intensive diabetes therapy was associated with potentially favorable changes in LDL and HDL subclasses in sera. Further research will determine whether these changes contribute to the beneficial effects of intensive diabetes management on vascular complications.

  14. Elucidation of ethanol tolerance mechanisms in Saccharomyces cerevisiae by global metabolite profiling.

    PubMed

    Kim, Sooah; Kim, Jungyeon; Song, Ju Hwan; Jung, Young Hoon; Choi, Il-Sup; Choi, Wonja; Park, Yong-Cheol; Seo, Jin-Ho; Kim, Kyoung Heon

    2016-09-01

    Ethanol, the major fermentation product of yeast, is a stress factor in yeast. We previously constructed an ethanol-tolerant mutant yeast iETS3 by using the global transcriptional machinery engineering. However, the ethanol-tolerance mechanism has not been systematically investigated. In this study, global metabolite profiling was carried out, mainly by gas chromatography/time-of-flight mass spectrometry (GC/TOF MS), to investigate the mechanisms of ethanol tolerance in iETS3. A total of 108 intracellular metabolites were identified by GC/TOF MS and high performance liquid chromatography, and these metabolites were mostly intermediates of the central carbon metabolism. The metabolite profiles of iETS3 and BY4741, cultured with or without ethanol, were significantly different based on principal component and hierarchical clustering analyses. Our metabolomic analyses identified the compositional changes in cell membranes and the activation of glutamate metabolism and the trehalose synthetic pathway as the possible mechanisms for the ethanol tolerance. These metabolic traits can be considered possible targets for further improvement of ethanol tolerance in the mutant. For example, the KGD1 deletion mutant, with up-regulated glutamate metabolism, showed increased tolerance to ethanol. This study has demonstrated that metabolomics can be a useful tool for strain improvement and phenotypic analysis of microorganisms under stress. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Biomarkers are used to predict quantitative metabolite concentration profiles in human red blood cells

    DOE PAGES

    Yurkovich, James T.; Yang, Laurence; Palsson, Bernhard O.; ...

    2017-03-06

    Deep-coverage metabolomic profiling has revealed a well-defined development of metabolic decay in human red blood cells (RBCs) under cold storage conditions. A set of extracellular biomarkers has been recently identified that reliably defines the qualitative state of the metabolic network throughout this metabolic decay process. Here, we extend the utility of these biomarkers by using them to quantitatively predict the concentrations of other metabolites in the red blood cell. We are able to accurately predict the concentration profile of 84 of the 91 (92%) measured metabolites (p < 0.05) in RBC metabolism using only measurements of these five biomarkers. Themore » median of prediction errors (symmetric mean absolute percent error) across all metabolites was 13%. Furthermore, the ability to predict numerous metabolite concentrations from a simple set of biomarkers offers the potential for the development of a powerful workflow that could be used to evaluate the metabolic state of a biological system using a minimal set of measurements.« less

  16. Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time-of-flight mass spectrometry.

    PubMed

    Chan, Eric C Y; New, Lee Sun; Yap, Chun Wei; Goh, Lin Tang

    2009-02-01

    The use of hybrid quadrupole ion mobility spectrometry time-of-flight mass spectrometry (Q/IMS/TOFMS) in the metabolite profiling of leflunomide (LEF) and acetaminophen (APAP) is presented. The IMS drift times (T(d)) of the drugs and their metabolites were determined in the IMS/TOFMS experiments and correlated with their exact monoisotopic masses and other in silico generated structural properties, such as connolly molecular area (CMA), connolly solvent-excluded volume (CSEV), principal moments of inertia along the X, Y and Z Cartesian coordinates (MI-X, MI-Y and MI-Z), inverse mobility and collision cross-section (CCS). The correlation of T(d) with these parameters is presented and discussed. IMS/TOF tandem mass spectrometry experiments (MS(2) and MS(3)) were successfully performed on the N-acetyl-p-benzoquinoneimine glutathione (NAPQI-GSH) adduct derived from the in vitro microsomal metabolism of APAP. As comparison, similar experiments were also performed using hybrid triple quadrupole linear ion trap mass spectrometry (QTRAPMS) and quadrupole time-of-flight mass spectrometry (QTOFMS). The abilities to resolve the product ions of the metabolite within the drift tube and fragment the ion mobility resolved product ions in the transfer travelling wave-enabled stacked ring ion guide (TWIG) demonstrated the potential applicability of the Q/IMS/TOFMS technique in pharmaceutical metabolite profiling.

  17. Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling.

    PubMed

    Wase, Nishikant; Black, Paul N; Stanley, Bruce A; DiRusso, Concetta C

    2014-03-07

    Nitrogen starvation induces a global stress response in microalgae that results in the accumulation of lipids as a potential source of biofuel. Using GC-MS-based metabolite and iTRAQ-labeled protein profiling, we examined and correlated the metabolic and proteomic response of Chlamydomonas reinhardtii under nitrogen stress. Key amino acids and metabolites involved in nitrogen sparing pathways, methyl group transfer reactions, and energy production were decreased in abundance, whereas certain fatty acids, citric acid, methionine, citramalic acid, triethanolamine, nicotianamine, trehalose, and sorbitol were increased in abundance. Proteins involved in nitrogen assimilation, amino acid metabolism, oxidative phosphorylation, glycolysis, TCA cycle, starch, and lipid metabolism were elevated compared with nonstressed cultures. In contrast, the enzymes of the glyoxylate cycle, one carbon metabolism, pentose phosphate pathway, the Calvin cycle, photosynthetic and light harvesting complex, and ribosomes were reduced. A noteworthy observation was that citrate accumulated during nitrogen stress coordinate with alterations in the enzymes that produce or utilize this metabolite, demonstrating the value of comparing protein and metabolite profiles to understand complex patterns of metabolic flow. Thus, the current study provides unique insight into the global metabolic adjustments leading to lipid storage during N starvation for application toward advanced biofuel production technologies.

  18. An approach to the simultaneous quantitative analysis of metabolites in table wines by (1)H NMR self-constructed three-dimensional spectra.

    PubMed

    Li, Bao Qiong; Xu, Min Li; Wang, Xue; Zhai, Hong Lin; Chen, Jing; Liu, Jin Jin

    2017-02-01

    Wine consists of several hundred components with different concentrations, including water, ethanol, glycerol, organic acids and sugars. Accurate quantification of target compounds in such complex samples is a difficult task based on conventional (1)H NMR spectra due to some challenges. In this paper, the three-dimensional spectrum was constructed firstly by simply repeating (1)H NMR spectrum itself so as to extract the features of target compounds by Tchebichef moment method. A proof-of-concept model system, the determination of five metabolites in wines was utilized to evaluate the performance of the proposed strategy. The results indicate that the proposed approach can provide accurate and reliable concentration predictions, probably the best results ever achieved using PLS and interval-PLS methods. Our novel strategy has not only good performance but also does not require laborious multi-step and subjective pretreatments. Therefore, it is expected that the proposed method could extend the application of conventional (1)H NMR.

  19. Characterization of metabolites of leonurine (SCM-198) in rats after oral administration by liquid chromatography/tandem mass spectrometry and NMR spectrometry.

    PubMed

    Zhu, Qing; Zhang, Jinlian; Yang, Ping; Tan, Bo; Liu, Xinhua; Zheng, Yuanting; Cai, Weimin; Zhu, Yizhun

    2014-01-01

    Leonurine, a major bioactive component from Herba Leonuri, shows therapeutic potential for cardiovascular disease and stroke prevention in some preclinical experiments. The aim of this study is to characterize metabolites of leonurine in rats using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC/MS/MS). The chromatographic separation was performed on an Agilent ZORBAX SB-C18 column using a gradient elution with acetonitrile/ammonium acetate buffer (10 mM, pH 4.0) solvent system. An information dependent acquisition (IDA) method was developed for screening and identifying metabolites of leonurine under positive ion mode. Compared with control, the interesting compound in the extracted ion chromatogram (XIC) of the in vivo samples was chosen and further identified by analyzing their retention times, changes in observed mass (Δm/z), and spectral patterns of product ion utilizing advanced software tool. For the first time, a total of three metabolites were identified, including two phase II metabolites generated by glucuronidation (M1) and sulfation (M2) and one phase I metabolite formed by O-demethylation (M3). Finally, the lead metabolite M1 was isolated from urine and its structure was characterized as leonurine-10-O- β-D-glucuronide by NMR spectroscopy (¹H, ¹³C, HMBC, and HSQC).

  20. Profiling of Intracellular Metabolites: An Approach to Understanding the Characteristic Physiology of Mycobacterium leprae.

    PubMed

    Miyamoto, Yuji; Mukai, Tetsu; Matsuoka, Masanori; Kai, Masanori; Maeda, Yumi; Makino, Masahiko

    2016-08-01

    Mycobacterium leprae is the causative agent of leprosy and also known to possess unique features such as inability to proliferate in vitro. Among the cellular components of M. leprae, various glycolipids present on the cell envelope are well characterized and some of them are identified to be pathogenic factors responsible for intracellular survival in host cells, while other intracellular metabolites, assumed to be associated with basic physiological feature, remain largely unknown. In the present study, to elucidate the comprehensive profile of intracellular metabolites, we performed the capillary electrophoresis-mass spectrometry (CE-MS) analysis on M. leprae and compared to that of M. bovis BCG. Interestingly, comparison of these two profiles showed that, in M. leprae, amino acids and their derivatives are significantly accumulated, but most of intermediates related to central carbon metabolism markedly decreased, implying that M. leprae possess unique metabolic features. The present study is the first report demonstrating the unique profiles of M. leprae metabolites and these insights might contribute to understanding undefined metabolism of M. leprae as well as pathogenic characteristics related to the manifestation of the disease.

  1. Water and salinity stress in grapevines: early and late changes in transcript and metabolite profiles.

    PubMed

    Cramer, Grant R; Ergül, Ali; Grimplet, Jerome; Tillett, Richard L; Tattersall, Elizabeth A R; Bohlman, Marlene C; Vincent, Delphine; Sonderegger, Justin; Evans, Jason; Osborne, Craig; Quilici, David; Schlauch, Karen A; Schooley, David A; Cushman, John C

    2007-04-01

    Grapes are grown in semiarid environments, where drought and salinity are common problems. Microarray transcript profiling, quantitative reverse transcription-PCR, and metabolite profiling were used to define genes and metabolic pathways in Vitis vinifera cv. Cabernet Sauvignon with shared and divergent responses to a gradually applied and long-term (16 days) water-deficit stress and equivalent salinity stress. In this first-of-a-kind study, distinct differences between water deficit and salinity were revealed. Water deficit caused more rapid and greater inhibition of shoot growth than did salinity at equivalent stem water potentials. One of the earliest responses to water deficit was an increase in the transcript abundance of RuBisCo activase (day 4), but this increase occurred much later in salt-stressed plants (day 12). As water deficit progressed, a greater number of affected transcripts were involved in metabolism, transport, and the biogenesis of cellular components than did salinity. Salinity affected a higher percentage of transcripts involved in transcription, protein synthesis, and protein fate than did water deficit. Metabolite profiling revealed that there were higher concentrations of glucose, malate, and proline in water-deficit-treated plants as compared to salinized plants. The metabolite differences were linked to differences in transcript abundance of many genes involved in energy metabolism and nitrogen assimilation, particularly photosynthesis, gluconeogenesis, and photorespiration. Water-deficit-treated plants appear to have a higher demand than salinized plants to adjust osmotically, detoxify free radicals (reactive oxygen species), and cope with photoinhibition.

  2. NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations.

    PubMed

    Helmling, Christina; Wacker, Anna; Wolfinger, Michael T; Hofacker, Ivo L; Hengesbach, Martin; Fürtig, Boris; Schwalbe, Harald

    2017-02-22

    Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

  3. NMR-based metabolic profiling of rice wines by F(2)-selective total correlation spectra.

    PubMed

    Koda, Masanori; Furihata, Kazuo; Wei, Feifei; Miyakawa, Takuya; Tanokura, Masaru

    2012-05-16

    In this study, we performed NMR-based metabolic profiling of major rice wines (Japanese sake, Chinese Shaoxing wine, and Korean makgeolli). In the (1)H NMR spectra, the rice wines showed broad resonances in the region of about 7.9-9.0 ppm. These resonances showed many and complex correlations with approximately 0.5-4.5 ppm in the F(2)-selective TOCSY (total correlation spectroscopy) spectra, and these correlations were attributed mainly to peptides. These spectral patterns were characteristic of individual rice wines, and the combination of F(2)-selective TOCSY spectra and principal component analysis enabled us to classify the rice wine species. Furthermore, it also provided information about raw materials, namely, what type of koji (rice koji or wheat koji) was used. These spectra may be useful as a new "fingerprint" for quality control or food authentication.

  4. 1H NMR profiling as an approach to differentiate conventionally and organically grown tomatoes.

    PubMed

    Hohmann, Monika; Christoph, Norbert; Wachter, Helmut; Holzgrabe, Ulrike

    2014-08-20

    This study describes the approach of (1)H NMR profiling for the authentication of organically produced tomatoes (Solanum lycopersicum). Overall, 361 tomato samples of two different cultivars and four different producers were regularly analyzed during a 7 month period. The results of principal component analysis showed a significant trend for the separation between organically and conventionally produced tomatoes (p < 0.001 using the t test). Linear discriminant analysis demonstrated good discrimination between the growing regimens, and external validation showed 100% correctly classified tomato samples. Further validation studies, however, also disclosed unexpected differences between individual producers, which interfere with the aim of predicting the cultivation method, yet the results indicate significant differences between (1)H NMR spectra of organically and conventionally grown tomatoes.

  5. NMR and GC-MS Based Metabolic Profiling and Free-Radical Scavenging Activities of Cordyceps pruinosa Mycelia Cultivated under Different Media and Light Conditions

    PubMed Central

    Oh, Taek-Joo; Hyun, Sun-Hee; Lee, Seul-Gi; Chun, Young-Jin; Sung, Gi-Ho; Choi, Hyung-Kyoon

    2014-01-01

    Variation of metabolic profiles in Cordyceps pruinosa mycelia cultivated under various media and light conditions was investigated using 1H nuclear magnetic resonance (NMR) analysis and gas chromatography mass spectrometry (GC-MS) with multivariate statistical analysis. A total of 71 metabolites were identified (5 alcohols, 21 amino acids, 15 organic acids, 4 purines, 3 pyrimidines, 7 sugars, 11 fatty acids, and 5 other metabolites) by NMR and GC-MS analysis. The mycelia grown in nitrogen media and under dark conditions showed the lowest growth and ergosterol levels, essential to a functional fungal cell membrane; these mycelia, however, had the highest levels of putrescine, which is involved in abiotic stress tolerance. In contrast, mycelia cultivated in sabouraud dextrose agar with yeast extract (SDAY) media and under light conditions contained relatively higher levels of fatty acids, including valeric acid, stearic acid, lignoceric acid, myristic acid, oleic acid, palmitoleic acid, hepadecenoic acid, and linoleic acid. These mycelia also had the highest phenolic content and antioxidant activity, and did not exhibit growth retardation due to enhanced asexual development caused by higher levels of linoleic acid. Therefore, we suggested that a light-enriched environment with SDAY media was more optimal than dark condition for cultivation of C. pruinosa mycelia as biopharmaceutical or nutraceutical resources. PMID:24608751

  6. Quantitative Metabolomic Profiling of Plasma, Urine, and Liver Extracts by (1)H NMR Spectroscopy Characterizes Different Stages of Atherosclerosis in Hamsters.

    PubMed

    Guo, Wei; Jiang, Chunying; Yang, Liu; Li, Tianqi; Liu, Xia; Jin, Mengxia; Qu, Kai; Chen, Huili; Jin, Xiangju; Liu, Hongyue; Zhu, Haibo; Wang, Yinghong

    2016-10-07

    Atherosclerosis (AS) is a progressive disease that contributes to cardiovascular disease and shows a complex etiology, including genetic and environmental factors. To understand systemic metabolic changes and to identify potential biomarkers correlated with the occurrence and perpetuation of diet-induced AS, we applied (1)H NMR-based metabolomics to detect the time-related metabolic profiles of plasma, urine, and liver extracts from male hamsters fed a high fat and high cholesterol (HFHC) diet. Conventional biochemical assays and histopathological examinations as well as protein expression analyses were performed to provide complementary information. We found that diet treatment caused obvious aortic lesions, lipid accumulation, and inflammatory infiltration in hamsters. Downregulation of proteins related to cholesterol metabolism, including hepatic SREBP2, LDL-R, CYP7A1, SR-BI, HMGCR, LCAT, and SOAT1 was detected, which elucidated the perturbation of cholesterol homeostasis during the HFHC diet challenge. Using "targeted analysis", we quantified 40 plasma, 80 urine, and 60 liver hydrophilic extract metabolites. Multivariate analyses of the identified metabolites elucidated sophisticated metabolic disturbances in multiple matrices, including energy homeostasis, intestinal microbiota functions, inflammation, and oxidative stress coupled with the metabolisms of cholesterol, fatty acids, saccharides, choline, amino acids, and nucleotides. For the first time, our results demonstrate a time-dependent metabolic progression of multiple biological matrices in hamsters from physiological status to early AS and further to late-stage AS, demonstrating that (1)H NMR-based metabolomics is a reliable tool for early diagnosis and monitoring of the process of AS.

  7. Metabolite profiling on wheat grain to enable a distinction of samples from organic and conventional farming systems.

    PubMed

    Bonte, Anja; Neuweger, Heiko; Goesmann, Alexander; Thonar, Cécile; Mäder, Paul; Langenkämper, Georg; Niehaus, Karsten

    2014-10-01

    Identification of biomarkers capable of distinguishing organic and conventional products would be highly welcome to improve the strength of food quality assurance. Metabolite profiling was used for biomarker search in organic and conventional wheat grain (Triticum aestivum L.) of 11 different old and new bread wheat cultivars grown in the DOK system comparison trial. Metabolites were extracted using methanol and analysed by gas chromatography-mass spectrometry. Altogether 48 metabolites and 245 non-identified metabolites (TAGs) were detected in the cultivar Runal. Principal component analysis showed a sample clustering according to farming systems and significant differences in peak areas between the farming systems for 10 Runal metabolites. Results obtained from all 11 cultivars indicated a greater influence of the cultivar than the farming system on metabolite concentrations. Nevertheless, a t-test on data of all cultivars still detected 5 metabolites and 11 TAGs with significant differences between the farming systems. Based on individual cultivars, metabolite profiling showed promising results for the categorization of organic and conventional wheat. Further investigations are necessary with wheat from more growing seasons and locations before definite conclusions can be drawn concerning the feasibility to evolve a combined set of biomarkers for organically grown wheat using metabolite profiles. © 2014 Society of Chemical Industry.

  8. Fully Automated Trimethylsilyl (TMS) Derivatisation Protocol for Metabolite Profiling by GC-MS

    PubMed Central

    Zarate, Erica; Boyle, Veronica; Rupprecht, Udo; Green, Saras; Villas-Boas, Silas G.; Baker, Philip; Pinu, Farhana R.

    2016-01-01

    Gas Chromatography-Mass Spectrometry (GC-MS) has long been used for metabolite profiling of a wide range of biological samples. Many derivatisation protocols are already available and among these, trimethylsilyl (TMS) derivatisation is one of the most widely used in metabolomics. However, most TMS methods rely on off-line derivatisation prior to GC-MS analysis. In the case of manual off-line TMS derivatisation, the derivative created is unstable, so reduction in recoveries occurs over time. Thus, derivatisation is carried out in small batches. Here, we present a fully automated TMS derivatisation protocol using robotic autosamplers and we also evaluate a commercial software, Maestro available from Gerstel GmbH. Because of automation, there was no waiting time of derivatised samples on the autosamplers, thus reducing degradation of unstable metabolites. Moreover, this method allowed us to overlap samples and improved throughputs. We compared data obtained from both manual and automated TMS methods performed on three different matrices, including standard mix, wine, and plasma samples. The automated TMS method showed better reproducibility and higher peak intensity for most of the identified metabolites than the manual derivatisation method. We also validated the automated method using 114 quality control plasma samples. Additionally, we showed that this online method was highly reproducible for most of the metabolites detected and identified (RSD < 20) and specifically achieved excellent results for sugars, sugar alcohols, and some organic acids. To the very best of our knowledge, this is the first time that the automated TMS method has been applied to analyse a large number of complex plasma samples. Furthermore, we found that this method was highly applicable for routine metabolite profiling (both targeted and untargeted) in any metabolomics laboratory. PMID:28036063

  9. Fully Automated Trimethylsilyl (TMS) Derivatisation Protocol for Metabolite Profiling by GC-MS.

    PubMed

    Zarate, Erica; Boyle, Veronica; Rupprecht, Udo; Green, Saras; Villas-Boas, Silas G; Baker, Philip; Pinu, Farhana R

    2016-12-29

    Gas Chromatography-Mass Spectrometry (GC-MS) has long been used for metabolite profiling of a wide range of biological samples. Many derivatisation protocols are already available and among these, trimethylsilyl (TMS) derivatisation is one of the most widely used in metabolomics. However, most TMS methods rely on off-line derivatisation prior to GC-MS analysis. In the case of manual off-line TMS derivatisation, the derivative created is unstable, so reduction in recoveries occurs over time. Thus, derivatisation is carried out in small batches. Here, we present a fully automated TMS derivatisation protocol using robotic autosamplers and we also evaluate a commercial software, Maestro available from Gerstel GmbH. Because of automation, there was no waiting time of derivatised samples on the autosamplers, thus reducing degradation of unstable metabolites. Moreover, this method allowed us to overlap samples and improved throughputs. We compared data obtained from both manual and automated TMS methods performed on three different matrices, including standard mix, wine, and plasma samples. The automated TMS method showed better reproducibility and higher peak intensity for most of the identified metabolites than the manual derivatisation method. We also validated the automated method using 114 quality control plasma samples. Additionally, we showed that this online method was highly reproducible for most of the metabolites detected and identified (RSD < 20) and specifically achieved excellent results for sugars, sugar alcohols, and some organic acids. To the very best of our knowledge, this is the first time that the automated TMS method has been applied to analyse a large number of complex plasma samples. Furthermore, we found that this method was highly applicable for routine metabolite profiling (both targeted and untargeted) in any metabolomics laboratory.

  10. Changes in metabolite profiles caused by genetically determined obesity in mice.

    PubMed

    Schäfer, Nadine; Yu, Zhonghao; Wagener, Asja; Millrose, Marion K; Reissmann, Monika; Bortfeldt, Ralf; Dieterich, Christoph; Adamski, Jerzy; Wang-Sattler, Rui; Illig, Thomas; Brockmann, Gudrun A

    2014-01-01

    The Berlin Fat Mouse Inbred (BFMI) line harbors a major recessive gene defect on chromosome 3 (jobes1) leading to juvenile obesity and metabolic syndrome. The present study aimed at the identification of metabolites that might be linked to recessively acting genes in the obesity locus. Firstly, serum metabolites were analyzed between obese BFMI and lean B6 and BFMI × B6 F1 mice to identify metabolites that are different. In a second step, a metabolite-protein network analysis was performed linking metabolites typical for BFMI mice with genes of the jobes1 region. The levels of 22 diacyl-phosphatidylcholines (PC aa), two lyso-PC and three carnitines were found to be significantly lower in obese mice compared with lean mice, while serine, glycine, arginine and hydroxysphingomyelin were higher for the same comparison. The network analysis identified PC aa C42:1 as functionally linked with the genes Ccna2 and Trpc3 via the enzymes choline kinase alpha and phospholipase A2 group 1B (PLA2G1B), respectively. Gene expression analysis revealed elevated Ccna2 expression in adipose tissue of BFMI mice. Furthermore, unique mutations were found in the Ccna2 promoter of BFMI mice which are located in binding sites for transcription factors or micro RNAs and could cause differential Ccna2 mRNA levels between BFMI and B6 mice. Increased expression of Ccna2 was consistent with higher mitotic activity of adipose tissue in BFMI mice. Therefore, we suggest a higher demand for PC necessary for adipose tissue growth and remodeling. This study highlights the relationship between metabolite profiles and the underlying genetics of obesity in the BFMI line.

  11. Two new flavonol glycosides and a metabolite profile of Bryophyllum pinnatum, a phytotherapeutic used in obstetrics and gynaecology.

    PubMed

    Fürer, Karin; Raith, Melanie; Brenneisen, Rudolf; Mennet, Monica; Simões-Wüst, Ana Paula; von Mandach, Ursula; Hamburger, Matthias; Potterat, Olivier

    2013-11-01

    Bryophyllum pinnatum is a succulent perennial plant native to Madagascar which is used in anthroposophical medicine to treat psychiatric disorders and as a tocolytic agent to prevent premature labour. We performed a metabolite profiling study in order to obtain a comprehensive picture of the constituents in B. pinnatum leaves and to identify chromatographic markers for quality control and safety assessment of medicinal preparations. Preliminary HPLC-PDA-ESIMS analyses revealed that flavonoid glycosides were the main UV-absorbing constituents in the MeOH extract of B. pinnatum. Two phenolic glucosides, syringic acid β-D-glucopyranosyl ester (1) and 4'-O-β-D-glucopyranosyl-cis-p-coumaric acid (2), as well as nine flavonoids (3-11) including kaempferol, quercetin, myricetin, acacetin, and diosmetin glycosides were unambiguously identified by 1H and 2D NMR analysis after isolation from a MeOH extract. The flavonol glycosides quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside 7-O-β-D-glucopyranoside (3) and myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (4) were new natural products. With the aid of HPLC-PDA-APCIMS and authentic references isolated from the related species B. daigremontianum, the presence of four bufadienolides, bersaldegenin-1-acetate (12), bryophyllin A (13), bersaldegenin-3-acetate (14), and bersaldegenin-1,3,5-orthoacetate (15) was detected in B. pinnatum.

  12. Metabolite and Lipid Profiling of Biobank Plasma Samples Collected Prior to Onset of Rheumatoid Arthritis

    PubMed Central

    Surowiec, Izabella; Ärlestig, Lisbeth

    2016-01-01

    Objective The early diagnosis of rheumatoid arthritis (RA) is desirable to install treatment to prevent disease progression and joint destruction. Autoantibodies and immunological markers pre-date the onset of symptoms by years albeit not all patients will present these factors, even at disease onset. Additional biomarkers would be of high value to improve early diagnosis and understanding of the process, leading to disease development. Methods Plasma samples donated before the onset of RA were identified in the Biobank of Northern Sweden, a collection within national health survey programs. Thirty samples from pre-symptomatic individuals and nineteen from controls were subjected to liquid chromatography-mass spectrometry (LCMS) metabolite and lipid profiling. Lipid and metabolite profiles discriminating samples from pre-symptomatic individuals from controls were identified after univariate and multivariate OPLS-DA based analyses. Results The OPLS-DA models including pre-symptomatic individuals and controls identified profiles differentiating between the groups that was characterized by lower levels of acyl-carnitines and fatty acids, with higher levels of lysophospatidylcholines (LPCs) and metabolites from tryptophan metabolism in pre-symptomatic individuals compared with controls. Lipid profiling showed that the majority of phospholipids and sphingomyelins were at higher levels in pre-symptomatic individuals in comparison with controls. Conclusions Our LCMS based approach demonstrated that there are changes in small molecule and lipid profiles detectable in plasma samples collected from the pre-symptomatic individuals who subsequently developed RA, which point to an up-regulation of levels of lysophospatidylcholines, and of tryptophan metabolism, perturbation of fatty acid beta-oxidation and increased oxidative stress in pre-symptomatic individuals’ years before onset of symptoms. PMID:27755546

  13. Metabolite Profiling of Diverse Rice Germplasm and Identification of Conserved Metabolic Markers of Rice Roots in Response to Long-Term Mild Salinity Stress

    PubMed Central

    Nam, Myung Hee; Bang, Eunjung; Kwon, Taek Yun; Kim, Yuran; Kim, Eun Hee; Cho, Kyungwon; Park, Woong June; Kim, Beom-Gi; Yoon, In Sun

    2015-01-01

    The sensitivity of rice to salt stress greatly depends on growth stages, organ types and cultivars. Especially, the roots of young rice seedlings are highly salt-sensitive organs that limit plant growth, even under mild soil salinity conditions. In an attempt to identify metabolic markers of rice roots responding to salt stress, metabolite profiling was performed by 1H-NMR spectroscopy in 38 rice genotypes that varied in biomass accumulation under long-term mild salinity condition. Multivariate statistical analysis showed separation of the control and salt-treated rice roots and rice genotypes with differential growth potential. By quantitative analyses of 1H-NMR data, five conserved salt-responsive metabolic markers of rice roots were identified. Sucrose, allantoin and glutamate accumulated by salt stress, whereas the levels of glutamine and alanine decreased. A positive correlation of metabolite changes with growth potential and salt tolerance of rice genotypes was observed for allantoin and glutamine. Adjustment of nitrogen metabolism in rice roots is likely to be closely related to maintain the growth potential and increase the stress tolerance of rice. PMID:26378525

  14. Alteration of the fecal microbiota and serum metabolite profiles in dogs with idiopathic inflammatory bowel disease

    PubMed Central

    Minamoto, Yasushi; Otoni, Cristiane C; Steelman, Samantha M; Büyükleblebici, Olga; Steiner, Jörg M; Jergens, Albert E; Suchodolski, Jan S

    2015-01-01

    Idiopathic inflammatory bowel disease (IBD) is a common cause of chronic gastrointestinal (GI) disease in dogs. The combination of an underlying host genetic susceptibility, an intestinal dysbiosis, and dietary/environmental factors are suspected as main contributing factors in the pathogenesis of canine IBD. However, actual mechanisms of the host-microbe interactions remain elusive. The aim of this study was to compare the fecal microbiota and serum metabolite profiles between healthy dogs (n = 10) and dogs with IBD before and after 3 weeks of medical therapy (n = 12). Fecal microbiota and metabolite profiles were characterized by 454-pyrosequencing of 16 S rRNA genes and by an untargeted metabolomics approach, respectively. Significantly lower bacterial diversity and distinct microbial communities were observed in dogs with IBD compared to the healthy control dogs. While Gammaproteobacteria were overrepresented, Erysipelotrichia, Clostridia, and Bacteroidia were underrepresented in dogs with IBD. The functional gene content was predicted from the 16 S rRNA gene data using PICRUSt, and revealed overrepresented bacterial secretion system and transcription factors, and underrepresented amino acid metabolism in dogs with IBD. The serum metabolites 3-hydroxybutyrate, hexuronic acid, ribose, and gluconic acid lactone were significantly more abundant in dogs with IBD. Although a clinical improvement was observed after medical therapy in all dogs with IBD, this was not accompanied by significant changes in the fecal microbiota or in serum metabolite profiles. These results suggest the presence of oxidative stress and a functional alteration of the GI microbiota in dogs with IBD, which persisted even in the face of a clinical response to medical therapy. PMID:25531678

  15. Alteration of the fecal microbiota and serum metabolite profiles in dogs with idiopathic inflammatory bowel disease.

    PubMed

    Minamoto, Yasushi; Otoni, Cristiane C; Steelman, Samantha M; Büyükleblebici, Olga; Steiner, Jörg M; Jergens, Albert E; Suchodolski, Jan S

    2015-01-01

    Idiopathic inflammatory bowel disease (IBD) is a common cause of chronic gastrointestinal (GI) disease in dogs. The combination of an underlying host genetic susceptibility, an intestinal dysbiosis, and dietary/environmental factors are suspected as main contributing factors in the pathogenesis of canine IBD. However, actual mechanisms of the host-microbe interactions remain elusive. The aim of this study was to compare the fecal microbiota and serum metabolite profiles between healthy dogs (n = 10) and dogs with IBD before and after 3 weeks of medical therapy (n = 12). Fecal microbiota and metabolite profiles were characterized by 454-pyrosequencing of 16 S rRNA genes and by an untargeted metabolomics approach, respectively. Significantly lower bacterial diversity and distinct microbial communities were observed in dogs with IBD compared to the healthy control dogs. While Gammaproteobacteria were overrepresented, Erysipelotrichia, Clostridia, and Bacteroidia were underrepresented in dogs with IBD. The functional gene content was predicted from the 16 S rRNA gene data using PICRUSt, and revealed overrepresented bacterial secretion system and transcription factors, and underrepresented amino acid metabolism in dogs with IBD. The serum metabolites 3-hydroxybutyrate, hexuronic acid, ribose, and gluconic acid lactone were significantly more abundant in dogs with IBD. Although a clinical improvement was observed after medical therapy in all dogs with IBD, this was not accompanied by significant changes in the fecal microbiota or in serum metabolite profiles. These results suggest the presence of oxidative stress and a functional alteration of the GI microbiota in dogs with IBD, which persisted even in the face of a clinical response to medical therapy.

  16. Metabolite profiling of two novel low phytic acid (lpa) soybean mutants.

    PubMed

    Frank, Thomas; Nörenberg, Svenja; Engel, Karl-Heinz

    2009-07-22

    A GC-based approach was applied to compare the metabolite profiles of two low phytic acid (lpa) soybean mutants and their respective wild-types. The lpa mutants (Gm-lpa-TW75-1 and Gm-lpa-ZC-2) were grown together with the wild-types (Taiwan 75 and Zhechun no. 3) in three and four field trials, respectively. HPLC analysis revealed a phytic acid reduction of -53% for Gm-lpa-TW75-1 and of -46% for Gm-lpa-ZC-2. For Gm-lpa-TW75-1, no accumulation of lower inositol phosphates was observed, whereas Gm-lpa-ZC-2 exhibited significantly increased contents of the lower inositol phosphates InsP(3), InsP(4), and InsP(5) compared to the corresponding wild-type. The metabolite profiling revealed that compared to the wild-types, 40% (Gm-lpa-TW75-1) and 21% (Gm-lpa-ZC-2) of the detected peaks were statistically significantly different in the lpa mutants grown at one field trial. However, the majority of these differences were shown to be related to environmental impact and natural variability rather than to the mutation event. Identification of consistent metabolic changes in the lpa mutants revealed decreased contents of myo-inositol, galactinol, raffinose, stachyose, and the galactosyl cyclitols galactopinitol A, galactopinitol B, and fagopyritol B1 compared to the wild-type. These consistently pronounced changes in Gm-lpa-TW75-1 confirmed the suggested mutation target. Consideration of the metabolic changes observed for Gm-lpa-ZC-2 (accumulation of lower inositol phosphates and increased myo-inositol contents) indicated a mutation event affecting the latter biosynthetic steps leading to phytic acid. The study demonstrated the applicability of metabolite profiling for the detection of changes in the metabolite phenotype induced by mutation breeding and its power in assisting in the elucidation of mutation events.

  17. Gut flora profiling and fecal metabolite composition of colorectal cancer patients and healthy individuals.

    PubMed

    Wang, Xiaoxue; Wang, Jianping; Rao, Benqiang; Deng, Li

    2017-06-01

    Colorectal cancer is one of the most common types of cancer in the world and its morbidity and mortality rates are increasing due to alterations to human lifestyle and dietary habits. The relationship between human gut flora and colorectal cancer has attracted increasing attention. In the present study, a metabolic fingerprinting technique that combined pyrosequencing with gas chromatography-mass spectrometry was utilized to compare the differences in gut flora profiling and fecal metabolites between healthy individuals and patients with colorectal cancer. The results demonstrated that there were no significant differences in the abundance and diversity of gut flora between healthy individuals and patients with colorectal cancer (P>0.05) and the dominant bacterial phyla present in the gut of both groups included Firmicutes, Bacteroidetes and Verrucomicrobia. At the bacterial strain/genus level, significant differences were observed in the relative abundance of 18 species of bacteria (P<0.05). Analysis of fecal metabolites demonstrated that the metabolic profiles of healthy individuals and patients with colorectal cancer were distinct. The levels of short-chain fatty acid metabolites, including acetic acid, valeric acid, isobutyric acid and isovaleric acid, and of nine amino acids in patients with colorectal cancer were significantly higher than those in healthy individuals (P<0.05). However, the levels of butyrate, oleic acid, trans-oleic acid, linoleic acid, glycerol, monoacyl glycerol, myristic acid, ursodesoxycholic acid and pantothenic acid in patients with colorectal cancer were significantly lower than those in healthy individuals (P<0.05). Pearson rank correlation analysis demonstrated that there was a correlation between gut flora profiling and metabolite composition. These findings suggest that gut flora disorder results in the alteration of bacterial metabolism, which may be associated with the pathogenesis of colorectal cancer. The results of the present

  18. Metabolite profiling of symbiont and host during thermal stress and bleaching in the coral Acropora aspera

    NASA Astrophysics Data System (ADS)

    Hillyer, Katie E.; Dias, Daniel A.; Lutz, Adrian; Wilkinson, Shaun P.; Roessner, Ute; Davy, Simon K.

    2017-03-01

    Rising seawater temperatures pose a significant threat to the persistence of coral reefs. Despite the importance of these systems, major gaps remain in our understanding of how thermal stress and bleaching affect the metabolic networks that underpin holobiont function. We applied gas chromatography-mass spectrometry (GC-MS) metabolomics to detect changes in the intracellular free metabolite pools (polar and semi-polar compounds) of in hospite dinoflagellate symbionts and their coral hosts (and any associated microorganisms) during early- and late-stage thermal bleaching (a reduction of approximately 50 and 70% in symbiont density, respectively). We detected characteristic changes to the metabolite profiles of each symbiotic partner associated with individual cellular responses to thermal, oxidative and osmotic stress, which progressed with the severity of bleaching. Alterations were also indicative of changes to energy-generating and biosynthesis pathways in both partners, with a shift to the increased catabolism of lipid stores. Specifically, in symbiont intracellular metabolite pools, we observed accumulations of multiple free fatty acids, plus the chloroplast-associated antioxidant alpha-tocopherol. In the host, we detected a decline in the abundance of pools of multiple carbohydrates, amino acids and intermediates, in addition to the antioxidant ascorbate. These findings further our understanding of the metabolic changes that occur to symbiont and host (and its associated microorganisms) during thermal bleaching. These findings also provide further insight into the largely undescribed roles of free metabolite pools in cellular homeostasis, signalling and acclimation to thermal stress in the cnidarian-dinoflagellate symbiosis.

  19. Ultrahigh Pressure Processing Produces Alterations in the Metabolite Profiles of Panax ginseng.

    PubMed

    Lee, Mee Youn; Singh, Digar; Kim, Sung Han; Lee, Sang Jun; Lee, Choong Hwan

    2016-06-22

    Ultrahigh pressure (UHP) treatments are non-thermal processing methods that have customarily been employed to enhance the quality and productivity of plant consumables. We aimed to evaluate the effects of UHP treatments on ginseng samples (white ginseng: WG; UHP-treated WG: UWG; red ginseng: RG; UHP-treated RG: URG; ginseng berries: GB; and UHP-treated GB: UGB) using metabolite profiling based on ultrahigh performance liquid chromatography-linear trap quadrupole-ion trap-tandem mass spectrometry (UHPLC-LTQ-IT-MS/MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Multivariate data analyses revealed a clear demarcation among the GB and UGB samples, and the phenotypic evaluations correlated the highest antioxidant activities and the total phenolic and flavonoid compositions with the UGB samples. Overall, eight amino acids, seven organic acids, seven sugars and sugar derivatives, two fatty acids, three notoginsenosides, three malonylginsenosides, and three ginsenosides, were identified as significantly discriminant metabolites between the GB and UGB samples, with relatively higher proportions in the latter. Ideally, these metabolites can be used as quality biomarkers for the assessment of ginseng products and our results indicate that UHP treatment likely led to an elevation in the proportions of total extractable metabolites in ginseng samples.

  20. Profile of urinary and fecal proanthocyanidin metabolites from common cinnamon (Cinnamomum zeylanicum L.) in rats.

    PubMed

    Mateos-Martín, María Luisa; Pérez-Jiménez, Jara; Fuguet, Elisabet; Torres, Josep Lluís

    2012-04-01

    Cinnamon (Cinnamomum zeylanicum L.) bark is widely used as a spice and in traditional medicine. Its oligomeric and polymeric proanthocyanidins are believed to be partly responsible for the beneficial properties of the plant. We describe here the metabolic fate of cinnamon proanthocyanidins in the urine and feces of rats fed a suspension of the whole bark. The metabolites include ten mono-, di-, and tri- conjugated (epi)catechin phase II metabolites and more than 20 small phenolic acids from intestinal microbial fermentation. Some of these are sulfated conjugates. Feces contain intact (epi)catechin and dimers. This suggests that free radical scavenging species are in contact with the intestinal walls for hours after ingestion of cinnamon. The phenolic metabolite profile of cinnamon bark in urine is consistent with a mixture of proanthocyanidins that are depolymerized into their constitutive (epi)catechin units as well as cleaved into smaller phenolic acids during their transit along the intestinal tract, with subsequent absorption and conjugation into bioavailable metabolites. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Quantification of amounts and (13)C content of metabolites in brain tissue using high- resolution magic angle spinning (13)C NMR spectroscopy.

    PubMed

    Risa, Oystein; Melø, Torun Margareta; Sonnewald, Ursula

    2009-04-01

    Metabolic pathway mapping using (13)C NMR spectroscopy has been used extensively to study interactions between neurons and glia in the brain. Established extraction procedures of brain tissue are time consuming and may result in degradation of labile substances. We examined the potential of mapping (13)C-enriched compounds in intact brain tissue using high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. Sprague-Dawley rats received an intraperitoneal injection of [1,6-(13)C]glucose, and 15 min later the animals were subjected to microwave fixation of the brain. Quantification of concentration and (13)C labelling of metabolites in intact rat thalamus were carried out based on exogenous ethylene glycol concentrations measured from (1)H NMR spectra using an ERETIC (Electronic REference To access In vivo Concentrations) signal. The results from intact tissue were compared with those from perchloric acid-extracted brain tissue. Amounts of (13)C labelling at different positions (C2, C3 and C4) in glutamate, glutamine, gamma-aminobutyric acid and aspartate measured in either intact tissue or perchloric acid extracts were not significantly different. Proton NMR spectra were used for quantification of six different amino acids plus lactate, inositol, N-acetylaspartate, creatine and phosphocreatine. Again, results were very similar when comparing the methods. To our knowledge, this is the first time quantitative (13)C NMR spectroscopy measurements have been carried out on intact brain tissue ex vivo using the HR-MAS technique. The results show that HR-MAS (13)C NMR spectroscopy in combination with (1)H NMR spectroscopy and the ERETIC method is useful for metabolic studies of intact brain tissue ex vivo.

  2. Screening for wound-induced oxylipins in Arabidopsis thaliana by differential HPLC-APCI/MS profiling of crude leaf extracts and subsequent characterisation by capillary-scale NMR.

    PubMed

    Thiocone, Aly; Farmer, Edward E; Wolfender, Jean-Luc

    2008-01-01

    A simple non-targeted differential HPLC-APCI/MS approach has been developed in order to survey metabolome modifications that occur in the leaves of Arabidopsis thaliana following wound-induced stress. The wound-induced accumulation of metabolites, particularly oxylipins, was evaluated by HPLC-MS analysis of crude leaf extracts. A generic, rapid and reproducible pressure liquid extraction procedure was developed for the analysis of restricted leaf samples without the need for specific sample preparation. The presence of various oxylipins was determined by head-to-head comparison of the HPLC-MS data, filtered with a component detection algorithm, and automatically compared with the aid of software searching for small differences in similar HPLC-MS profiles. Repeatability was verified in several specimens belonging to different series. Wound-inducible jasmonates were efficiently highlighted by this non-targeted approach without the need for complex sample preparation as is the case for the 'oxylipin signature' procedure based on GC-MS. Furthermore this HPLC-MS screening technique allowed the isolation of induced compounds for further characterisation by capillary-scale NMR (CapNMR) after HPLC scale-up. In this paper, the screening method is described and applied to illustrate its potential for monitoring polar and non-polar stress-induced constituents as well as its use in combination with CapNMR for the structural assignment of wound-induced compounds of interest.

  3. Metabolite Profiling of Plastidial Deoxyxylulose-5-Phosphate Pathway Intermediates by Liquid Chromatography and Mass Spectrometry

    PubMed Central

    Baidoo, Edward E.K.; Xiao, Yanmei; Dehesh, Katayoon; Keasling, Jay D.

    2016-01-01

    Metabolite profiling is a powerful tool that enhances our understanding of complex regulatory processes and extends to the comparative analysis of plant gene function. However, at present, there are relatively few examples of metabolite profiling being used to characterize the regulatory aspects of the plastidial deoxyxylulose-5-phosphate (DXP) pathway in plants. Since the DXP pathway is one of two pathways in plants that are essential for isoprenoid biosynthesis, it is imperative that robust analytical methods be employed for the characterization of this metabolic pathway. Recently, liquid chromatography-mass spectrometry (LC-MS), in conjunction with traditional molecular biology approaches, established that the DXP pathway metabolite, methylerythritol cyclodiphosphate (MEcPP), previously known solely as an intermediate in the isoprenoid biosynthetic pathway, is a stress sensor that communicates environmental perturbations sensed by plastids to the nucleus, a process referred to as retrograde signaling. In this chapter, we describe two LC-MS methods from this study that can be broadly used to characterize DXP pathway intermediates. PMID:24777790

  4. Artificially decreased vapour pressure deficit in field conditions modifies foliar metabolite profiles in birch and aspen

    PubMed Central

    Lihavainen, Jenna; Keinänen, Markku; Keski-Saari, Sarita; Kontunen-Soppela, Sari; Sõber, Anu; Oksanen, Elina

    2016-01-01

    Relative air humidity (RH) is expected to increase in northern Europe due to climate change. Increasing RH reduces the difference of water vapour pressure deficit (VPD) between the leaf and the atmosphere, and affects the gas exchange of plants. Little is known about the effects of decreased VPD on plant metabolism, especially under field conditions. This study was conducted to determine the effects of artificially decreased VPD on silver birch (Betula pendula Roth.) and hybrid aspen (Populus tremula L.×P. tremuloides Michx.) foliar metabolite and nutrient profiles in a unique free air humidity manipulation (FAHM) field experiment during the fourth season of humidity manipulation, in 2011. Long-term exposure to decreased VPD modified nutrient homeostasis in tree leaves, as demonstrated by a lower N concentration and N:P ratio in aspen leaves, and higher Na concentration and lower K:Na ratio in the leaves of both species in decreased VPD than in ambient VPD. Decreased VPD caused a shift in foliar metabolite profiles of both species, affecting primary and secondary metabolites. Metabolic adjustment to decreased VPD included elevated levels of starch and heptulose sugars, sorbitol, hemiterpenoid and phenolic glycosides, and α-tocopherol. High levels of carbon reserves, phenolic compounds, and antioxidants under decreased VPD may modify plant resistance to environmental stresses emerging under changing climate. PMID:27255929

  5. Taxonomic Characterization and Secondary Metabolite Profiling of Aspergillus Section Aspergillus Contaminating Feeds and Feedstuffs

    PubMed Central

    Greco, Mariana; Kemppainen, Minna; Pose, Graciela; Pardo, Alejandro

    2015-01-01

    Xerophilic fungal species of the genus Aspergillus are economically highly relevant due to their ability to grow on low water activity substrates causing spoilage of stored goods and animal feeds. These fungi can synthesize a variety of secondary metabolites, many of which show animal toxicity, creating a health risk for food production animals and to humans as final consumers, respectively. Animal feeds used for rabbit, chinchilla and rainbow trout production in Argentina were analysed for the presence of xerophilic Aspergillus section Aspergillus species. High isolation frequencies (>60%) were detected in all the studied rabbit and chinchilla feeds, while the rainbow trout feeds showed lower fungal charge (25%). These section Aspergillus contaminations comprised predominantly five taxa. Twenty isolates were subjected to taxonomic characterization using both ascospore SEM micromorphology and two independent DNA loci sequencing. The secondary metabolite profiles of the isolates were determined qualitatively by HPLC-MS. All the isolates produced neoechinulin A, 17 isolates were positive for cladosporin and echinulin, and 18 were positive for neoechinulin B. Physcion and preechinulin were detected in a minor proportion of the isolates. This is the first report describing the detailed species composition and the secondary metabolite profiles of Aspergillus section Aspergillus contaminating animal feeds. PMID:26364643

  6. Metabolite Profiling of Preneoplastic and Neoplastic Lesions of Oral Cavity Tissue Samples Revealed a Biomarker Pattern

    PubMed Central

    Musharraf, Syed Ghulam; Shahid, Najia; Naqvi, Syed Muhammad Ali; Saleem, Mahwish; Siddiqui, Amna Jabbar; Ali, Anwar

    2016-01-01

    Oral cancer is a major health challenge in the Indian subcontinent and a dreadful form of cancers worldwide. The current study is focused on the identification of distinguished metabolites of oral cancer tissue samples in comparison with precancerous and control tissue samples using gas chromatography coupled with triple quadrupole tandem mass spectrometry and chemometric analyses. Metabolites obtained were identified through National Institute of Standards and Technology (NIST) mass spectral (Wiley registry) library. Mass Profiler Professional (MPP) software was used for the alignment and for all the statistical analysis. 31 compounds out of 735 found distinguishing among oral cancer, precancerous and control group samples using p-value ≤ 0.05. Partial Least Square Discriminant Analysis (PLSDA) model was generated using statistically significant metabolites gave an overall accuracy of 90.2%. Down-regulated amino acid levels appear to be the result of enhanced energy metabolism or up-regulation of the appropriate biosynthetic pathways, and required cell proliferation in cancer tissues. These results suggest that tissue metabolic profiles have great potential in detecting oral cancer and may aid in understanding its underlying mechanisms. PMID:27958349

  7. Prediction of future risk of insulin resistance and metabolic syndrome based on Korean boy's metabolite profiling.

    PubMed

    Lee, AeJin; Jang, Han Byul; Ra, Moonjin; Choi, Youngshim; Lee, Hye-Ja; Park, Ju Yeon; Kang, Jae Heon; Park, Kyung-Hee; Park, Sang Ick; Song, Jihyun

    2015-01-01

    Childhood obesity is strongly related to future insulin resistance and metabolic syndrome. Thus, identifying early biomarkers of obesity-related diseases based on metabolic profiling is useful to control future metabolic disorders. We compared metabolic profiles between obese and normal-weight children and investigated specific biomarkers of future insulin resistance and metabolic syndrome. In all, 186 plasma metabolites were analysed at baseline and after 2 years in 109 Korean boys (age 10.5±0.4 years) from the Korean Child Obesity Cohort Study using the AbsoluteIDQ™ p180 Kit. We observed that levels of 41 metabolites at baseline and 40 metabolites at follow-up were significantly altered in obese children (p<0.05). Obese children showed significantly higher levels of branched-chain amino acids (BCAAs) and several acylcarnitines and lower levels of acyl-alkyl phosphatidylcholines. Also, baseline BCAAs were significantly positively correlated with both homeostasis model assessment for insulin resistance (HOMA-IR) and continuous metabolic risk score at the 2-year follow-up. In logistic regression analyses with adjustments for degree of obesity at baseline, baseline BCAA concentration, greater than the median value, was identified as a predictor of future risk of insulin resistance and metabolic syndrome. High BCAA concentration could be "early" biomarkers for predicting future metabolic diseases. Copyright © 2014 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

  8. Taxonomic Characterization and Secondary Metabolite Profiling of Aspergillus Section Aspergillus Contaminating Feeds and Feedstuffs.

    PubMed

    Greco, Mariana; Kemppainen, Minna; Pose, Graciela; Pardo, Alejandro

    2015-09-02

    Xerophilic fungal species of the genus Aspergillus are economically highly relevant due to their ability to grow on low water activity substrates causing spoilage of stored goods and animal feeds. These fungi can synthesize a variety of secondary metabolites, many of which show animal toxicity, creating a health risk for food production animals and to humans as final consumers, respectively. Animal feeds used for rabbit, chinchilla and rainbow trout production in Argentina were analysed for the presence of xerophilic Aspergillus section Aspergillus species. High isolation frequencies (>60%) were detected in all the studied rabbit and chinchilla feeds, while the rainbow trout feeds showed lower fungal charge (25%). These section Aspergillus contaminations comprised predominantly five taxa. Twenty isolates were subjected to taxonomic characterization using both ascospore SEM micromorphology and two independent DNA loci sequencing. The secondary metabolite profiles of the isolates were determined qualitatively by HPLC-MS. All the isolates produced neoechinulin A, 17 isolates were positive for cladosporin and echinulin, and 18 were positive for neoechinulin B. Physcion and preechinulin were detected in a minor proportion of the isolates. This is the first report describing the detailed species composition and the secondary metabolite profiles of Aspergillus section Aspergillus contaminating animal feeds.

  9. Metabolite profiling stratifies pancreatic ductal adenocarcinomas into subtypes with distinct sensitivities to metabolic inhibitors

    PubMed Central

    Daemen, Anneleen; Peterson, David; Sahu, Nisebita; McCord, Ron; Du, Xiangnan; Liu, Bonnie; Kowanetz, Katarzyna; Hong, Rebecca; Moffat, John; Gao, Min; Boudreau, Aaron; Mroue, Rana; Corson, Laura; O’Brien, Thomas; Qing, Jing; Sampath, Deepak; Merchant, Mark; Yauch, Robert; Manning, Gerard; Settleman, Jeffrey; Hatzivassiliou, Georgia; Evangelista, Marie

    2015-01-01

    Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional ∼200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors. PMID:26216984

  10. Plant secondary metabolite profiling evidences strain-dependent effect in the Azospirillum-Oryza sativa association.

    PubMed

    Chamam, Amel; Sanguin, Hervé; Bellvert, Floriant; Meiffren, Guillaume; Comte, Gilles; Wisniewski-Dyé, Florence; Bertrand, Cédric; Prigent-Combaret, Claire

    2013-03-01

    Azospirillum is a plant growth-promoting rhizobacterium (PGPR) able to enhance growth and yield of cereals such as rice, maize and wheat. The growth-promoting ability of some Azospirillum strains appears to be highly specific to certain plant species and cultivars. In order to ascertain the specificity of the associative symbiosis between rice and Azospirillum, the physiological response of two rice cultivars, Nipponbare and Cigalon, inoculated with two rice-associated Azospirillum was analyzed at two levels: plant growth response and plant secondary metabolic response. Each strain of Azospirillum (Azospirillum lipoferum 4B isolated from Cigalon and Azospirillum sp. B510 isolated from Nipponbare) preferentially increased growth of the cultivar from which it was isolated. This specific effect is not related to a defect in colonization of host cultivar as each strain colonizes effectively both rice cultivars, either at the rhizoplane (for 4B and B510) and inside the roots (for B510). The metabolic profiling approach showed that, in response to PGPR inoculation, profiles of rice secondary metabolites were modified, with phenolic compounds such as flavonoids and hydroxycinnamic derivatives being the main metabolites affected. Moreover, plant metabolic changes differed according to Azospirillum strain×cultivar combinations; indeed, 4B induced major secondary metabolic profile modifications only on Cigalon roots, while B510, probably due to its endophytic feature, induced metabolic variations on shoots and roots of both cultivars, triggering a systemic response. Plant secondary metabolite profiling thereby evidences the specific interaction between an Azospirillum strain and its original host cultivar. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Comparative LC-MS-based metabolite profiling of the ancient tropical rainforest tree Symphonia globulifera.

    PubMed

    Cottet, Kévin; Genta-Jouve, Grégory; Fromentin, Yann; Odonne, Guillaume; Duplais, Christophe; Laprévote, Olivier; Michel, Sylvie; Lallemand, Marie-Christine

    2014-12-01

    In the last few years, several phytochemical studies have been undertaken on the tropical tree Symphonia globulifera leading to the isolation and characterisation of several compounds exhibiting antiparasitic activities against Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani. The comparative LC-MS based metabolite profiling study conducted on the tree led to the identification of compounds originating from specific tissues. The results showed that renewable organs/tissues can be used as the starting material for the production of polycyclic poly-prenylated-acylphloroglucinols, therefore reducing impacts on biodiversity. This study also underlined the lack of knowledge on the secondary metabolites produced by S. globulifera since only a small number of the total detected features were putatively identified using the database of known compounds for the species. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Factors influencing annual fecal testosterone metabolite profiles in captive male polar bears (Ursus maritimus).

    PubMed

    Curry, E; Roth, T L; MacKinnon, K M; Stoops, M A

    2012-12-01

    The objectives of this study were to assess the effects of season, breeding activity, age and latitude on fecal testosterone metabolite concentrations in captive, adult male polar bears (Ursus maritimus). Fourteen polar bears from 13 North American zoos were monitored for 12-36 months, producing 25-year-long testosterone profiles. Results indicated that testosterone was significantly higher during the breeding season (early January through the end of May) compared with the non-breeding season with the highest concentrations excreted from early January through late March. Variations in excretion patterns were observed among individuals and also between years within an individual, with testosterone peaks closely associated with breeding activity. Results indicate that fecal testosterone concentrations are influenced by season, breeding activity and age, but not by latitude. This is the first report describing longitudinal fecal testosterone metabolite concentrations in individual adult male polar bears. © 2012 Blackwell Verlag GmbH.

  13. Polyphenol metabolite profile of artichoke is modulated by agronomical practices and cooking method.

    PubMed

    Palermo, Mariantonella; Colla, Giuseppe; Barbieri, Giancarlo; Fogliano, Vincenzo

    2013-08-21

    In this paper artichoke phenolic pattern was characterized using an Orbitrap Exactive Mass Spectrometer at high mass accuracy and conventional HPLC MS/MS. Twenty four phenolic acids and 40 flavonoids were identified, many of them not previously reported in artichoke. Variations in phenolic compounds were investigated in relation to mycorrhization: results showed that inoculation with mycorrhizae greatly influences metabolite profile proving to be a good strategy to enhance the biosynthesis of secondary metabolites in this plant. This practice also caused a different distribution of the main phenolic compounds within head parts. Both steaming and microwaving cooking treatments caused an increase in antioxidant activity: the lower the initial concentration the higher the effect. A similar trend was observed looking at the phenolic compounds concentration: it increased because of cooking treatments the lower the initial content, the highest the increase. Steamed artichoke showed higher phenols content than microwaved ones.

  14. Response to the Letter to the Editor regarding "Determination of the fatty acid profile by 1H-NMR spectroscopy."

    USDA-ARS?s Scientific Manuscript database

    In expansion of previous work (G. Knothe, J.A. Kenar, Determination of the fatty acid profile by 1H-NMR spectroscopy, Eur. J. Lipid Sci. Technol. 2004, 106, 88-96), an additional approach is discussed for quantitating saturated fatty acids in the fatty acid profiles of common vegetable oils by 1H-NM...

  15. Peripheral blood gene expression profiles linked to monoamine metabolite levels in cerebrospinal fluid

    PubMed Central

    Luykx, J J; Olde Loohuis, L M; Neeleman, M; Strengman, E; Bakker, S C; Lentjes, E; Borgdorff, P; van Dongen, E P A; Bruins, P; Kahn, R S; Horvath, S; de Jong, S; Ophoff, R A

    2016-01-01

    The blood–brain barrier separates circulating blood from the central nervous system (CNS). The scope of this barrier is not fully understood which limits our ability to relate biological measurements from peripheral to central phenotypes. For example, it is unknown to what extent gene expression levels in peripheral blood are reflective of CNS metabolism. In this study, we examine links between central monoamine metabolite levels and whole-blood gene expression to better understand the connection between peripheral systems and the CNS. To that end, we correlated the prime monoamine metabolites in cerebrospinal fluid (CSF) with whole-genome gene expression microarray data from blood (N=240 human subjects). We additionally applied gene-enrichment analysis and weighted gene co-expression network analyses (WGCNA) to identify modules of co-expressed genes in blood that may be involved with monoamine metabolite levels in CSF. Transcript levels of two genes were significantly associated with CSF serotonin metabolite levels after Bonferroni correction for multiple testing: THAP7 (P=2.8 × 10−8, β=0.08) and DDX6 (P=2.9 × 10−7, β=0.07). Differentially expressed genes were significantly enriched for genes expressed in the brain tissue (P=6.0 × 10−52). WGCNA revealed significant correlations between serotonin metabolism and hub genes with known functions in serotonin metabolism, for example, HTR2A and COMT. We conclude that gene expression levels in whole blood are associated with monoamine metabolite levels in the human CSF. Our results, including the strong enrichment of brain-expressed genes, illustrate that gene expression profiles in peripheral blood can be relevant for quantitative metabolic phenotypes in the CNS. PMID:27959337

  16. NMR-metabolomics profiling of mammary gland secretory tissue and milk serum in two goat breeds with different levels of tolerance to seasonal weight loss.

    PubMed

    Palma, Mariana; Hernández-Castellano, Lorenzo E; Castro, Noemí; Arguëllo, Anastasio; Capote, Juan; Matzapetakis, Manolis; de Almeida, André Martinho

    2016-06-21

    Goats are of special importance in the Mediterranean and tropical regions for producing a variety of dairy products. The scarcity of pastures during the dry season leads to seasonal weight loss (SWL), which affects milk production. In this work, we studied the effect of feed-restriction on two dairy goat breeds, with different tolerance levels to SWL: the Majorera breed (tolerant) and the Palmera breed (susceptible). Nuclear magnetic resonance (NMR) was used to compare the metabolome of an aqueous fraction of the mammary gland and milk serum from both breeds. Goats in mid-lactation were divided by breed, and each in two feed-regime groups: the control group and the restricted-fed group (to achieve 15-20% reduction of body weight at the end of the experiment). Milk and mammary gland samples were collected at the end of the experimental period (23rd day). (1)H NMR spectra were collected from the aqueous extract of the mammary gland biopsies and the milk serum. Profiling analysis has led to the identification of 46 metabolites in the aqueous extract of the mammary gland. Lactose, glutamate, glycine and lactate were found to be the most abundant. Analysis of milk serum allowed the identification of 50 metabolites, the most abundant being lactose, citrate and creatine. Significant differences were observed, in mammary gland biopsies and milk serum, between control and restricted-fed groups in both breeds, albeit with no differences between the breeds. Variations seem to be related to metabolism adaptation to the low-energy diet and are indicative of breed-specific microflora. Milk serum showed more metabolites varying between control and restricted groups, than the mammary gland. The Majorera breed also showed more variations than the Palmera breed in milk samples, which could be an indication of a prompt adaptation to SWL by the Majorera breed.

  17. ProAlgaZyme subfraction improves the lipoprotein profile of hypercholesterolemic hamsters, while inhibiting production of betaine, carnitine, and choline metabolites

    PubMed Central

    2013-01-01

    Background Previously, we reported that ProAlgaZyme (PAZ) and its biologically active fraction improved plasma lipids in hypercholesterolemic hamsters, by significantly increasing the high density lipoprotein cholesterol (HDL-C) while reducing non-HDL cholesterol and the ratio of total cholesterol/HDL-C. Moreover, hepatic mRNA expression of genes involved in HDL/reverse cholesterol transport were significantly increased, while cholesteryl ester transfer protein (CETP) expression was partially inhibited. In the current study, we investigated the therapeutic efficacy of the biologically active fraction of PAZ (BaP) on the plasma lipid and plasma metabolomic profiles in diet induced hypercholesterolemic hamsters. Methods Fifty male Golden Syrian hamsters were fed a high fat diet for 4 weeks prior to randomization into 6 groups, based on the number of days they received subsequent treatment. Thus animals in T0, T3, T7, T10, T14, and T21 groups received BaP for 0, 3, 7, 10, 14, and 21 days, respectively, as their drinking fluid. Plasma lipids were assayed enzymatically, while real-time reverse transcriptase polymerase chain reaction (RT-PCR) provided the transcription levels of the Apolipoprotein (Apo) A1 gene. The plasma metabolomic profile was determined using 1H nuclear magnetic resonance (NMR) spectroscopy in conjunction with multivariate analysis. Results Plasma HDL-C was significantly increased in T3 (P < 0.05) and T21 (P < 0.001), while non-HDL cholesterol was significantly reduced in T3, T7, T10 (P < 0.001) and T14, T21 (P < 0.01). Moreover, the ratio of total cholesterol/HDL-C was significantly lower in all BaP treated groups (P < 0.001) as compared with T0. Quantitative RT-PCR showed an increase in Apo A1 expression in T10 (3-fold) and T21 (6-fold) groups. NMR data followed by multivariate analysis showed a clear separation between T0 and T21 groups, indicating a difference in their metabolomic profiles. Plasma concentrations of

  18. The gut microbiota modulates glycaemic control and serum metabolite profiles in non-obese diabetic mice.

    PubMed

    Greiner, Thomas U; Hyötyläinen, Tuulia; Knip, Mikael; Bäckhed, Fredrik; Orešič, Matej

    2014-01-01

    Islet autoimmunity in children who later progress to type 1 diabetes is preceded by dysregulated serum metabolite profiles, but the origin of these metabolic changes is unknown. The gut microbiota affects host metabolism and changes in its composition contribute to several immune-mediated diseases; however, it is not known whether the gut microbiota is involved in the early metabolic disturbances in progression to type 1 diabetes. We rederived non-obese diabetic (NOD) mice as germ free to explore the potential role of the gut microbiota in the development of diabetic autoimmunity and to directly investigate whether the metabolic profiles associated with the development of type 1 diabetes can be modulated by the gut microbiota. The absence of a gut microbiota in NOD mice did not affect the overall diabetes incidence but resulted in increased insulitis and levels of interferon gamma and interleukin 12; these changes were counterbalanced by improved peripheral glucose metabolism. Furthermore, we observed a markedly increased variation in blood glucose levels in the absence of a microbiota in NOD mice that did not progress to diabetes. Additionally, germ-free NOD mice had a metabolite profile similar to that of pre-diabetic children. Our data suggest that germ-free NOD mice have reduced glycaemic control and dysregulated immunologic and metabolic responses.

  19. Understanding developmental and adaptive cues in pine through metabolite profiling and co-expression network analysis

    PubMed Central

    Cañas, Rafael A.; Canales, Javier; Muñoz-Hernández, Carmen; Granados, Jose M.; Ávila, Concepción; García-Martín, María L.; Cánovas, Francisco M.

    2015-01-01

    Conifers include long-lived evergreen trees of great economic and ecological importance, including pines and spruces. During their long lives conifers must respond to seasonal environmental changes, adapt to unpredictable environmental stresses, and co-ordinate their adaptive adjustments with internal developmental programmes. To gain insights into these responses, we examined metabolite and transcriptomic profiles of needles from naturally growing 25-year-old maritime pine (Pinus pinaster L. Aiton) trees over a year. The effect of environmental parameters such as temperature and rain on needle development were studied. Our results show that seasonal changes in the metabolite profiles were mainly affected by the needles’ age and acclimation for winter, but changes in transcript profiles were mainly dependent on climatic factors. The relative abundance of most transcripts correlated well with temperature, particularly for genes involved in photosynthesis or winter acclimation. Gene network analysis revealed relationships between 14 co-expressed gene modules and development and adaptation to environmental stimuli. Novel Myb transcription factors were identified as candidate regulators during needle development. Our systems-based analysis provides integrated data of the seasonal regulation of maritime pine growth, opening new perspectives for understanding the complex regulatory mechanisms underlying conifers’ adaptive responses. Taken together, our results suggest that the environment regulates the transcriptome for fine tuning of the metabolome during development. PMID:25873654

  20. Nutrient Intake Is Associated with Longevity Characterization by Metabolites and Element Profiles of Healthy Centenarians

    PubMed Central

    Cai, Da; Zhao, Shancang; Li, Danlei; Chang, Fang; Tian, Xiangxu; Huang, Guohong; Zhu, Zhenjun; Liu, Dong; Dou, Xiaowei; Li, Shubo; Zhao, Mouming; Li, Quanyang

    2016-01-01

    The relationships between diet and metabolites as well as element profiles in healthy centenarians are important but remain inconclusive. Therefore, to test the interesting hypothesis that there would be distinctive features of metabolites and element profiles in healthy centenarians, and that these would be associated with nutrient intake; the short chain fatty acids (SCFAs), total bile acids and ammonia in feces, phenol, p-cresol, uric acid, urea, creatinine and ammonia in urine, and element profiles in fingernails were determined in 90 healthy elderly people, including centenarians from Bama county (China)—a famous longevous region—and elderly people aged 80–99 from the longevous region and a non-longevous region. The partial least squares-discriminant analysis was used for pattern recognition. As a result, the centenarians showed a distinct metabolic pattern. Seven characteristic components closely related to the centenarians were identified, including acetic acid, total SCFA, Mn, Co, propionic acid, butyric acid and valeric acid. Their concentrations were significantly higher in the centenarians group (p < 0.05). Additionally, the dietary fiber intake was positively associated with butyric acid contents in feces (r = 0.896, p < 0.01), and negatively associated with phenol in urine (r = −0.326, p < 0.01). The results suggest that the specific metabolic pattern of centenarians may have an important and positive influence on the formation of the longevity phenomenon. Elevated dietary fiber intake should be a path toward health and longevity. PMID:27657115

  1. Nutrient Intake Is Associated with Longevity Characterization by Metabolites and Element Profiles of Healthy Centenarians.

    PubMed

    Cai, Da; Zhao, Shancang; Li, Danlei; Chang, Fang; Tian, Xiangxu; Huang, Guohong; Zhu, Zhenjun; Liu, Dong; Dou, Xiaowei; Li, Shubo; Zhao, Mouming; Li, Quanyang

    2016-09-19

    The relationships between diet and metabolites as well as element profiles in healthy centenarians are important but remain inconclusive. Therefore, to test the interesting hypothesis that there would be distinctive features of metabolites and element profiles in healthy centenarians, and that these would be associated with nutrient intake; the short chain fatty acids (SCFAs), total bile acids and ammonia in feces, phenol, p-cresol, uric acid, urea, creatinine and ammonia in urine, and element profiles in fingernails were determined in 90 healthy elderly people, including centenarians from Bama county (China)-a famous longevous region-and elderly people aged 80-99 from the longevous region and a non-longevous region. The partial least squares-discriminant analysis was used for pattern recognition. As a result, the centenarians showed a distinct metabolic pattern. Seven characteristic components closely related to the centenarians were identified, including acetic acid, total SCFA, Mn, Co, propionic acid, butyric acid and valeric acid. Their concentrations were significantly higher in the centenarians group (p < 0.05). Additionally, the dietary fiber intake was positively associated with butyric acid contents in feces (r = 0.896, p < 0.01), and negatively associated with phenol in urine (r = -0.326, p < 0.01). The results suggest that the specific metabolic pattern of centenarians may have an important and positive influence on the formation of the longevity phenomenon. Elevated dietary fiber intake should be a path toward health and longevity.

  2. Identification of some Bioactive Metabolites in a Fractionated Methanol Extract from Ipomoea aquatica (Aerial Parts) through TLC, HPLC, UPLC-ESI-QTOF-MS and LC-SPE-NMR Fingerprints Analyses.

    PubMed

    Hefny Gad, Mahmoud; Tuenter, Emmy; El-Sawi, Nagwa; Younes, Sabry; El-Ghadban, El-Mewafy; Demeyer, Kristiaan; Pieters, Luc; Vander Heyden, Yvan; Mangelings, Debby

    2017-08-04

    The plant species Ipomoea aquatica contains various bioactive constituents, e.g. phenols and flavonoids, which have several medical uses. All previous studies were executed in Asia; however, no reports are available from Africa, and the secondary metabolites of this plant species from Africa are still unknown. The present study aims finding suitable conditions to identify the bioactive compounds from different fractions. Chromatographic fingerprint profiles of different fractions were developed using high-performance liquid chromatography (HPLC) and then these conditions were transferred to thin-layer chromatography (TLC). Subsequently, the chemical structure of some bioactive compounds was elucidated using ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-QTOF-MS) and liquid chromatography-solid phase extraction-nuclear magnetic resonance (LC-SPE-NMR) spectroscopy. The HPLC fingerprints, developed on two coupled Chromolith RP-18e columns, using a gradient mobile phase (methanol/water/trifluoroacetic acid, 5:95:0.05, v/v/v), showed more peaks than the TLC profile. The TLC fingerprint allows the identification of the types of chemical constituents, e.g. flavonoids. Two flavonoids (nicotiflorin and ramnazin-3-O-rutinoside) and two phenolic compounds (dihydroxybenzoic acid pentoside and di-pentoside) were tentatively identified by QTOF-MS, while NMR confirmed the structure of rutin and nicotiflorin. The HPLC and TLC results showed that HPLC fingerprints give more and better separated peaks, but TLC helped in determining the class of the active compounds in some fractions. Bioactive constituents were identified as well using MS and NMR analyses. Two flavonoids and two phenolic compounds were tentatively identified in this species for the first time, to the best of our knowledge. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  3. Metabolic profiling of stages of healthy pregnancy in Hu sheep using nuclear magnetic resonance (NMR).

    PubMed

    Sun, Lingwei; Guo, Yixuan; Fan, Yixuan; Nie, Haitao; Wang, Ruocheng; Wang, Feng

    2017-04-01

    Nutrition is one of the most important factors affecting the reproductive performance of animals. Changes in the ovine maternal metabolism during pregnancy are critical to fetal development. To understand the differences in ovine metabolic changes that occur during normal pregnancy, pregnant ewes carrying twin fetuses (n = 8) were selected at 35 days of gestation (dG). All ewes received 100% of National Research Council (NRC) requirements of all nutrients and energy during this experiment. At 50, 70, 90, and 110 dG, maternal plasma samples were collected and designated as one of four corresponding time points (T1, T2, T3, and T4, respectively). Maternal plasma samples were analyzed using (1)H nuclear magnetic resonance spectroscopy to compare their metabolomic profiles among time points. We used multivariate pattern recognition to screen for different metabolites in the plasma of the ewes. The body weight and food intake of the ewes were significantly (P < 0.05) different at the four time points, and increased with the passage of pregnancy time. The principal component analysis model results showed that the metabolic states at time points T2 and T3 moved gradually further away from that at T1 and were furthest away from that at T1 at time point T4. Among the different time points, there were thirteen significantly differential metabolites in the maternal plasma (P < 0.05). These metabolites were closely related to amino acid metabolism and lipid metabolism, which might occur at different time points in pregnant ewes. In particular, newly observed changes in 1-methylhistidine and malonate were the first such changes found in maternal plasma. These results demonstrate that the metabolomics approach has value for evaluating metabolism in pregnancy with advancing gestation. In conclusion, during normal pregnancy in Hu sheep, related metabolites play an important role in amino acid and lipid metabolism for meeting the nutritional demands of pregnant ewes.

  4. Metabolite Profiling Reveals Abiotic Stress Tolerance in Tn5 Mutant of Pseudomonas putida

    PubMed Central

    Chaudhry, Vasvi; Bhatia, Anil; Bharti, Santosh Kumar; Mishra, Shashank Kumar; Chauhan, Puneet Singh; Mishra, Aradhana; Sidhu, Om Prakash; Nautiyal, Chandra Shekhar

    2015-01-01

    Pseudomonas is an efficient plant growth–promoting rhizobacteria (PGPR); however, intolerance to drought and high temperature limit its application in agriculture as a bioinoculant. Transposon 5 (Tn5) mutagenesis was used to generate a stress tolerant mutant from a PGPR Pseudomonas putida NBRI1108 isolated from chickpea rhizosphere. A mutant NBRI1108T, selected after screening of nearly 10,000 transconjugants, exhibited significant tolerance towards high temperature and drought. Southern hybridization analysis of EcoRI and XhoI restricted genomic DNA of NBRI1108T confirmed that it had a single Tn5 insertion. The metabolic changes in the polar and non-polar extracts of NBRI1108 and NBRI1108T were examined using 1H, 31P nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Thirty six chemically diverse metabolites consisting of amino acids, fatty acids and phospholipids were identified and quantified. Insertion of Tn5 influenced amino acid and phospholipid metabolism and resulted in significantly higher concentration of aspartic acid, glutamic acid, glycinebetaine, glycerophosphatidylcholine (GPC) and putrescine in NBRI1108T as compared to that in NBRI1108. The concentration of glutamic acid, glycinebetaine and GPC increased by 34%, 95% and 100%, respectively in the NBRI1108T as compared to that in NBRI1108. High concentration of glycerophosphatidylethanolamine (GPE) and undetected GPC in NBRI1108 indicates that biosynthesis of GPE may have taken place via the methylation pathway of phospholipid biosynthesis. However, high GPC and low GPE concentration in NBRI1108T suggest that methylation pathway and phosphatidylcholine synthase (PCS) pathway of phospholipid biosynthesis are being followed in the NBRI1108T. Application of multivariate principal component analysis (PCA) on the quantified metabolites revealed clear variations in NBRI1108 and NBRI1108T in polar and non-polar metabolites. Identification of abiotic stress

  5. Can NMR solve some significant challenges in metabolomics?

    NASA Astrophysics Data System (ADS)

    Nagana Gowda, G. A.; Raftery, Daniel

    2015-11-01

    The field of metabolomics continues to witness rapid growth driven by fundamental studies, methods development, and applications in a number of disciplines that include biomedical science, plant and nutrition sciences, drug development, energy and environmental sciences, toxicology, etc. NMR spectroscopy is one of the two most widely used analytical platforms in the metabolomics field, along with mass spectrometry (MS). NMR's excellent reproducibility and quantitative accuracy, its ability to identify structures of unknown metabolites, its capacity to generate metabolite profiles using intact bio-specimens with no need for separation, and its capabilities for tracing metabolic pathways using isotope labeled substrates offer unique strengths for metabolomics applications. However, NMR's limited sensitivity and resolution continue to pose a major challenge and have restricted both the number and the quantitative accuracy of metabolites analyzed by NMR. Further, the analysis of highly complex biological samples has increased the demand for new methods with improved detection, better unknown identification, and more accurate quantitation of larger numbers of metabolites. Recent efforts have contributed significant improvements in these areas, and have thereby enhanced the pool of routinely quantifiable metabolites. Additionally, efforts focused on combining NMR and MS promise opportunities to exploit the combined strength of the two analytical platforms for direct comparison of the metabolite data, unknown identification and reliable biomarker discovery that continue to challenge the metabolomics field. This article presents our perspectives on the emerging trends in NMR-based metabolomics and NMR's continuing role in the field with an emphasis on recent and ongoing research from our laboratory.

  6. Can NMR solve some significant challenges in metabolomics?

    PubMed Central

    Gowda, G.A. Nagana; Raftery, Daniel

    2015-01-01

    The field of metabolomics continues to witness rapid growth driven by fundamental studies, methods development, and applications in a number of disciplines that include biomedical science, plant and nutrition sciences, drug development, energy and environmental sciences, toxicology, etc. NMR spectroscopy is one of the two most widely used analytical platforms in the metabolomics field, along with mass spectrometry (MS). NMR's excellent reproducibility and quantitative accuracy, its ability to identify structures of unknown metabolites, its capacity to generate metabolite profiles using intact biospecimens with no need for separation, and its capabilities for tracing metabolic pathways using isotope labeled substrates offer unique strengths for metabolomics applications. However, NMR's limited sensitivity and resolution continue to pose a major challenge and have restricted both the number and the quantitative accuracy of metabolites analyzed by NMR. Further, the analysis of highly complex biological samples has increased the demand for new methods with improved detection, better unknown identification, and more accurate quantitation of larger numbers of metabolites. Recent efforts have contributed significant improvements in these areas, and have thereby enhanced the pool of routinely quantifiable metabolites. Additionally, efforts focused on combining NMR and MS promise opportunities to exploit the combined strength of the two analytical platforms for direct comparison of the metabolite data, unknown identification and reliable biomarker discovery that continue to challenge the metabolomics field. This article presents our perspectives on the emerging trends in NMR-based metabolomics and NMR's continuing role in the field with an emphasis on recent and ongoing research from our laboratory. PMID:26476597

  7. Metabolite Profiles of Diabetes Incidence and Intervention Response in the Diabetes Prevention Program.

    PubMed

    Walford, Geoffrey A; Ma, Yong; Clish, Clary; Florez, Jose C; Wang, Thomas J; Gerszten, Robert E

    2016-05-01

    Identifying novel biomarkers of type 2 diabetes risk may improve prediction and prevention among individuals at high risk of the disease and elucidate new biological pathways relevant to diabetes development. We performed plasma metabolite profiling in the Diabetes Prevention Program (DPP), a completed trial that randomized high-risk individuals to lifestyle, metformin, or placebo interventions. Previously reported markers, branched-chain and aromatic amino acids and glutamine/glutamate, were associated with incident diabetes (P < 0.05 for all), but these associations were attenuated upon adjustment for clinical and biochemical measures. By contrast, baseline levels of betaine, also known as glycine betaine (hazard ratio 0.84 per SD log metabolite level, P = 0.02), and three other metabolites were associated with incident diabetes even after adjustment. Moreover, betaine was increased by the lifestyle intervention, which was the most effective approach to preventing diabetes, and increases in betaine at 2 years were also associated with lower diabetes incidence (P = 0.01). Our findings indicate betaine is a marker of diabetes risk among high-risk individuals both at baseline and during preventive interventions and they complement animal models demonstrating a direct role for betaine in modulating metabolic health. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  8. Effects of dry-aging on meat quality attributes and metabolite profiles of beef loins.

    PubMed

    Kim, Yuan H Brad; Kemp, Robert; Samuelsson, Linda M

    2016-01-01

    The objectives of this study were to evaluate different dry-aging regimes and their impacts on quality attributes and metabolite profiles of beef loins. Thirty loins (M. longissimus lumborum) from 15 beef carcasses at 2 days post-mortem were obtained. Each loin was cut in half yielding 60 sections, which were randomly assigned to six treatments including 4 dry-aging (2 temperatures (1 or 3°C) × 2 air-velocities (0.2 or 0.5 m/s)) and 2 wet-aging regimes for 3 weeks; n=10/treatment. The sensory panel found that dry-aged loins had better flavour and overall liking (P<0.05), but there were no differences in tenderness and juiciness. No differences in drip/cook-loss and colour were observed. Metabolite analysis showed that 7 metabolites, including several flavour precursors, were more abundant in the dry-aged beef compared to the wet-aged beef, which may contribute to the enhanced flavours of the dry-aged beef. Overall, dry-aging loins at 3°C with 0.2m/s resulted in the greatest improvement in beef palatability.

  9. Rice bran fermented with saccharomyces boulardii generates novel metabolite profiles with bioactivity.

    PubMed

    Ryan, Elizabeth P; Heuberger, Adam L; Weir, Tiffany L; Barnett, Brittany; Broeckling, Corey D; Prenni, Jessica E

    2011-03-09

    Emerging evidence supporting chronic disease fighting properties of rice bran has advanced the development of stabilized rice bran for human use as a functional food and dietary supplement. A global and targeted metabolomic investigation of stabilized rice bran fermented with Saccharomyces boulardii was performed in three rice varieties. Metabolites from S. boulardii-fermented rice bran were detected by gas chromatography-mass spectrometry (GC-MS) and assessed for bioactivity compared to nonfermented rice bran in normal and malignant lymphocytes. Global metabolite profiling revealed significant differences in the metabolome that led to discovery of candidate compounds modulated by S. boulardii fermentation. Fermented rice bran extracts from three rice varieties reduced growth of human B lymphomas compared to each variety's nonfermented control and revealed that fermentation differentially altered bioactive compounds. These data support that integration of global and targeted metabolite analysis can be utilized for assessing health properties of rice bran phytochemicals that are enhanced by yeast fermentation and that differ across rice varieties.

  10. Rice Bran Fermented with Saccharomyces boulardii Generates Novel Metabolite Profiles with Bioactivity

    PubMed Central

    2011-01-01

    Emerging evidence supporting chronic disease fighting properties of rice bran has advanced the development of stabilized rice bran for human use as a functional food and dietary supplement. A global and targeted metabolomic investigation of stabilized rice bran fermented with Saccharomyces boulardii was performed in three rice varieties. Metabolites from S. boulardii-fermented rice bran were detected by gas chromatography−mass spectrometry (GC−MS) and assessed for bioactivity compared to nonfermented rice bran in normal and malignant lymphocytes. Global metabolite profiling revealed significant differences in the metabolome that led to discovery of candidate compounds modulated by S. boulardii fermentation. Fermented rice bran extracts from three rice varieties reduced growth of human B lymphomas compared to each variety’s nonfermented control and revealed that fermentation differentially altered bioactive compounds. These data support that integration of global and targeted metabolite analysis can be utilized for assessing health properties of rice bran phytochemicals that are enhanced by yeast fermentation and that differ across rice varieties. PMID:21306106

  11. Variable Cyanobacterial Toxin and Metabolite Profiles across Six Eutrophic Lakes of Differing Physiochemical Characteristics

    PubMed Central

    Beversdorf, Lucas J.; Weirich, Chelsea A.; Bartlett, Sarah L.; Miller, Todd. R.

    2017-01-01

    Future sustainability of freshwater resources is seriously threatened due to the presence of harmful cyanobacterial blooms, and yet, the number, extent, and distribution of most cyanobacterial toxins—including “emerging” toxins and other bioactive compounds—are poorly understood. We measured 15 cyanobacterial compounds—including four microcystins (MC), saxitoxin (SXT), cylindrospermopsin (CYL), anatoxin-a (ATX) and homo-anatoxin-a (hATX), two anabaenopeptins (Apt), three cyanopeptolins (Cpt), microginin (Mgn), and nodularin (NOD)—in six freshwater lakes that regularly experience noxious cHABs. MC, a human liver toxin, was present in all six lakes and was detected in 80% of all samples. Similarly, Apt, Cpt, and Mgn were detected in all lakes in roughly 86%, 50%, and 35% of all samples, respectively. Despite being a notable brackish water toxin, NOD was detected in the two shallowest lakes—Wingra (4.3 m) and Koshkonong (2.1 m). All compounds were highly variable temporally, and spatially. Metabolite profiles were significantly different between lakes suggesting lake characteristics influenced the cyanobacterial community and/or metabolite production. Understanding how cyanobacterial toxins are distributed across eutrophic lakes may shed light onto the ecological function of these metabolites, provide valuable information for their remediation and removal, and aid in the protection of public health. PMID:28208628

  12. Metabolite profiling reveals clear metabolic changes during somatic embryo development of Norway spruce (Picea abies).

    PubMed

    Businge, Edward; Brackmann, Klaus; Moritz, Thomas; Egertsdotter, Ulrika

    2012-02-01

    Progress on industrial-scale propagation of conifers by somatic embryogenesis has been hampered by the differences in developmental capabilities between cell lines, which are limiting the capture of genetic gains from breeding programs. In this study, we investigated the metabolic events occurring during somatic embryo development in Norway spruce to establish a better understanding of the fundamental metabolic events required for somatic embryo development. Three embryogenic cell lines of Norway spruce (Picea abies (L.) Karst) with different developmental capabilities were studied during somatic embryo development from proliferation of proembryogenic masses to mature somatic embryos. The three different cell lines displayed normal, aberrant and blocked somatic embryo development. Metabolite profiles from four development stages in each of the cell lines were obtained using combined gas chromatography-mass spectrometry. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P  ≤  0.05) for each development stage and transition. The results suggest that endogenous auxin and sugar signaling affects initial stages of somatic embryo development. Furthermore, the results highlight the importance of a timed stress response and the presence of stimulatory metabolites during late stages of embryo development.

  13. MS-Based Metabolite Profiling of Aboveground and Root Components of Zingiber mioga and Officinale.

    PubMed

    Han, Ji Soo; Lee, Sunmin; Kim, Hyang Yeon; Lee, Choong Hwan

    2015-09-03

    Zingiber species are members of the Zingiberaceae family, and are widely used for medicinal and food purposes. In this study aboveground and root parts of Zingiber mioga and Zingiber officinale were subjected to metabolite profiling by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) in order to characterize them by species and parts and also to measure bioactivities. Both primary and secondary metabolites showed clear discrimination in the PCA score plot and PLS-DA by species and parts. Tetrahydrocurcumin, diarylheptanoid, 8-gingerol, and 8-paradol were discriminating metabolites between Z. mioga and Z. officinale that were present in different quantities. Eleven flavonoids, six amino acids, six organic acids, four fatty acids, and gingerenone A were higher in the aboveground parts than the root parts. Antioxidant activities were measured and were highest in the root part of Z. officinale. The relatively high contents of tetrahydrocurcumin, diarylheptanoid, and galanganol C in the root part of Z. officinale showed highly positive correlation with bioactivities based on correlation assay. On the basis of these results, we can suggest different usages of structurally different parts of Zingiber species as food plants.

  14. Profiling of microbial-derived phenolic metabolites in human feces after moderate red wine intake.

    PubMed

    Muñoz-González, Irene; Jiménez-Girón, Ana; Martín-Álvarez, Pedro J; Bartolomé, Begoña; Moreno-Arribas, M Victoria

    2013-10-02

    A controlled and randomized trial study involving 41 healthy volunteers (33 intervention and 8 control subjects) was performed in order to establish changes in the microbial-derived phenolic metabolite profile of feces after moderate consumption of red wine (250 mL/day, 4 weeks). Out of the 35 phenolic metabolites identified, 10 compounds (mainly benzoic and 4-hydroxyvaleric acids) showed statistically significant increases (P < 0.05) after the wine intake. Also, the total phenolic metabolites content was significantly (P < 0.05) higher in the samples after the wine intake (625 ± 380 μg/g feces) in comparison to the samples before (358 ± 270 μg/g feces), and a tentative distribution of the volunteers into three groups could be established: <500, 500-1000, and >1000 μg/g feces. These results suggest that a different gut microbial capacity to metabolize wine polyphenols exists among the human population, as observed for polyphenols from other sources.

  15. Pentylindole/Pentylindazole Synthetic Cannabinoids and Their 5-Fluoro Analogs Produce Different Primary Metabolites: Metabolite Profiling for AB-PINACA and 5F-AB-PINACA.

    PubMed

    Wohlfarth, Ariane; Castaneto, Marisol S; Zhu, Mingshe; Pang, Shaokun; Scheidweiler, Karl B; Kronstrand, Robert; Huestis, Marilyn A

    2015-05-01

    Whereas non-fluoropentylindole/indazole synthetic cannabinoids appear to be metabolized preferably at the pentyl chain though without clear preference for one specific position, their 5-fluoro analogs' major metabolites usually are 5-hydroxypentyl and pentanoic acid metabolites. We determined metabolic stability and metabolites of N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and 5-fluoro-AB-PINACA (5F-AB-PINACA), two new synthetic cannabinoids, and investigated if results were similar. In silico prediction was performed with MetaSite (Molecular Discovery). For metabolic stability, 1 μmol/L of each compound was incubated with human liver microsomes for up to 1 h, and for metabolite profiling, 10 μmol/L was incubated with pooled human hepatocytes for up to 3 h. Also, authentic urine specimens from AB-PINACA cases were hydrolyzed and extracted. All samples were analyzed by liquid chromatography high-resolution mass spectrometry on a TripleTOF 5600+ (AB SCIEX) with gradient elution (0.1% formic acid in water and acetonitrile). High-resolution full-scan mass spectrometry (MS) and information-dependent acquisition MS/MS data were analyzed with MetabolitePilot (AB SCIEX) using different data processing algorithms. Both drugs had intermediate clearance. We identified 23 AB-PINACA metabolites, generated by carboxamide hydrolysis, hydroxylation, ketone formation, carboxylation, epoxide formation with subsequent hydrolysis, or reaction combinations. We identified 18 5F-AB-PINACA metabolites, generated by the same biotransformations and oxidative defluorination producing 5-hydroxypentyl and pentanoic acid metabolites shared with AB-PINACA. Authentic urine specimens documented presence of these metabolites. AB-PINACA and 5F-AB-PINACA produced suggested metabolite patterns. AB-PINACA was predominantly hydrolyzed to AB-PINACA carboxylic acid, carbonyl-AB-PINACA, and hydroxypentyl AB-PINACA, likely in 4-position. The most intense 5F

  16. Combination of UHPLC/Q-TOF-MS, NMR spectroscopy, and ECD calculation for screening and identification of reactive metabolites of gentiopicroside in humans.

    PubMed

    Han, Han; Xiong, Ai-Zhen; He, Chun-Yong; Liu, Qing; Yang, Li; Wang, Zheng-Tao

    2014-02-01

    The metabolic investigation of natural products is a great challenge because of unpredictable metabolic pathways, little knowledge on metabolic effects, and lack of recommended analytical methodology. Herein, a combined strategy based on ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), nuclear magnetic resonance (NMR) spectroscopy, and electronic circular dichroism (ECD) calculation was developed and employed for the human metabolism study of gentiopicroside (GPS), a naturally hepato-protective iridoid glycoside. The whole metabolic study consisted of three major procedures. First, an improved UHPLC/Q-TOF-MS method was used to separate and detect a total of 15 GPS metabolites that were obtained from urine samples (0 to 72 h) of 12 healthy male participants after a single 50-mg oral dose of GPS. Second, a developed "MS-NMR-MS" method was applied to accurately identify molecular structures of the observed metabolites. Finally, given that the associated stereochemistry may be a crucial factor of the metabolic activation, the absolute configuration of the reactive metabolites was revealed through chemical calculations. Based on the combined use, a pair of diastereoisomers (G05 and G06) were experimentally addressed as the bioreactive metabolites of GPS, and the stereochemical determination was completed. Whereas several novel metabolic transformations, occurring via oxidation, N-heterocyclization and glucuronidation after deglycosylation, were also observed. The results indicated that GPS has to undergo in vivo metabolism-based activation to generate reactive molecules capable of processing its hepato-protective activity.

  17. Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm, and testa

    PubMed Central

    Winkelmann, Traud; Ratjens, Svenja; Bartsch, Melanie; Rode, Christina; Niehaus, Karsten; Bednarz, Hanna

    2015-01-01

    Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified. Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos. PMID:26300898

  18. Metabolite profiling of bendamustine in urine of cancer patients after administration of [14C]bendamustine.

    PubMed

    Dubbelman, Anne-Charlotte; Jansen, Robert S; Rosing, Hilde; Darwish, Mona; Hellriegel, Edward; Robertson, Philmore; Schellens, Jan H M; Beijnen, Jos H

    2012-07-01

    Bendamustine is an alkylating agent consisting of a mechlorethamine derivative, a benzimidazole group, and a butyric acid substituent. A human mass balance study showed that bendamustine is extensively metabolized and subsequently excreted in urine. However, limited information is available on the metabolite profile of bendamustine in human urine. The objective of this study was to elucidate the metabolic pathways of bendamustine in humans by identification of its metabolites excreted in urine. Human urine samples were collected up to 168 h after an intravenous infusion of 120 mg/m(2) (80-95 μCi) [(14)C]bendamustine. Metabolites of [(14)C]bendamustine were identified using liquid chromatography (high-resolution)-tandem mass spectrometry with off-line radioactivity detection. Bendamustine and a total of 25 bendamustine-related compounds were detected. Observed metabolic conversions at the benzimidazole and butyric acid moiety were N-demethylation and γ-hydroxylation. In addition, various other combinations of these conversions with modifications at the mechlorethamine moiety were observed, including hydrolysis (the primary metabolic pathway), cysteine conjugation, and subsequent biotransformation to mercapturic acid and thiol derivatives, N-dealkylation, oxidation, and conjugation with phosphate, creatinine, and uric acid. Bendamustine-derived products containing phosphate, creatinine, and uric acid conjugates were also detected in control urine incubated with bendamustine. Metabolites that were excreted up to 168 h after the infusion included products of dihydrolysis and cysteine conjugation of bendamustine and γ-hydroxybendamustine. The range of metabolic reactions is generally consistent with those reported for rat urine and bile, suggesting that the overall processes involved in metabolic elimination are qualitatively the same in rats and humans.

  19. Analysis of Hypoxia and Hypoxia-Like States through Metabolite Profiling

    PubMed Central

    Cox, James E.; Reddi, Amit R.; McGinnis, Lauren A.; Culotta, Valeria C.

    2011-01-01

    Background In diverse organisms, adaptation to low oxygen (hypoxia) is mediated through complex gene expression changes that can, in part, be mimicked by exposure to metals such as cobalt. Although much is known about the transcriptional response to hypoxia and cobalt, little is known about the all-important cell metabolism effects that trigger these responses. Methods and Findings Herein we use a low molecular weight metabolome profiling approach to identify classes of metabolites in yeast cells that are altered as a consequence of hypoxia or cobalt exposures. Key findings on metabolites were followed-up by measuring expression of relevant proteins and enzyme activities. We find that both hypoxia and cobalt result in a loss of essential sterols and unsaturated fatty acids, but the basis for these changes are disparate. While hypoxia can affect a variety of enzymatic steps requiring oxygen and heme, cobalt specifically interferes with diiron-oxo enzymatic steps for sterol synthesis and fatty acid desaturation. In addition to diiron-oxo enzymes, cobalt but not hypoxia results in loss of labile 4Fe-4S dehydratases in the mitochondria, but has no effect on homologous 4Fe-4S dehydratases in the cytosol. Most striking, hypoxia but not cobalt affected cellular pools of amino acids. Amino acids such as aromatics were elevated whereas leucine and methionine, essential to the strain used here, dramatically decreased due to hypoxia induced down-regulation of amino acid permeases. Conclusions These studies underscore the notion that cobalt targets a specific class of iron proteins and provide the first evidence for hypoxia effects on amino acid regulation. This research illustrates the power of metabolite profiling for uncovering new adaptations to environmental stress. PMID:21931840

  20. Faecal metabolite profiling identifies medium-chain fatty acids as discriminating compounds in IBD.

    PubMed

    De Preter, Vicky; Machiels, Kathleen; Joossens, Marie; Arijs, Ingrid; Matthys, Christophe; Vermeire, Severine; Rutgeerts, Paul; Verbeke, Kristin

    2015-03-01

    Bacteria play a role in the onset and perpetuation of intestinal inflammation in IBD. Compositional alterations may also change the metabolic capacities of the gut bacteria. To examine the metabolic activity of the microbiota of patients with Crohn's disease (CD), UC or pouchitis compared with healthy controls (HC) and determine whether eventual differences might be related to the pathogenesis of the disease. Faecal samples were obtained from 40 HC, 83 patients with CD, 68 with UC and 13 with pouchitis. Disease activity was assessed in CD using the Harvey-Bradshaw Index, in UC using the UC Disease Activity Index and in pouchitis using the Pouchitis Disease Activity Index. Metabolite profiles were analysed using gas chromatography-mass spectrometry. The number of metabolites identified in HC (54) was significantly higher than in patients with CD (44, p<0.001), UC (47, p=0.042) and pouchitis (43, p=0.036). Multivariate discriminant analysis predicted HC, CD, UC and pouchitis group membership with high sensitivity and specificity. The levels of medium-chain fatty acids (MCFAs: pentanoate, hexanoate, heptanoate, octanoate and nonanoate), and of some protein fermentation metabolites, were significantly decreased in patients with CD, UC and pouchitis. Hexanoate levels were inversely correlated to disease activity in CD (correlation coefficient=-0.157, p=0.046), whereas a significant positive correlation was found between styrene levels and disease activity in UC (correlation coefficient=0.338, p=0.001). Faecal metabolic profiling in patients with IBD relative to healthy controls identified MCFAs as important metabolic biomarkers of disease-related changes. NCT 01666717. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  1. Multicomponent Analysis of the Differential Induction of Secondary Metabolite Profiles in Fungal Endophytes.

    PubMed

    González-Menéndez, Víctor; Pérez-Bonilla, Mercedes; Pérez-Victoria, Ignacio; Martín, Jesús; Muñoz, Francisca; Reyes, Fernando; Tormo, José R; Genilloud, Olga

    2016-02-18

    Small molecule histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors are commonly used to perturb the production of fungal metabolites leading to the induction of the expression of silent biosynthetic pathways. Several reports have described the variable effects observed in natural product profiles in fungi treated with HDAC and DNMT inhibitors, such as enhanced chemical diversity and/or the induction of new molecules previously unknown to be produced by the strain. Fungal endophytes are known to produce a wide variety of secondary metabolites (SMs) involved in their adaptation and survival within higher plants. The plant-microbe interaction may influence the expression of some biosynthetic pathways, otherwise cryptic in these fungi when grown in vitro. The aim of this study was to setup a systematic approach to evaluate and identify the possible effects of HDAC and DNMT inhibitors on the metabolic profiles of wild type fungal endophytes, including the chemical identification and characterization of the most significant SMs induced by these epigenetic modifiers.

  2. Mass Spectrometry-Based Metabolomics Identifies Longitudinal Urinary Metabolite Profiles Predictive of Radiation-Induced Cancer.

    PubMed

    Cook, John A; Chandramouli, Gadisetti V R; Anver, Miriam R; Sowers, Anastasia L; Thetford, Angela; Krausz, Kristopher W; Gonzalez, Frank J; Mitchell, James B; Patterson, Andrew D

    2016-03-15

    Nonlethal exposure to ionizing radiation (IR) is a public concern due to its known carcinogenic effects. Although latency periods for IR-induced neoplasms are relatively long, the ability to detect cancer as early as possible is highly advantageous for effective therapeutic intervention. Therefore, we hypothesized that metabolites in the urine from mice exposed to total body radiation (TBI) would predict for the presence of cancer before a palpable mass was detected. In this study, we exposed mice to 0 or 5.4 Gy TBI, collected urine samples periodically over 1 year, and assayed urine metabolites by using mass spectrometry. Longitudinal data analysis within the first year post-TBI revealed that cancers, including hematopoietic, solid, and benign neoplasms, could be distinguished by unique urinary signatures as early as 3 months post-TBI. Furthermore, a distinction among different types of malignancies could be clearly delineated as early as 3 months post-TBI for hematopoietic neoplasms, 6 months for solid neoplasms, and by 1 year for benign neoplasms. Moreover, the feature profile for radiation-exposed mice 6 months post-TBI was found to be similar to nonirradiated control mice at 18 months, suggesting that TBI accelerates aging. These results demonstrate that urine feature profiles following TBI can identify cancers in mice prior to macroscopic detection, with important implications for the early diagnosis and treatment. ©2016 American Association for Cancer Research.

  3. Comparative Metabolite Profiling of Triterpenoid Saponins and Flavonoids in Flower Color Mutations of Primula veris L.

    PubMed

    Apel, Lysanne; Kammerer, Dietmar R; Stintzing, Florian C; Spring, Otmar

    2017-01-13

    Primula veris L. is an important medicinal plant with documented use for the treatment of gout, headache and migraine reaching back to the Middle Ages. Triterpenoid saponins from roots and flowers are used in up-to-date phytotherapeutic treatment of bronchitis and colds due to their expectorant and secretolytic effects. In addition to the wild type plants with yellow petals, a red variant and an intermediate orange form of Primula veris L. have recently been found in a natural habitat. The secondary metabolite profiles of roots, leaves and flowers of these rare variants were investigated and compared with the wild type metabolome. Two flavonoids, six flavonoid glycosides, four novel methylated flavonoid glycosides, five anthocyanins and three triterpenoid saponins were identified in alcoholic extracts from the petals, leaves and roots of the three variants by high performance liquid chromatography (HPLC)-diode array detection (DAD)/mass spectrometry (MS(n)) analyses. Anthocyanins were detected in the petals of the red and orange variety, but not in the wild type. No other effects on the metabolite profiles of the three varieties have been observed. The possibility is discussed that a regulatory step of the anthocyanin biosynthetic pathway may have been affected by mutation thus triggering color polymorphism in the petals.

  4. Comparative Metabolite Profiling of Triterpenoid Saponins and Flavonoids in Flower Color Mutations of Primula veris L.

    PubMed Central

    Apel, Lysanne; Kammerer, Dietmar R.; Stintzing, Florian C.; Spring, Otmar

    2017-01-01

    Primula veris L. is an important medicinal plant with documented use for the treatment of gout, headache and migraine reaching back to the Middle Ages. Triterpenoid saponins from roots and flowers are used in up-to-date phytotherapeutic treatment of bronchitis and colds due to their expectorant and secretolytic effects. In addition to the wild type plants with yellow petals, a red variant and an intermediate orange form of Primula veris L. have recently been found in a natural habitat. The secondary metabolite profiles of roots, leaves and flowers of these rare variants were investigated and compared with the wild type metabolome. Two flavonoids, six flavonoid glycosides, four novel methylated flavonoid glycosides, five anthocyanins and three triterpenoid saponins were identified in alcoholic extracts from the petals, leaves and roots of the three variants by high performance liquid chromatography (HPLC)-diode array detection (DAD)/mass spectrometry (MSn) analyses. Anthocyanins were detected in the petals of the red and orange variety, but not in the wild type. No other effects on the metabolite profiles of the three varieties have been observed. The possibility is discussed that a regulatory step of the anthocyanin biosynthetic pathway may have been affected by mutation thus triggering color polymorphism in the petals. PMID:28098796

  5. Quantitative transcriptome, proteome, and sulfur metabolite profiling of the Saccharomyces cerevisiae response to arsenite.

    PubMed

    Thorsen, Michael; Lagniel, Gilles; Kristiansson, Erik; Junot, Christophe; Nerman, Olle; Labarre, Jean; Tamás, Markus J

    2007-06-19

    Arsenic is ubiquitously present in nature, and various mechanisms have evolved enabling cells to evade toxicity and acquire tolerance. Herein, we explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent arsenic (arsenite) by quantitative transcriptome, proteome, and sulfur metabolite profiling. Arsenite exposure affected transcription of genes encoding functions related to protein biosynthesis, arsenic detoxification, oxidative stress defense, redox maintenance, and proteolytic activity. Importantly, we observed that nearly all components of the sulfate assimilation and glutathione biosynthesis pathways were induced at both gene and protein levels. Kinetic metabolic profiling evidenced a significant increase in the pools of sulfur metabolites as well as elevated cellular glutathione levels. Moreover, the flux in the sulfur assimilation pathway as well as the glutathione synthesis rate strongly increased with a concomitant reduction of sulfur incorporation into proteins. By combining comparative genomics and molecular analyses, we pinpointed transcription factors that mediate the core of the transcriptional response to arsenite. Taken together, our data reveal that arsenite-exposed cells channel a large part of assimilated sulfur into glutathione biosynthesis, and we provide evidence that the transcriptional regulators Yap1p and Met4p control this response in concert.

  6. 1H NMR-based metabolic profiling for evaluating poppy seed rancidity and brewing.

    PubMed

    Jawień, Ewa; Ząbek, Adam; Deja, Stanisław; Łukaszewicz, Marcin; Młynarz, Piotr

    2015-12-01

    Poppy seeds are widely used in household and commercial confectionery. The aim of this study was to demonstrate the application of metabolic profiling for industrial monitoring of the molecular changes which occur during minced poppy seed rancidity and brewing processes performed on raw seeds. Both forms of poppy seeds were obtained from a confectionery company. Proton nuclear magnetic resonance (1H NMR) was applied as the analytical method of choice together with multivariate statistical data analysis. Metabolic fingerprinting was applied as a bioprocess control tool to monitor rancidity with the trajectory of change and brewing progressions. Low molecular weight compounds were found to be statistically significant biomarkers of these bioprocesses. Changes in concentrations of chemical compounds were explained relative to the biochemical processes and external conditions. The obtained results provide valuable and comprehensive information to gain a better understanding of the biology of rancidity and brewing processes, while demonstrating the potential for applying NMR spectroscopy combined with multivariate data analysis tools for quality control in food industries involved in the processing of oilseeds. This precious and versatile information gives a better understanding of the biology of these processes.

  7. Impurity profiling of carbocisteine by HPLC-CAD, qNMR and UV/vis spectroscopy.

    PubMed

    Wahl, Oliver; Holzgrabe, Ulrike

    2014-07-01

    For the impurity profiling of the mucolytic and anti-inflammatory drug carbocisteine a high performance liquid chromatographic (HPLC) method using corona charged aerosol detection (CAD) was developed and fully validated following the ICH guideline Q2(R1). The response was linear (R²>0.995) over a small concentration range (0.05-0.25 or 0.10-0.60% respectively) and a detection limit of at least 0.03% was registered. The separation was achieved on a mixed mode column combining hydrophobic C18 and strong cation exchange retention mechanisms using a mass spectrometer compatible volatile mobile phase consisting of trifluoroacetic acid 10 mM and acetonitrile 12% (V/V). Impurities, not assessable by HPLC-CAD such as the volatile chloroacetic acid and the unstable cysteine, were determined by quantitative NMR (qNMR) with maleic acid as internal standard and UV/vis spectroscopy after reaction with Ellman's reagent, respectively. Six batches of three different manufacturers were tested by means of those methods. The purity varied from below 99.0 to higher than 99.8 per cent. The major impurities of all batches were the starting material cystine and N,S-dicarboxymethylcysteine being a synthesis by-product.

  8. Metabolite profiling and cardiovascular event risk: a prospective study of 3 population-based cohorts.

    PubMed

    Würtz, Peter; Havulinna, Aki S; Soininen, Pasi; Tynkkynen, Tuulia; Prieto-Merino, David; Tillin, Therese; Ghorbani, Anahita; Artati, Anna; Wang, Qin; Tiainen, Mika; Kangas, Antti J; Kettunen, Johannes; Kaikkonen, Jari; Mikkilä, Vera; Jula, Antti; Kähönen, Mika; Lehtimäki, Terho; Lawlor, Debbie A; Gaunt, Tom R; Hughes, Alun D; Sattar, Naveed; Illig, Thomas; Adamski, Jerzy; Wang, Thomas J; Perola, Markus; Ripatti, Samuli; Vasan, Ramachandran S; Raitakari, Olli T; Gerszten, Robert E; Casas, Juan-Pablo; Chaturvedi, Nish; Ala-Korpela, Mika; Salomaa, Veikko

    2015-03-03

    High-throughput profiling of circulating metabolites may improve cardiovascular risk prediction over established risk factors. We applied quantitative nuclear magnetic resonance metabolomics to identify the biomarkers for incident cardiovascular disease during long-term follow-up. Biomarker discovery was conducted in the National Finnish FINRISK study (n=7256; 800 events). Replication and incremental risk prediction was assessed in the Southall and Brent Revisited (SABRE) study (n=2622; 573 events) and British Women's Health and Heart Study (n=3563; 368 events). In targeted analyses of 68 lipids and metabolites, 33 measures were associated with incident cardiovascular events at P<0.0007 after adjusting for age, sex, blood pressure, smoking, diabetes mellitus, and medication. When further adjusting for routine lipids, 4 metabolites were associated with future cardiovascular events in meta-analyses: higher serum phenylalanine (hazard ratio per standard deviation, 1.18; 95% confidence interval, 1.12-1.24; P=4×10(-10)) and monounsaturated fatty acid levels (1.17; 1.11-1.24; P=1×10(-8)) were associated with increased cardiovascular risk, while higher omega-6 fatty acids (0.89; 0.84-0.94; P=6×10(-5)) and docosahexaenoic acid levels (0.90; 0.86-0.95; P=5×10(-5)) were associated with lower risk. A risk score incorporating these 4 biomarkers was derived in FINRISK. Risk prediction estimates were more accurate in the 2 validation cohorts (relative integrated discrimination improvement, 8.8% and 4.3%), albeit discrimination was not enhanced. Risk classification was particularly improved for persons in the 5% to 10% risk range (net reclassification, 27.1% and 15.5%). Biomarker associations were further corroborated with mass spectrometry in FINRISK (n=671) and the Framingham Offspring Study (n=2289). Metabolite profiling in large prospective cohorts identified phenylalanine, monounsaturated fatty acids, and polyunsaturated fatty acids as biomarkers for cardiovascular risk

  9. UPLC-ESI-TOF MS-Based Metabolite Profiling of the Antioxidative Food Supplement Garcinia buchananii.

    PubMed

    Stark, Timo D; Lösch, Sofie; Wakamatsu, Junichiro; Balemba, Onesmo B; Frank, Oliver; Hofmann, Thomas

    2015-08-19

    Comparative antioxidative analyses of aqueous ethanolic extracts from leaf, root, and stem of Garcinia buchananii revealed high activity of all three organs. To investigate the metabolite composition of the different parts of G. buchananii, an untargeted metabolomics approach using UPLC-ESI-TOF MS with simultaneous acquisition of low- and high-collision energy mass spectra (MS(e)) was performed. Unsupervised statistics (PCA) highlighted clear differences in the metabolomes of the three organs. OPLS-DA revealed (2R,3S,2″R,3″R)-GB-1, (2R,3S)-morelloflavone, and (2R,3S)-volkensiflavone as the most decisive marker compounds discriminating leaf from root and stem extract. Leaves represent the best source to isolate GB-1, morelloflavone, and volkensiflavone. Root extract is the best organ to isolate xanthones and stem bark extract the best source to isolate (2R,3S,2″R,3″R)-manniflavanone; the identified polyisoprenylated benzophenones are characteristic compounds for the leaf organ. Morelloflavone, volkensiflavone, and garcicowin C were isolated for the first time from G. buchananii, identified via MS, NMR, and CD spectroscopy, and showed in H2O2 scavenging, H/L-TEAC, and H/L-ORAC assays moderate to strong in vitro antioxidative activities.

  10. Improving Assessment of Lipoprotein Profile in Type 1 Diabetes by 1H NMR Spectroscopy

    PubMed Central

    Brugnara, Laura; Mallol, Roger; Ribalta, Josep; Vinaixa, Maria; Murillo, Serafín; Casserras, Teresa; Guardiola, Montse; Vallvé, Joan Carles; Kalko, Susana G.; Correig, Xavier; Novials, Anna

    2015-01-01

    Patients with type 1 diabetes (T1D) present increased risk of cardiovascular disease (CVD). The aim of this study is to improve the assessment of lipoprotein profile in patients with T1D by using a robust developed method 1H nuclear magnetic resonance spectroscopy (1H NMR), for further correlation with clinical factors associated to CVD. Thirty patients with T1D and 30 non-diabetes control (CT) subjects, matched for gender, age, body composition (DXA, BMI, waist/hip ratio), regular physical activity levels and cardiorespiratory capacity (VO2peak), were analyzed. Dietary records and routine lipids were assessed. Serum lipoprotein particle subfractions, particle sizes, and cholesterol and triglycerides subfractions were analyzed by 1H NMR. It was evidenced that subjects with T1D presented lower concentrations of small LDL cholesterol, medium VLDL particles, large VLDL triglycerides, and total triglycerides as compared to CT subjects. Women with T1D presented a positive association with HDL size (p<0.005; R = 0.601) and large HDL triglycerides (p<0.005; R = 0.534) and negative (p<0.005; R = -0.586) to small HDL triglycerides. Body fat composition represented an important factor independently of normal BMI, with large LDL particles presenting a positive correlation to total body fat (p<0.005; R = 0.505), and total LDL cholesterol and small LDL cholesterol a positive correlation (p<0.005; R = 0.502 and R = 0.552, respectively) to abdominal fat in T1D subjects; meanwhile, in CT subjects, body fat composition was mainly associated to HDL subclasses. VO2peak was negatively associated (p<0.005; R = -0.520) to large LDL-particles only in the group of patients with T1D. In conclusion, patients with T1D with adequate glycemic control and BMI and without chronic complications presented a more favourable lipoprotein profile as compared to control counterparts. In addition, slight alterations in BMI and/or body fat composition showed to be relevant to provoking alterations in

  11. An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection

    PubMed Central

    Ward, Jane L.; Baker, John M.; Miller, Sonia J.; Deborde, Catherine; Maucourt, Mickael; Biais, Benoit; Rolin, Dominique; Moing, Annick; Moco, Sofia; Vervoort, Jacques; Lommen, Arjen; Schäfer, Hartmut; Humpfer, Eberhard

    2010-01-01

    In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [1H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [1H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories. PMID:20526352

  12. Automated Quantification of Human Brain Metabolites by Artificial Neural Network Analysis from in VivoSingle-Voxel 1H NMR Spectra

    NASA Astrophysics Data System (ADS)

    Kaartinen, Jouni; Mierisová, Šarka; Oja, Joni M. E.; Usenius, Jukka-Pekka; Kauppinen, Risto A.; Hiltunen, Yrjö

    1998-09-01

    A real-time automated way of quantifying metabolites fromin vivoNMR spectra using an artificial neural network (ANN) analysis is presented. The spectral training and test sets for ANN containing peaks at the chemical shift ranges resembling long echo time proton NMR spectra from human brain were simulated. The performance of the ANN constructed was compared with an established lineshape fitting (LF) analysis using both simulated and experimental spectral data as inputs. The correspondence between the ANN and LF analyses showed correlation coefficients of order of 0.915-0.997 for spectra with large variations in both signal-to-noise and peak areas. Water suppressed1H NMR spectra from 24 healthy subjects were collected and choline-containing compounds (Cho), total creatine (Cr), and N-acetyl aspartate (NAA) were quantified with both methods. The ANN quantified these spectra with an accuracy similar to LF analysis (correlation coefficients of 0.915-0.951). These results show that LF and ANN are equally good quantifiers; however, the ANN analyses are more easily automated than LF analyses.

  13. Qualitative Profiling and Quantification of Neonicotinoid Metabolites in Human Urine by Liquid Chromatography Coupled with Mass Spectrometry

    PubMed Central

    Taira, Kumiko; Fujioka, Kazutoshi; Aoyama, Yoshiko

    2013-01-01

    Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human

  14. Qualitative profiling and quantification of neonicotinoid metabolites in human urine by liquid chromatography coupled with mass spectrometry.

    PubMed

    Taira, Kumiko; Fujioka, Kazutoshi; Aoyama, Yoshiko

    2013-01-01

    Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human

  15. Liquid Chromatography-Mass Spectrometry-Based Rapid Secondary-Metabolite Profiling of Marine Pseudoalteromonas sp. M2

    PubMed Central

    Kim, Woo Jung; Kim, Young Ok; Kim, Jin Hee; Nam, Bo-Hye; Kim, Dong-Gyun; An, Cheul Min; Lee, Jun Sik; Kim, Pan Soo; Lee, Hye Min; Oh, Joa-Sup; Lee, Jong Suk

    2016-01-01

    The ocean is a rich resource of flora, fauna, and food. A wild-type bacterial strain showing confluent growth on marine agar with antibacterial activity was isolated from marine water, identified using 16S rDNA sequence analysis as Pseudoalteromonas sp., and designated as strain M2. This strain was found to produce various secondary metabolites including quinolone alkaloids. Using high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis, we identified nine secondary metabolites of 4-hydroxy-2-alkylquinoline (pseudane-III, IV, V, VI, VII, VIII, IX, X, and XI). Additionally, this strain produced two novel, closely related compounds, 2-isopentylqunoline-4-one and 2-(2,3-dimetylbutyl)qunoline-4-(1H)-one, which have not been previously reported from marine bacteria. From the metabolites produced by Pseudoalteromonas sp. M2, 2-(2,3-dimethylbutyl)quinolin-4-one, pseudane-VI, and pseudane-VII inhibited melanin synthesis in Melan-A cells by 23.0%, 28.2%, and 42.7%, respectively, wherein pseudane-VII showed the highest inhibition at 8 µg/mL. The results of this study suggest that liquid chromatography (LC)-MS/MS-based metabolite screening effectively improves the efficiency of novel metabolite discovery. Additionally, these compounds are promising candidates for further bioactivity development. PMID:26805856

  16. Liquid Chromatography-Mass Spectrometry-Based Rapid Secondary-Metabolite Profiling of Marine Pseudoalteromonas sp. M2.

    PubMed

    Kim, Woo Jung; Kim, Young Ok; Kim, Jin Hee; Nam, Bo-Hye; Kim, Dong-Gyun; An, Cheul Min; Lee, Jun Sik; Kim, Pan Soo; Lee, Hye Min; Oh, Joa-Sup; Lee, Jong Suk

    2016-01-20

    The ocean is a rich resource of flora, fauna, and food. A wild-type bacterial strain showing confluent growth on marine agar with antibacterial activity was isolated from marine water, identified using 16S rDNA sequence analysis as Pseudoalteromonas sp., and designated as strain M2. This strain was found to produce various secondary metabolites including quinolone alkaloids. Using high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis, we identified nine secondary metabolites of 4-hydroxy-2-alkylquinoline (pseudane-III, IV, V, VI, VII, VIII, IX, X, and XI). Additionally, this strain produced two novel, closely related compounds, 2-isopentylqunoline-4-one and 2-(2,3-dimetylbutyl)qunoline-4-(1H)-one, which have not been previously reported from marine bacteria. From the metabolites produced by Pseudoalteromonas sp. M2, 2-(2,3-dimethylbutyl)quinolin-4-one, pseudane-VI, and pseudane-VII inhibited melanin synthesis in Melan-A cells by 23.0%, 28.2%, and 42.7%, respectively, wherein pseudane-VII showed the highest inhibition at 8 µg/mL. The results of this study suggest that liquid chromatography (LC)-MS/MS-based metabolite screening effectively improves the efficiency of novel metabolite discovery. Additionally, these compounds are promising candidates for further bioactivity development.

  17. Synergistic Hepatoprotective and Antioxidant Effect of Artichoke, Fig, Mulberry Herbal Mixture on HepG2 Cells and their Metabolic Profiling Using NMR Coupled with Chemometrics.

    PubMed

    Youssef, Fadia S; Labib, Rola M; Eldahshan, Omayma A; Singab, Abdel Nasser B

    2017-09-12

    The edible plants have long been reported to possess a lot of biological activities. Herein, the hepatoprotective and the antioxidant activity of the aqueous infusion of the edible parts of Cynara cardunculus, Ficus carica and Morus nigra and their herbal mixture (CFM) was investigated in vitro using CCl4 induced damage in HepG2 cells. The highest amelioration was observed via the consumption of CFM at 1 mg/ml showing 47.00, 37.09% decline in AST (Aspartate Transaminase) and ALT (Alanine Transaminase) and 77.32 and 101.02% increase in GSH (Reduced Glutathione) and SOD (Superoxide dismutase) comparable to CCl4 treated cells. Metabolic profiling of their aqueous infusions was done using nuclear magnetic resonance spectroscopic experiments coupled with chemometrics particularly HCA (Hierarchical Cluster Analysis) and PCA (Principal Component Analysis). The structural closeness of the various metabolites existing in black berry and the mixture as reflected in the PCA score plot and HCA processed from the (1) H NMR spectral data could eventually explained the close values in their biological behavior. For fig and artichoke the existence of different phenolic metabolites that act synergistically could greatly interpret their potent biological behavior. Thus, it can be concluded that a herbal mixture composed of black berry, artichoke and fig could afford an excellent natural candidate to combat oxidative stress and counteract hepatic toxins owing to its phenolic compounds. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  18. NMR methodologies in the analysis of blueberries.

    PubMed

    Capitani, Donatella; Sobolev, Anatoly P; Delfini, Maurizio; Vista, Silvia; Antiochia, Riccarda; Proietti, Noemi; Bubici, Salvatore; Ferrante, Gianni; Carradori, Simone; De Salvador, Flavio Roberto; Mannina, Luisa

    2014-06-01

    An NMR analytical protocol based on complementary high and low field measurements is proposed for blueberry characterization. Untargeted NMR metabolite profiling of blueberries aqueous and organic extracts as well as targeted NMR analysis focused on anthocyanins and other phenols are reported. Bligh-Dyer and microwave-assisted extractions were carried out and compared showing a better recovery of lipidic fraction in the case of microwave procedure. Water-soluble metabolites belonging to different classes such as sugars, amino acids, organic acids, and phenolic compounds, as well as metabolites soluble in organic solvent such as triglycerides, sterols, and fatty acids, were identified. Five anthocyanins (malvidin-3-glucoside, malvidin-3-galactoside, delphinidin-3-glucoside, delphinidin-3-galactoside, and petunidin-3-glucoside) and 3-O-α-l-rhamnopyranosyl quercetin were identified in solid phase extract. The water status of fresh and withered blueberries was monitored by portable NMR and fast-field cycling NMR. (1) H depth profiles, T2 transverse relaxation times and dispersion profiles were found to be sensitive to the withering.

  19. Metabolite Profile of Cervicovaginal Fluids from Early Pregnancy Is Not Predictive of Spontaneous Preterm Birth

    PubMed Central

    Thomas, Melinda M.; Sulek, Karolina; McKenzie, Elizabeth J.; Jones, Beatrix; Han, Ting-Li; Villas-Boas, Silas G.; Kenny, Louise C.; McCowan, Lesley M. E.; Baker, Philip N.

    2015-01-01

    In our study, we used a mass spectrometry-based metabolomic approach to search for biomarkers that may act as early indicators of spontaneous preterm birth (sPTB). Samples were selected as a nested case-control study from the Screening for Pregnancy Endpoints (SCOPE) biobank in Auckland, New Zealand. Cervicovaginal swabs were collected at 20 weeks from women who were originally assessed as being at low risk of sPTB. Samples were analysed using gas chromatography-mass spectrometry (GC-MS). Despite the low amount of biomass (16–23 mg), 112 compounds were detected. Statistical analysis showed no significant correlations with sPTB. Comparison of reported infection and plasma inflammatory markers from early pregnancy showed two inflammatory markers were correlated with reported infection, but no correlation with any compounds in the metabolite profile was observed. We hypothesise that the lack of biomarkers of sPTB in the cervicovaginal fluid metabolome is simply because it lacks such markers in early pregnancy. We propose alternative biofluids be investigated for markers of sPTB. Our results lead us to call for greater scrutiny of previously published metabolomic data relating to biomarkers of sPTB in cervicovaginal fluids, as the use of small, high risk, or late pregnancy cohorts may identify metabolite biomarkers that are irrelevant for predicting risk in normal populations. PMID:26610472

  20. Integration of deep transcript and targeted metabolite profiles for eight cultivars of opium poppy.

    PubMed

    Desgagné-Penix, Isabel; Farrow, Scott C; Cram, Dustin; Nowak, Jacek; Facchini, Peter J

    2012-06-01

    Recent advances in DNA sequencing technology and analytical mass spectrometry are providing unprecedented opportunities to develop the functional genomics resources required to investigate complex biological processes in non-model plants. Opium poppy produces a wide variety of benzylisoquinoline alkaloids (BIAs), including the pharmaceutical compounds codeine, morphine, noscapine and papaverine. A functional genomics platform to identify novel BIA biosynthetic and regulatory genes in opium poppy has been established based on the differential metabolite profile of eight selected cultivars. Stem cDNA libraries from each of the eight opium poppy cultivars were subjected to 454 pyrosequencing and searchable expressed sequence tag databases were created from the assembled reads. These deep and integrated metabolite and transcript databases provide a nearly complete representation of the genetic and metabolic variances responsible for the differential occurrence of specific BIAs in each cultivar as demonstrated using the biochemically well characterized pathway from tyrosine to morphine. Similar correlations between the occurrence of specific transcripts and alkaloids effectively reveals candidate genes encoding uncharacterized biosynthetic enzymes as shown using cytochromes P450 potentially involved in the formation of papaverine and noscapine.

  1. Arabidopsis transcript and metabolite profiles: ecotype-specific responses to open-air elevated [CO2].

    PubMed

    Li, Pinghua; Ainsworth, Elizabeth A; Leakey, Andrew D B; Ulanov, Alexander; Lozovaya, Vera; Ort, Donald R; Bohnert, Hans J

    2008-11-01

    A Free-Air CO(2) Enrichment (FACE) experiment compared the physiological parameters, transcript and metabolite profiles of Arabidopsis thaliana Columbia-0 (Col-0) and Cape Verde Island (Cvi-0) at ambient (approximately 0.375 mg g(-1)) and elevated (approximately 0.550 mg g(-1)) CO(2) ([CO(2)]). Photoassimilate pool sizes were enhanced in high [CO(2)] in an ecotype-specific manner. Short-term growth at elevated [CO(2)] stimulated carbon gain irrespective of down-regulation of plastid functions and altered expression of genes involved in nitrogen metabolism resembling patterns observed under N-deficiency. The study confirmed well-known characteristics, but the use of a time course, ecotypic genetic differences, metabolite analysis and the focus on clusters of functional categories provided new aspects about responses to elevated [CO(2)]. Longer-term Cvi-0 responded by down-regulating functions favouring carbon accumulation, and both ecotypes showed altered expression of genes for defence, redox control, transport, signalling, transcription and chromatin remodelling. Overall, carbon fixation with a smaller commitment of resources in elevated [CO(2)] appeared beneficial, with the extra C only partially utilized possibly due to disturbance of the C : N ratio. To different degrees, both ecotypes perceived elevated [CO(2)] as a metabolic perturbation that necessitated increased functions consuming or storing photoassimilate, with Cvi-0 emerging as more capable of acclimating. Elevated [CO(2)] in Arabidopsis favoured adjustments in reactive oxygen species (ROS) homeostasis and signalling that defined genotypic markers.

  2. Discovery metabolite profiling--forging functional connections between the proteome and metabolome.

    PubMed

    Saghatelian, Alan; Cravatt, Benjamin F

    2005-08-19

    Of primary interest for every enzyme is the identification of its physiological substrates. However, the vast structural diversity of endogenous metabolites, coupled with the overlapping activities of numerous enzymes, makes it difficult to deduce the identity of natural substrates for a given enzyme based on in vitro experiments. To address this challenge, we recently introduced an LC-MS based analytical method termed discovery metabolite profiling (DMP) to evaluate the global metabolic effects of enzyme inactivation in vivo. We have applied DMP to study mice lacking the enzyme fatty acid amide hydrolase (FAAH), which degrades the endocannabinoid family of signaling lipids. DMP identified several previously uncharacterized FAAH substrates, including a structurally novel class of brain lipids that represent conjugates of very long chain fatty acids with the amino acid derivative taurine [N-acyl taurines (NATs)]. These findings show that DMP can establish direct connections between the proteome and metabolome and thus offers a powerful strategy to assign physiological functions to enzymes in the post-genomic era.

  3. Penicillium strains isolated from Slovak grape berries taxonomy assessment by secondary metabolite profile.

    PubMed

    Santini, Antonello; Mikušová, Petra; Sulyok, Michael; Krska, Rudolf; Labuda, Roman; Srobárová, Antónia

    2014-11-01

    The secondary metabolite profiles of microfungi of the genus Penicillium isolated from samples of grape berries collected in two different phases during two vegetative seasons in Slovakia is described to assess the taxonomy. Three Slovak vine regions have been selected for this study, based on their climatic differences and national economic importance. Cultures of microfungi isolated from berries were incubated on different selective media for macro and micromorphology identification. The species Penicillium brevicompactum, Penicillium crustosum, Penicillium chrysogenum, Penicillium expansum, Penicillium palitans and Penicillium polonicum were identified according to growth and morphology. The related strains were found to produce a broad spectrum of fungal metabolites, including roquefortine C, chaetoglobosin A, penitrem A, cyclopeptin, cyclopenin, viridicatin, methylviridicatin, verrucofortine, secalonic acid D, cyclopiazonic acid, fumigaclavine and mycophenolic acid. Chemotaxonomy was performed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Dried grape berries were also analyzed allowing to assess the presence of patulin, roquefortine C and penicillic acid; this last one has been identified in dried berries but not in vitro.

  4. Molecular dynamics and information on possible sites of interaction of intramyocellular metabolites in vivo from resolved dipolar couplings in localized 1H NMR spectra

    NASA Astrophysics Data System (ADS)

    Schröder, Leif; Schmitz, Christian; Bachert, Peter

    2004-12-01

    Proton NMR resonances of the endogenous metabolites creatine and phosphocreatine ((P)Cr), taurine (Tau), and carnosine (Cs, β-alanyl- L-histidine) were studied with regard to residual dipolar couplings and molecular mobility. We present an analysis of the direct 1H- 1H interaction that provides information on motional reorientation of subgroups in these molecules in vivo. For this purpose, localized 1H NMR experiments were performed on m. gastrocnemius of healthy volunteers using a 1.5-T clinical whole-body MR scanner. We evaluated the observable dipolar coupling strength SD0 ( S = order parameter) of the (P)Cr-methyl triplet and the Tau-methylene doublet by means of the apparent line splitting. These were compared to the dipolar coupling strength of the (P)Cr-methylene doublet. In contrast to the aliphatic protons of (P)Cr and Tau, the aromatic H2 ( δ = 8 ppm) and H4 ( δ = 7 ppm) protons of the imidazole ring of Cs exhibit second-order spectra at 1.5 T. This effect is the consequence of incomplete transition from Zeeman to Paschen-Back regime and allows a determination of SD0 from H2 and H4 of Cs as an alternative to evaluating the multiplet splitting which can be measured directly in high-resolution 1H NMR spectra. Experimental data showed striking differences in the mobility of the metabolites when the dipolar coupling constant D0 (calculated with the internuclear distance known from molecular geometry in the case of complete absence of molecular dynamics and motion) is used for comparison. The aliphatic signals involve very small order parameters S ≈ (1.4 - 3) × 10 -4 indicating rapid reorientation of the corresponding subgroups in these metabolites. In contrast, analysis of the Cs resonances yielded S ≈ (113 - 137) × 10 -4. Thus, the immobilization of the Cs imidazole ring owing to an anisotropic cellular substructure in human m. gastrocnemius is much more effective than for (P)Cr and Tau subgroups. Furthermore, 1H NMR experiments on aqueous model

  5. Profiling a gut microbiota-generated catechin metabolite's fate in human blood cells using a metabolomic approach.

    PubMed

    Mülek, Melanie; Fekete, Agnes; Wiest, Johannes; Holzgrabe, Ulrike; Mueller, Martin J; Högger, Petra

    2015-10-10

    The microbial catechin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1) has been found in human plasma samples after intake of maritime pine bark extract (Pycnogenol). M1 has been previously shown to accumulate in endothelial and blood cells in vitro after facilitated uptake and to exhibit anti-inflammatory activity. The purpose of the present research approach was to systematically and comprehensively analyze the metabolism of M1 in human blood cells in vitro and in vivo. A metabolomic approach that had been successfully applied for drug metabolite profiling was chosen to detect 19 metabolite peaks of M1 which were subsequently further analyzed and validated. The metabolites were categorized into three levels of identification according to the Metabolomics Standards Initiative with six compounds each confirmed at levels 1 and 2 and seven putative metabolites at level 3. The predominant metabolites were glutathione conjugates which were rapidly formed and revealed prolonged presence within the cells. Although a formation of an intracellular conjugate of M1 and glutathione (M1-GSH) was already known two GSH conjugate isomers, M1-S-GSH and M1-N-GSH were observed in the current study. Additionally detected organosulfur metabolites were conjugates with oxidized glutathione and cysteine. Other biotransformation products constituted the open-chained ester form of M1 and a methylated M1. Six of the metabolites determined in in vitro assays were also detected in blood cells in vivo after ingestion of the pine bark extract by two volunteers. The present study provides the first evidence that multiple and structurally heterogeneous polyphenol metabolites can be generated in human blood cells. The bioactivity of the M1 metabolites and their contribution to the previously determined anti-inflammatory effects of M1 now need to be elucidated.

  6. Systematic review A systematic review of metabolite profiling in gestational diabetes mellitus

    PubMed Central

    Huynh, Jennifer; Xiong, Grace; Bentley-Lewis, Rhonda

    2014-01-01

    Aims/hypothesis Gestational diabetes mellitus is associated with adverse maternal and fetal outcomes during, as well as subsequent to, pregnancy, including increased risk of type 2 diabetes and cardiovascular disease. Because of the importance of early risk stratification in preventing these complications, improved first-trimester biomarker determination for diagnosing gestational diabetes would enhance our ability to optimise both maternal and fetal health. Metabolomic profiling, the systematic study of small molecule products of biochemical pathways, has shown promise in the identification of key metabolites associated with the pathogenesis of several metabolic diseases, including gestational diabetes. This article provides a systematic review of the current state of research on biomarkers and gestational diabetes and discusses the clinical relevance of metabolomics in the prediction, diagnosis and management of gestational diabetes. Methods We conducted a systematic search of MEDLINE (PubMed) up to the end of February 2014 using the key term combinations of ‘metabolomics,’ ‘metabonomics,’ ‘nuclear magnetic spectroscopy,’ ‘mass spectrometry,’ ‘metabolic profiling’ and ‘amino acid profile’ combined (AND) with ‘gestational diabetes’. Additional articles were identified through searching the reference lists from included studies. Quality assessment of included articles was conducted through the use of QUADOMICS. Results This systematic review included 17 articles. The biomarkers most consistently associated with gestational diabetes were asymmetric dimethylarginine and NEFAs. After QUADOMICS analysis, 13 of the 17 included studies were classified as ‘high quality’. Conclusions/interpretation Existing metabolomic studies of gestational diabetes present inconsistent findings regarding metabolite profile characteristics. Further studies are needed in larger, more racially/ethnically diverse populations. PMID:25193282

  7. Metabolite Analysis and Histology on the Exact Same Tissue: Comprehensive Metabolomic Profiling and Metabolic Classification of Prostate Cancer

    NASA Astrophysics Data System (ADS)

    Huan, Tao; Troyer, Dean A.; Li, Liang

    2016-08-01

    We report a method of metabolomic profiling of intact tissue based on molecular preservation by extraction and fixation (mPREF) and high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS). mPREF extracts metabolites by aqueous methanol from tissue biopsies without altering tissue architecture and thus conventional histology can be performed on the same tissue. In a proof-of-principle study, we applied dansylation LC-MS to profile the amine/phenol submetabolome of prostate needle biopsies from 25 patient samples derived from 16 subjects. 2900 metabolites were consistently detected in more than 50% of the samples. This unprecedented coverage allowed us to identify significant metabolites for differentiating tumor and normal tissues. The panel of significant metabolites was refined using 36 additional samples from 18 subjects. Receiver Operating Characteristic (ROC) analysis showed area-under-the-curve (AUC) of 0.896 with sensitivity of 84.6% and specificity of 83.3% using 7 metabolites. A blind study of 24 additional validation samples gave a specificity of 90.9% at the same sensitivity of 84.6%. The mPREF extraction can be readily implemented into the existing clinical workflow. Our method of combining mPREF with CIL LC-MS offers a powerful and convenient means of performing histopathology and discovering or detecting metabolite biomarkers in the same tissue biopsy.

  8. Metabolite Analysis and Histology on the Exact Same Tissue: Comprehensive Metabolomic Profiling and Metabolic Classification of Prostate Cancer

    PubMed Central

    Huan, Tao; Troyer, Dean A.; Li, Liang

    2016-01-01

    We report a method of metabolomic profiling of intact tissue based on molecular preservation by extraction and fixation (mPREF) and high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS). mPREF extracts metabolites by aqueous methanol from tissue biopsies without altering tissue architecture and thus conventional histology can be performed on the same tissue. In a proof-of-principle study, we applied dansylation LC-MS to profile the amine/phenol submetabolome of prostate needle biopsies from 25 patient samples derived from 16 subjects. 2900 metabolites were consistently detected in more than 50% of the samples. This unprecedented coverage allowed us to identify significant metabolites for differentiating tumor and normal tissues. The panel of significant metabolites was refined using 36 additional samples from 18 subjects. Receiver Operating Characteristic (ROC) analysis showed area-under-the-curve (AUC) of 0.896 with sensitivity of 84.6% and specificity of 83.3% using 7 metabolites. A blind study of 24 additional validation samples gave a specificity of 90.9% at the same sensitivity of 84.6%. The mPREF extraction can be readily implemented into the existing clinical workflow. Our method of combining mPREF with CIL LC-MS offers a powerful and convenient means of performing histopathology and discovering or detecting metabolite biomarkers in the same tissue biopsy. PMID:27578275

  9. Meat, the metabolites: an integrated metabolite profiling and lipidomics approach for the detection of the adulteration of beef with pork

    PubMed Central

    Trivedi, Drupad K.; Hollywood, Katherine A.; Rattray, Nicholas J. W.; Ward, Holli; Trivedi, Dakshat K.; Greenwood, Joseph; Ellis, David I.

    2016-01-01

    Adulteration of high quality food products with sub-standard and cheaper grades is a world-wide problem taxing the global economy. Currently, many traditional tests suffer from poor specificity, highly complex outputs and a lack of high-throughput processing. Metabolomics has been successfully used as an accurate discriminatory technique in a number of applications including microbiology, cancer research and environmental studies and certain types of food fraud. In this study, we have developed metabolomics as a technique to assess the adulteration of meat as an improvement on current methods. Different grades of beef mince and pork mince, purchased from a national retail outlet were combined in a number of percentage ratios and analysed using GC-MS and UHPLC-MS. These techniques were chosen because GC-MS enables investigations of metabolites involved in primary metabolism whilst UHPLC-MS using reversed phase chromatography provides information on lipophilic species. With the application of chemometrics and statistical analyses, a panel of differential metabolites were found for identification of each of the two meat types. Additionally, correlation was observed between metabolite content and percentage of fat declared on meat products’ labelling. PMID:26911805

  10. Meat, the metabolites: an integrated metabolite profiling and lipidomics approach for the detection of the adulteration of beef with pork.

    PubMed

    Trivedi, Drupad K; Hollywood, Katherine A; Rattray, Nicholas J W; Ward, Holli; Trivedi, Dakshat K; Greenwood, Joseph; Ellis, David I; Goodacre, Royston

    2016-04-07

    Adulteration of high quality food products with sub-standard and cheaper grades is a world-wide problem taxing the global economy. Currently, many traditional tests suffer from poor specificity, highly complex outputs and a lack of high-throughput processing. Metabolomics has been successfully used as an accurate discriminatory technique in a number of applications including microbiology, cancer research and environmental studies and certain types of food fraud. In this study, we have developed metabolomics as a technique to assess the adulteration of meat as an improvement on current methods. Different grades of beef mince and pork mince, purchased from a national retail outlet were combined in a number of percentage ratios and analysed using GC-MS and UHPLC-MS. These techniques were chosen because GC-MS enables investigations of metabolites involved in primary metabolism whilst UHPLC-MS using reversed phase chromatography provides information on lipophilic species. With the application of chemometrics and statistical analyses, a panel of differential metabolites were found for identification of each of the two meat types. Additionally, correlation was observed between metabolite content and percentage of fat declared on meat products' labelling.

  11. Quantification of (1) H-MRS signals based on sparse metabolite profiles in the time-frequency domain.

    PubMed

    Parto Dezfouli, Mohammad Ali; Parto Dezfouli, Mohsen; Ahmadian, Alireza; Frangi, Alejandro F; Esmaeili Rad, Melika; Saligheh Rad, Hamidreza

    2017-02-01

    MRS is an analytical approach used for both quantitative and qualitative analysis of human body metabolites. The accurate and robust quantification capability of proton MRS ((1) H-MRS) enables the accurate estimation of living tissue metabolite concentrations. However, such methods can be efficiently employed for quantification of metabolite concentrations only if the overlapping nature of metabolites, existing static field inhomogeneity and low signal-to-noise ratio (SNR) are taken into consideration. Representation of (1) H-MRS signals in the time-frequency domain enables us to handle the baseline and noise better. This is possible because the MRS signal of each metabolite is sparsely represented, with only a few peaks, in the frequency domain, but still along with specific time-domain features such as distinct decay constant associated with T2 relaxation rate. The baseline, however, has a smooth behavior in the frequency domain. In this study, we proposed a quantification method using continuous wavelet transformation of (1) H-MRS signals in combination with sparse representation of features in the time-frequency domain. Estimation of the sparse representations of MR spectra is performed according to the dictionaries constructed from metabolite profiles. Results on simulated and phantom data show that the proposed method is able to quantify the concentration of metabolites in (1) H-MRS signals with high accuracy and robustness. This is achieved for both low SNR (5 dB) and low signal-to-baseline ratio (-5 dB) regimes.

  12. Identification of altered brain metabolites associated with TNAP activity in a mouse model of hypophosphatasia using untargeted NMR-based metabolomics analysis

    PubMed Central

    Cruz, Thomas; Gleizes, Marie; Balayssac, Stéphane; Mornet, Etienne; Marsal, Grégory; Millán, José Luis; Martino, Myriam Malet; Nowak, Lionel G; Gilard, Véronique; Fonta, Caroline

    2017-01-01

    Tissue Nonspecific Alkaline Phosphatase (TNAP) is a key player of bone mineralization and TNAP gene (ALPL) mutations in human are responsible for hypophosphatasia (HPP), a rare heritable disease affecting the mineralization of bones and teeth. Moreover, TNAP is also expressed by brain cells and the severe forms of HPP are associated with neurological disorders, including epilepsy and brain morphological anomalies. However TNAP’s role in the nervous system remains poorly understood. In order to investigate its neuronal functions, we aimed to identify without any a priori the metabolites regulated by TNAP in the nervous tissue. For this purpose we used 1H- and 31P NMR to analyze the brain metabolome of Alpl (Akp2) mice null for TNAP function, a well-described model of infantile HPP. Among 39 metabolites identified in brain extracts of one week-old animals, 8 displayed significantly different concentration in Akp2−/− compared to Akp2+/+ and Akp2+/− mice: cystathionine, adenosine, GABA, methionine, histidine, 3-methylhistidine, N-acetylaspartate (NAA) and N-acetyl-aspartyl-glutamate (NAAG), with cystathionine and adenosine levels displaying the strongest alteration. These metabolites identify several biochemical processes that directly or indirectly involve TNAP function, in particular through the regulation of ecto-nucleotide levels and of pyridoxal phosphate-dependent enzymes. Some of these metabolites are involved in neurotransmission (GABA, adenosine), in myelin synthesis (NAA, NAAG), and in the methionine cycle and transsulfuration pathway (cystathionine, methionine). Their disturbances may contribute to the neurodevelopmental and neurological phenotype of HPP. PMID:28072448

  13. Establishing the Secondary Metabolite Profile of the Marine Fungus: Tolypocladium geodes sp. MF458 and Subsequent Optimisation of Bioactive Secondary Metabolite Production.

    PubMed

    Kebede, Bethlehem; Wrigley, Stephen K; Prashar, Anjali; Rahlff, Janina; Wolf, Markus; Reinshagen, Jeanette; Gribbon, Philip; Imhoff, Johannes F; Silber, Johanna; Labes, Antje; Ellinger, Bernhard

    2017-03-23

    As part of an international research project, the marine fungal strain collection of the Helmholtz Centre for Ocean Research (GEOMAR) research centre was analysed for secondary metabolite profiles associated with anticancer activity. Strain MF458 was identified as Tolypocladium geodes, by internal transcribed spacer region (ITS) sequence similarity and its natural product production profile. By using five different media in two conditions and two time points, we were able to identify eight natural products produced by MF458. As well as cyclosporin A (1), efrapeptin D (2), pyridoxatin (3), terricolin A (4), malettinins B and E (5 and 6), and tolypocladenols A1/A2 (8), we identified a new secondary metabolite which we termed tolypocladenol C (7). All compounds were analysed for their anticancer potential using a selection of the NCI60 cancer cell line panel, with malettinins B and E (5 and 6) being the most promising candidates. In order to obtain sufficient quantities of these compounds to start preclinical development, their production was transferred from a static flask culture to a stirred tank reactor, and fermentation medium development resulted in a nearly eight-fold increase in compound production. The strain MF458 is therefore a producer of a number of interesting and new secondary metabolites and their production levels can be readily improved to achieve higher yields.

  14. Establishing the Secondary Metabolite Profile of the Marine Fungus: Tolypocladium geodes sp. MF458 and Subsequent Optimisation of Bioactive Secondary Metabolite Production

    PubMed Central

    Kebede, Bethlehem; Wrigley, Stephen K.; Prashar, Anjali; Rahlff, Janina; Wolf, Markus; Reinshagen, Jeanette; Gribbon, Philip; Imhoff, Johannes F.; Silber, Johanna; Labes, Antje; Ellinger, Bernhard

    2017-01-01

    As part of an international research project, the marine fungal strain collection of the Helmholtz Centre for Ocean Research (GEOMAR) research centre was analysed for secondary metabolite profiles associated with anticancer activity. Strain MF458 was identified as Tolypocladium geodes, by internal transcribed spacer region (ITS) sequence similarity and its natural product production profile. By using five different media in two conditions and two time points, we were able to identify eight natural products produced by MF458. As well as cyclosporin A (1), efrapeptin D (2), pyridoxatin (3), terricolin A (4), malettinins B and E (5 and 6), and tolypocladenols A1/A2 (8), we identified a new secondary metabolite which we termed tolypocladenol C (7). All compounds were analysed for their anticancer potential using a selection of the NCI60 cancer cell line panel, with malettinins B and E (5 and 6) being the most promising candidates. In order to obtain sufficient quantities of these compounds to start preclinical development, their production was transferred from a static flask culture to a stirred tank reactor, and fermentation medium development resulted in a nearly eight-fold increase in compound production. The strain MF458 is therefore a producer of a number of interesting and new secondary metabolites and their production levels can be readily improved to achieve higher yields. PMID:28333084

  15. A Systematic Approach to Time-series Metabolite Profiling and RNA-seq Analysis of Chinese Hamster Ovary Cell Culture.

    PubMed

    Hsu, Han-Hsiu; Araki, Michihiro; Mochizuki, Masao; Hori, Yoshimi; Murata, Masahiro; Kahar, Prihardi; Yoshida, Takanobu; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-03-02

    Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput "omics" methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture.

  16. A Systematic Approach to Time-series Metabolite Profiling and RNA-seq Analysis of Chinese Hamster Ovary Cell Culture

    PubMed Central

    Hsu, Han-Hsiu; Araki, Michihiro; Mochizuki, Masao; Hori, Yoshimi; Murata, Masahiro; Kahar, Prihardi; Yoshida, Takanobu; Hasunuma, Tomohisa; Kondo, Akihiko

    2017-01-01

    Chinese hamster ovary (CHO) cells are the primary host used for biopharmaceutical protein production. The engineering of CHO cells to produce higher amounts of biopharmaceuticals has been highly dependent on empirical approaches, but recent high-throughput “omics” methods are changing the situation in a rational manner. Omics data analyses using gene expression or metabolite profiling make it possible to identify key genes and metabolites in antibody production. Systematic omics approaches using different types of time-series data are expected to further enhance understanding of cellular behaviours and molecular networks for rational design of CHO cells. This study developed a systematic method for obtaining and analysing time-dependent intracellular and extracellular metabolite profiles, RNA-seq data (enzymatic mRNA levels) and cell counts from CHO cell cultures to capture an overall view of the CHO central metabolic pathway (CMP). We then calculated correlation coefficients among all the profiles and visualised the whole CMP by heatmap analysis and metabolic pathway mapping, to classify genes and metabolites together. This approach provides an efficient platform to identify key genes and metabolites in CHO cell culture. PMID:28252038

  17. Comparison of the circulating metabolite profile of PF-04991532, a hepatoselective glucokinase activator, across preclinical species and humans: potential implications in metabolites in safety testing assessment.

    PubMed

    Sharma, Raman; Litchfield, John; Bergman, Arthur; Atkinson, Karen; Kazierad, David; Gustavson, Stephanie M; Di, Li; Pfefferkorn, Jeffrey A; Kalgutkar, Amit S

    2015-02-01

    A previous report from our laboratory disclosed the identification of PF-04991532 [(S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid] as a hepatoselective glucokinase activator for the treatment of type 2 diabetes mellitus. Lack of in vitro metabolic turnover in microsomes and hepatocytes from preclinical species and humans suggested that metabolism would be inconsequential as a clearance mechanism of PF-04991532 in vivo. Qualitative examination of human circulating metabolites using plasma samples from a 14-day multiple ascending dose clinical study, however, revealed a glucuronide (M1) and monohydroxylation products (M2a and M2b/M2c) whose abundances (based on UV integration) were greater than 10% of the total drug-related material. Based on this preliminary observation, mass balance/excretion studies were triggered in animals, which revealed that the majority of circulating radioactivity following the oral administration of [¹⁴C]PF-04991532 was attributed to an unchanged parent (>70% in rats and dogs). In contrast with the human circulatory metabolite profile, the monohydroxylated metabolites were not detected in circulation in either rats or dogs. Available mass spectral evidence suggested that M2a and M2b/M2c were diastereomers derived from cyclopentyl ring oxidation in PF-04991532. Because cyclopentyl ring hydroxylation on the C-2 and C-3 positions can generate eight possible diastereomers, it was possible that additional diastereomers may have also formed and would need to be resolved from the M2a and M2b/M2c peaks observed in the current chromatography conditions. In conclusion, the human metabolite scouting study in tandem with the animal mass balance study allowed early identification of PF-04991532 oxidative metabolites, which were not predicted by in vitro methods and may require additional scrutiny in the development phase of PF-04991532.

  18. Dieldrin stimulates biliary excretion of 14C-benzo[a]pyrene polar metabolites but does not change the biliary metabolite profile in rainbow trout (Oncorhyncus mykiss).

    PubMed

    Barnhill, Melanie L; Rosemond, Marie V M; Curtis, Lawrence R

    2003-10-01

    Activities of hepatic microsomal and cytosolic epoxide hydrolases, accumulation of dieldrin in liver, and in vivo metabolism and disposition of the polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene (BP), were examined in rainbow trout pretreated with dieldrin, a chlorinated cyclodiene insecticide. Rainbow trout were fed 0.3 mg dieldrin/kg/day for 9 weeks and the same dose of dieldrin for 9 weeks, followed by 3 weeks on control diet (12 weeks). Fish then received an intraperitoneal (ip) challenge dose of 14C-BP (10 micromol/kg). Dieldrin pretreatment significantly elevated the concentration of 14C-BP in bile (142% and 200% at 9 and 12 weeks, respectively), but not liver or fat. Extraction of bile subsamples confirmed dieldrin pretreatment significantly stimulated total biliary excretion of 14C-BP polar metabolites (244% and 221% at week 9 and 12, respectively). The complex metabolism of BP characterized the in vivo state of the CYP system, UDP-glucuronyltransferases, and sulfotransferases. Bile was extracted and then hydrolyzed by beta-glucuronidase and arylsulfatase to regenerate BP metabolites conjugated by phase II enzymes. Evaluation of biliary polar metabolite profiles of 14C-BP revealed no significant differences between control and dieldrin-fed fish. There was no selective enhancement of any particular metabolite, or formation of a novel metabolite with dieldrin pretreatment. This research confirmed that enhanced biliary excretion, following chronic dieldrin exposure, was not explained by induction of xenobiotic metabolizing enzymes. The results are consistent with induction of hepatic intracellular trafficking proteins in dieldrin-fed fish.

  19. Metabolite Profiling of Low-P Tolerant and Low-P Sensitive Maize Genotypes under Phosphorus Starvation and Restoration Conditions.

    PubMed

    Ganie, Arshid Hussain; Ahmad, Altaf; Pandey, Renu; Aref, Ibrahim M; Yousuf, Peerzada Yasir; Ahmad, Sayeed; Iqbal, Muhammad

    2015-01-01

    Maize (Zea mays L.) is one of the most widely cultivated crop plants. Unavoidable economic and environmental problems associated with the excessive use of phosphatic fertilizers demands its better management. The solution lies in improving the phosphorus (P) use efficiency to sustain productivity even at low P levels. Untargeted metabolomic profiling of contrasting genotypes provides a snap shot of whole metabolome which differs under specific conditions. This information provides an understanding of the mechanisms underlying tolerance to P stress and the approach for increasing P-use-efficiency. A comparative metabolite-profiling approach based on gas chromatography-mass spectrometry (GC/MS) was applied to investigate the effect of P starvation and its restoration in low-P sensitive (HM-4) and low-P tolerant (PEHM-2) maize genotypes. A comparison of the metabolite profiles of contrasting genotypes in response to P-deficiency revealed distinct differences among low-P sensitive and tolerant genotypes. Another set of these genotypes were grown under P-restoration condition and sampled at different time intervals (3, 5 and 10 days) to investigate if the changes in metabolite profile under P-deficiency was restored. Significant variations in the metabolite pools of these genotypes were observed under P-deficiency which were genotype specific. Out of 180 distinct analytes, 91 were identified. Phosphorus-starvation resulted in accumulation of di- and trisaccharides and metabolites of ammonium metabolism, specifically in leaves, but decreased the levels of phosphate-containing metabolites and organic acids. A sharp increase in the concentrations of glutamine, asparagine, serine and glycine was observed in both shoots and roots under low-P condition. The new insights generated on the maize metabolome in response to P-starvation and restoration would be useful towards improvement of the P-use efficiency in maize.

  20. Metabolite Profiling of Low-P Tolerant and Low-P Sensitive Maize Genotypes under Phosphorus Starvation and Restoration Conditions

    PubMed Central

    Ganie, Arshid Hussain; Ahmad, Altaf; Pandey, Renu; Aref, Ibrahim M.; Yousuf, Peerzada Yasir; Ahmad, Sayeed; Iqbal, Muhammad

    2015-01-01

    Background Maize (Zea mays L.) is one of the most widely cultivated crop plants. Unavoidable economic and environmental problems associated with the excessive use of phosphatic fertilizers demands its better management. The solution lies in improving the phosphorus (P) use efficiency to sustain productivity even at low P levels. Untargeted metabolomic profiling of contrasting genotypes provides a snap shot of whole metabolome which differs under specific conditions. This information provides an understanding of the mechanisms underlying tolerance to P stress and the approach for increasing P-use-efficiency. Methodology/Principal Findings A comparative metabolite-profiling approach based on gas chromatography-mass spectrometry (GC/MS) was applied to investigate the effect of P starvation and its restoration in low-P sensitive (HM-4) and low-P tolerant (PEHM-2) maize genotypes. A comparison of the metabolite profiles of contrasting genotypes in response to P-deficiency revealed distinct differences among low-P sensitive and tolerant genotypes. Another set of these genotypes were grown under P-restoration condition and sampled at different time intervals (3, 5 and 10 days) to investigate if the changes in metabolite profile under P-deficiency was restored. Significant variations in the metabolite pools of these genotypes were observed under P-deficiency which were genotype specific. Out of 180 distinct analytes, 91 were identified. Phosphorus-starvation resulted in accumulation of di- and trisaccharides and metabolites of ammonium metabolism, specifically in leaves, but decreased the levels of phosphate-containing metabolites and organic acids. A sharp increase in the concentrations of glutamine, asparagine, serine and glycine was observed in both shoots and roots under low-P condition. Conclusion The new insights generated on the maize metabolome in resposne to P-starvation and restoration would be useful towards improvement of the P-use efficiency in maize. PMID

  1. Arsenate Impact on the Metabolite Profile, Production, and Arsenic Loading of Xylem Sap in Cucumbers (Cucumis sativus L.).

    PubMed

    Uroic, M Kalle; Salaün, Pascal; Raab, Andrea; Feldmann, Jörg

    2012-01-01

    Arsenic uptake and translocation studies on xylem sap focus generally on the concentration and speciation of arsenic in the xylem. Arsenic impact on the xylem sap metabolite profile and its production during short term exposure has not been reported in detail. To investigate this, cucumbers were grown hydroponically and arsenate (As(V)) and DMA were used for plant treatment for 24 h. Total arsenic and arsenic speciation in xylem sap was analyzed including a metabolite profiling under As(V) stress. Produced xylem sap was quantified and absolute arsenic transported was determined. As(V) exposure had a significant impact on the metabolite profile of xylem sap. Four m/z values corresponding to four compounds were up-regulated, one compound down-regulated by As(V) exposure. The compound down-regulated was identified to be isoleucine. Furthermore, As(V) exposure had a significant influence on sap production, leading to a reduction of up to 96% sap production when plants were exposed to 1000 μg kg(-1) As(V). No difference to control plants was observed when plants were exposed to 1000 μg kg(-1) DMA. Absolute arsenic amount in xylem sap was the lowest at high As(V) exposure. These results show that As(V) has a significant impact on the production and metabolite profile of xylem sap. The physiological importance of isoleucine needs further attention.

  2. Establishing high temperature gas chromatographic profiles of non-polar metabolites for quality assessment of African traditional herbal medicinal products.

    PubMed

    Bony, Nicaise F; Libong, Danielle; Solgadi, Audrey; Bleton, Jean; Champy, Pierre; Malan, Anglade K; Chaminade, Pierre

    2014-01-01

    The quality assessment of African traditional herbal medicinal products is a difficult challenge since they are complex mixtures of several herbal drug or herbal drug preparations. The plant source is also often unknown and/or highly variable. Plant metabolites chromatographic profiling is therefore an important tool for quality control of such herbal products. The objective of this work is to propose a protocol for sample preparation and gas chromatographic profiling of non-polar metabolites for quality control of African traditional herbal medicinal products. The methodology is based on the chemometric assessment of chromatographic profiles of non-polar metabolites issued from several batches of leaves of Combretum micranthum and Mitracarpus scaber by high temperature gas chromatography coupled to mass spectrometry, performed on extracts obtained in refluxed dichloromethane, after removal of chlorophyll pigments. The method using high temperature gas chromatography after dichloromethane extraction allows detection of most non-polar bioactive and non-bioactive metabolites already identified in leaves of both species. Chemometric data analysis using Principal Component Analysis and Partial Least Squares after Orthogonal Signal Correction applied to chromatographic profiles of leaves of Combretum micranthum and Mitracarpus scaber showed slight batch to batch differences, and allowed clear differentiation of the two herbal extracts.

  3. Arsenate Impact on the Metabolite Profile, Production, and Arsenic Loading of Xylem Sap in Cucumbers (Cucumis sativus L.)

    PubMed Central

    Uroic, M. Kalle; Salaün, Pascal; Raab, Andrea; Feldmann, Jörg

    2012-01-01

    Arsenic uptake and translocation studies on xylem sap focus generally on the concentration and speciation of arsenic in the xylem. Arsenic impact on the xylem sap metabolite profile and its production during short term exposure has not been reported in detail. To investigate this, cucumbers were grown hydroponically and arsenate (AsV) and DMA were used for plant treatment for 24 h. Total arsenic and arsenic speciation in xylem sap was analyzed including a metabolite profiling under AsV stress. Produced xylem sap was quantified and absolute arsenic transported was determined. AsV exposure had a significant impact on the metabolite profile of xylem sap. Four m/z values corresponding to four compounds were up-regulated, one compound down-regulated by AsV exposure. The compound down-regulated was identified to be isoleucine. Furthermore, AsV exposure had a significant influence on sap production, leading to a reduction of up to 96% sap production when plants were exposed to 1000 μg kg−1 AsV. No difference to control plants was observed when plants were exposed to 1000 μg kg−1 DMA. Absolute arsenic amount in xylem sap was the lowest at high AsV exposure. These results show that AsV has a significant impact on the production and metabolite profile of xylem sap. The physiological importance of isoleucine needs further attention. PMID:22536187

  4. Identification of bioactive metabolites dihydrocanadensolide, Kojic acid, and vanillic acid in soy sauce using GC-MS, NMR spectroscopy, and single-crystal X-ray diffraction.

    PubMed

    Li, Ying; Teng, Zi; Parkin, Kirk L; Wang, Qin; Zhang, Qingli; Luo, Wei; Ma, Deyun; Zhao, Mouming

    2014-08-20

    Microbial transformations of intrinsic substrates offer immense potential for generating new bioactive compounds in fermented food products. The aim of this work was to characterize the secondary metabolites in soy sauce, one of the oldest fermented condiments. Ethyl acetate extract (EAE) of soy sauce was separated using flash column chromatography, crystallized, and analyzed by nuclear magnetic resonance (NMR), single-crystal X-ray diffraction (SC-XRD), and mass spectroscopy. Dihydrocanadensolide (DHC), an antiulcer agent, was identified in a food for the first time. The natural stereostructure of DHC, which remained controversial for several decades, was determined as (3S,3aS,6R,6aR)-6-butyl-3-methyltetrahydrofuro[3,4-b]furan-2,4-dione using SC-XRD analysis. Kojic acid (KA) and vanillic acid (VA) were also identified from EAE as bioactive metabolic products of fungi and yeasts. Moreover, a new polymorphic form of KA was determined by SC-XRD.

  5. Nonanthocyanin