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Metabolite Analysis (metabolite + analysis)
Selected AbstractsGas chromatographic,mass spectrometric urinary metabolome analysis to study mutations of inborn errors of metabolismMASS SPECTROMETRY REVIEWS, Issue 6 2005Tomiko Kuhara Abstract Urine contains numerous metabolites, and can provide evidence for the screening or molecular diagnosis of many inborn errors of metabolism (IEMs). The metabolomic analysis of urine by the combined use of urease pretreatment, stable-isotope dilution, and capillary gas chromatography/mass spectrometry offers reliable and quantitative data for the simultaneous screening or molecular diagnosis of more than 130 IEMs. Those IEMs include hyperammonemias and lactic acidemias, and the IEMs of amino acids, pyrimidines, purines, carbohydrates, and others including primary hyperoxalurias, hereditary fructose intolerance, propionic acidemia, and methylmalonic acidemia. Metabolite analysis is comprehensive for mutant genotypes. Enzyme dysfunction,either by the abnormal structure of an enzyme/apoenzyme, the reduced quantity of a normal enzyme/apoenzyme, or the lack of a coenzyme,is involved. Enzyme dysfunction,either by an abnormal regulatory gene, abnormal sub-cellular localization, or by abnormal post-transcriptional or post-translational modification,is included. Mutations,either known or unknown, common or uncommon,are involved. If the urine metabolome approach can accurately observe quantitative abnormality for hundreds of metabolites, reflecting 100 different disease-causing reactions in a body, then it is possible to simultaneously detect different mutant genotypes of far more than tens of thousands. © 2004 Wiley Periodicals, Inc., Mass Spec Rev 24:814,827, 2005 [source] General Method for the 11C-Labeling of 2-Arylpropionic Acids and Their Esters: Construction of a PET Tracer Library for a Study of Biological Events Involved in COXs ExpressionCHEMISTRY - A EUROPEAN JOURNAL, Issue 14 2010Misato Takashima-Hirano Abstract Cyclooxygenase (COX) is a critical enzyme in prostaglandin biosynthesis that modulates a wide range of biological functions, such as pain, fever, and so on. To perform in vivo COX imaging by positron emission tomography (PET), we developed a method to incorporate 11C radionuclide into various 2-arylpropionic acids that have a common methylated structure, particularly among nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, we developed a novel 11C-radiolabeling methodology based on rapid C -[11C]methylation by the reaction of [11C]CH3I with enolate intermediates generated from the corresponding esters under basic conditions. One-pot hydrolysis of the above [11C]methylation products also allows the synthesis of desired 11C-incorporated acids. We demonstrated the utility of this method in the syntheses of six PET tracers, [11C]Ibuprofen, [11C]Naproxen, [11C]Flurbiprofen, [11C]Fenoprofen, [11C]Ketoprofen, and [11C]Loxoprofen. Notably, we found that their methyl esters were particularly useful as proradiotracers for a study of neuroinflammation. The microPET studies of rats with lipopolysaccharide (LPS)-induced brain inflammation clearly showed that the radioactivity of PET tracers accumulated in the inflamed region. Among these PET tracers, the specificity of [11C]Ketoprofen methyl ester was demonstrated by a blocking study. Metabolite analysis in the rat brain revealed that the methyl esters were initially taken up in the brain and then underwent hydrolysis to form pharmacologically active forms of the corresponding acids. Thus, we succeeded in general 11C-labeling of 2-arylpropionic acids and their methyl esters as PET tracers of NSAIDs to construct a potentially useful PET tracer library for in vivo imaging of inflammation involved in COXs expression. [source] Nitric oxide synthase inhibition reduces O2 cost of force development and spares high-energy phosphates following contractions in pump-perfused rat hindlimb musclesEXPERIMENTAL PHYSIOLOGY, Issue 3 2006David J. Baker The purpose of the present experiments was to test the hypotheses that: (i) nitric oxide synthase (NOS) inhibition reduces the O2 cost of force development across a range of contractile demands; and (ii) this reduced O2 cost of force development would be reflected in a sparing of intramuscular higher energy phosphates. Rat distal hindlimb muscles were pump perfused in situ and electrically stimulated (200 ms trains with pulses at 100 Hz, each pulse 0.05 ms duration) for 1 min each at 15, 30 and 60 tetani min,1 and for 2 min at 90 tetani min,1 in three groups: 0.01 mm adenosine; 1 mm d -NAME and 0.01 mm adenosine (d -NAME); and 1 mm l -NAME and 0.01 mm adenosine (l -NAME). The gastrocnemius,plantaris,soleus muscle group was freeze clamped post-contractions for metabolite analyses. Force was 19% higher and oxygen uptake was 20% lower with l -NAME versus adenosine, and there was a 35% reduction in /time-integrated tension versus adenosine and 24% versusd -NAME that was independent of contraction frequency. l -NAME treatment produced a 33% sparing of muscle phosphocreatine (PCr), and intramuscular lactate was no different between groups. In contrast, d -NAME reduced force by 30%, by 29% and the O2 cost of force development by 15% compared with adenosine, but had no effect on the degree of intramuscular ATP and PCr depletion. These results show that NOS inhibition improved the metabolic efficiency of force development, either by improving the ATP yield for a given O2 consumption or by reducing the ATP cost of force development. In addition, these effects were independent of contraction frequency. [source] Severe Epilepsy in X-Linked Creatine Transporter Defect (CRTR-D)EPILEPSIA, Issue 6 2007Maria Margherita Mancardi Disorders of creatine synthesis or its transporter resulting in neurological impairment with mental retardation and epilepsy have only been recognized in recent years. To date, the epileptic disorder observed in creatine transporter deficiency (CRTR-D) has been described as a mild phenotype with infrequent seizures and favorable response to common antiepileptic drugs. We report on a 5 year-old boy with known speech delay who presented with severe and refractory epilepsy. After extensive investigations, metabolite analysis and brain 1H-MRS suggested CRTR-D, which was confirmed by the detection of a known pathogenic mutation in the SLC6A8 gene (c.1631C>T; p.Pro544Leu). [source] Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 13 2009Damien L. Callahan Abstract 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. [source] Quantification of metabolites in breast cancer patients with different clinical prognosis using HR MAS MR spectroscopyNMR IN BIOMEDICINE, Issue 4 2010Beathe Sitter Abstract Absolute quantitative measures of breast cancer tissue metabolites can increase our understanding of biological processes. Electronic REference To access In vivo Concentrations (ERETIC) was applied to high resolution magic angle spinning MR spectroscopy (HR MAS MRS) to quantify metabolites in intact breast cancer samples. The ERETIC signal was calibrated using solutions of creatine and TSP. The largest relative errors of the ERETIC method were 8.4%, compared to 4.4% for the HR MAS MRS method using TSP as a standard. The same MR experimental procedure was applied to intact tissue samples from breast cancer patients with clinically defined good (n,=,13) and poor (n,=,16) prognosis. All samples were examined by histopathology for relative content of different tissue types and proliferation index (MIB-1) after MR analysis. The resulting spectra were analyzed by quantification of tissue metabolites (,-glucose, lactate, glycine, myo-inositol, taurine, glycerophosphocholine, phosphocholine, choline and creatine), by peak area ratios and by principal component analysis. We found a trend toward lower concentrations of glycine in patients with good prognosis (1.1,µmol/g) compared to patients with poor prognosis (1.9,µmol/g, p,=,0.067). Tissue metabolite concentrations (except for ,-glucose) were also found to correlate to the fraction of tumor, connective, fat or glandular tissue by Pearson correlation analysis. Tissue concentrations of ,-glucose correlated to proliferation index (MIB-1) with a negative correlation factor (,0.45, p,=,0.015), consistent with increased energy demand in proliferating tumor cells. By analyzing several metabolites simultaneously, either in ratios or by metabolic profiles analyzed by PCA, we found that tissue metabolites correlate to patients' prognoses and health status five years after surgery. This study shows that the diagnostic and prognostic potential in MR metabolite analysis of breast cancer tissue is greater when combining multiple metabolites (MR Metabolomics). Copyright © 2010 John Wiley & Sons, Ltd. [source] Arabidopsis transcript and metabolite profiles: ecotype-specific responses to open-air elevated [CO2]PLANT CELL & ENVIRONMENT, Issue 11 2008PINGHUA LI ABSTRACT A Free-Air CO2 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 (,0.375 mg g,1) and elevated (,0.550 mg g,1) CO2 ([CO2]). Photoassimilate pool sizes were enhanced in high [CO2] in an ecotype-specific manner. Short-term growth at elevated [CO2] 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 [CO2]. 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 [CO2] 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 [CO2] as a metabolic perturbation that necessitated increased functions consuming or storing photoassimilate, with Cvi-0 emerging as more capable of acclimating. Elevated [CO2] in Arabidopsis favoured adjustments in reactive oxygen species (ROS) homeostasis and signalling that defined genotypic markers. [source] Proteolysis during long-term glucose starvation in Staphylococcus aureus COLPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2009Stephan Michalik Abstract A combination of pulse-chase experiments and 2-D PAGE revealed that protein degradation appears to play a crucial role for the cell physiology of Staphylococcus aureus COL during extended periods of glucose starvation. The synthesis rate of virtually all cytosolic and radioactively labeled proteins from growing cells seemed dramatically reduced in the first 3.5,h of glucose starvation. The stability of proteins synthesized in growing cells was monitored by a pulse-chase approach on a proteome wide scale. Especially, enzymes involved in nucleic acid and amino acid biosyntheses, energy metabolism and biosynthesis of cofactors were found rather rapidly degraded within the onset of the stationary phase, whereas the majority of glycolytic and tricarboxylic acid cycle enzymes remained more stable. Furthermore, single enzymes of biosynthetic pathways were differentially degraded. A metabolite analysis revealed that glucose completely depleted from the medium in the transient phase, and amino acids such as alanine and glycine were taken up by the cells in the stationary phase. We suggest that vegetative proteins no longer required in non-growing cells and thus no longer protected by integration into functional complexes were degraded. Proteolysis of putative non-substrate-bound or "unemployed" proteins appears to be a characteristic feature of S. aureus in order to access nutrients as an important survival strategy under starvation conditions. [source] Proteomic and selected metabolite analysis of grape berry tissues under well-watered and water-deficit stress conditionsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2009Jérôme Grimplet Abstract In order to investigate the unique contribution of individual wine grape (Vitis vinifera) berry tissues and water-deficit to wine quality traits, a survey of tissue-specific differences in protein and selected metabolites was conducted using pericarp (skin and pulp) and seeds of berries from vines grown under well-watered and water-deficit stress conditions. Of 1047 proteins surveyed from pericarp by 2-D PAGE, 90 identified proteins showed differential expression between the skin and pulp. Of 695 proteins surveyed from seed tissue, 163 were identified and revealed that the seed and pericarp proteomes were nearly completely distinct from one another. Water-deficit stress altered the abundance of approximately 7% of pericarp proteins, but had little effect on seed protein expression. Comparison of protein and available mRNA expression patterns showed that 32% pericarp and 69% seed proteins exhibited similar quantitative expression patterns indicating that protein accumulation patterns are strongly influenced by post-transcriptional processes. About half of the 32 metabolites surveyed showed tissue-specific differences in abundance with water-deficit stress affecting the accumulation of seven of these compounds. These results provide novel insights into the likely tissue-specific origins and the influence of water-deficit stress on the accumulation of key flavor and aroma compounds in wine. [source] Translational and transcriptional analysis of Sulfolobus solfataricus P2 to provide insights into alcohol and ketone utilisationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2007Poh Kuan Chong Abstract The potential of Sulfolobus solfataricus P2 for alcohol or ketone bioconversion was explored in this study. S. solfataricus was grown in different concentrations (0.1,0.8% w/v) of alcohols or ketones (ethanol, iso-propanol, n -propanol, acetone, phenol and hexanol) in the presence of 0.4% w/v glucose. Consequently, the addition of these alcohols or ketones into the growth media had an inhibitory effect on biomass production, whereby lag times increased and specific growth rates decreased when compared to a glucose control. Complete glucose utilisation was observed in all cultures, although slower rates of glucose consumption were observed in experimental cultures (average of 14.9,mg/L/h compared to 18.9,mg/L/h in the control). On the other hand, incomplete solvent utilisation was observed, with the highest solvent consumption being approximately 51% of the initial concentration in acetone cultures. Translational responses of S. solfataricus towards these alcohols or ketones were then investigated using the isobaric tags for relative and absolute quantitation (iTRAQ) technique. The majority (>80%) of proteins identified and quantified showed no discernable changes in regulation compared to the control. These results, along with those obtained from transcriptional analysis of key genes involved within this catabolic process using quantitative RT-PCR and metabolite analysis, demonstrate successful alcohol or ketone conversion in S. solfataricus. [source] The use of turbulent flow chromatography and the isocratic focusing effect to achieve on-line cleanup and concentration of neat biological samples for low-level metabolite analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2005J. L. Herman The use of turbulent flow chromatography in conjunction with column switching isocratic focusing was used to perform on-line sample cleanup and concentration of neat rat plasma for the identification of low-level metabolites. The concentration was achieved by focusing multiple injections, which were cleaned by a turbulent flow column, onto an analytical column prior to elution into the mass spectrometer. In addition, the first application of turbulent flow chromatography for on-line sample cleanup of neat bile samples is reported. The on-line cleanup and concentration method extracts and concentrates a sample 20-fold in 1,h, and is completely automated. Copyright © 2005 John Wiley & Sons, Ltd. [source] Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesisTHE PLANT JOURNAL, Issue 2 2006Sara Andersson-Gunnerås Summary Stems and branches of angiosperm trees form tension wood (TW) when exposed to a gravitational stimulus. One of the main characteristics of TW, which distinguishes it from normal wood, is the formation of fibers with a thick inner gelatinous cell wall layer mainly composed of crystalline cellulose. Hence TW is enriched in cellulose, and deficient in lignin and hemicelluloses. An expressed sequence tag library made from TW-forming tissues in Populus tremula (L.) × tremuloides (Michx.) and data from transcript profiling using microarray and metabolite analysis were obtained during TW formation in Populus tremula (L.) in two growing seasons. The data were examined with the aim of identifying the genes responsible for the change in carbon (C) flow into various cell wall components, and the mechanisms important for the formation of the gelatinous cell wall layer (G-layer). A specific effort was made to identify carbohydrate-active enzymes with a putative function in cell wall biosynthesis. An increased C flux to cellulose was suggested by a higher abundance of sucrose synthase transcripts. However, genes related to the cellulose biosynthetic machinery were not generally affected, although the expression of secondary wall-specific CesA genes was modified in both directions. Other pathways for which the data suggested increased activity included lipid and glucosamine biosynthesis and the pectin degradation machinery. In addition, transcripts encoding fasciclin-like arabinogalactan proteins were particularly increased and found to lack true Arabidopsis orthologs. Major pathways for which the transcriptome and metabolome analysis suggested decreased activity were the pathway for C flux through guanosine 5,-diphosphate (GDP) sugars to mannans, the pentose phosphate pathway, lignin biosynthesis, and biosynthesis of cell wall matrix carbohydrates. Several differentially expressed auxin- and ethylene-related genes and transcription factors were also identified. [source] Folinic acid,responsive seizures are identical to pyridoxine-dependent epilepsy,ANNALS OF NEUROLOGY, Issue 5 2009Renata C. Gallagher MD Objective Folinic acid,responsive seizures and pyridoxine-dependent epilepsy are two treatable causes of neonatal epileptic encephalopathy. The former is diagnosed by characteristic peaks on cerebrospinal fluid (CSF) monoamine metabolite analysis; its genetic basis has remained elusive. The latter is due to ,-aminoadipic semialdehyde (,-AASA) dehydrogenase deficiency, associated with pathogenic mutations in the ALDH7A1 (antiquitin) gene. We report two patients whose CSF showed the marker of folinic acid,responsive seizures, but who responded clinically to pyridoxine. We performed genetic and biochemical testing of samples from these patients, and seven others, to determine the relation between these two disorders. Methods CSF samples were analyzed for the presence of ,-AASA and pipecolic acid. DNA sequencing of the ALDH7A1 gene was performed. Results Both patients reported here had increased CSF ,-AASA, CSF pipecolic acid, and known or likely pathogenic mutations in the ALDH7A1 gene, consistent with ,-AASA dehydrogenase deficiency. Analysis of CSF samples from seven other anonymous individuals diagnosed with folinic acid,responsive seizures showed similar results. Interpretation These results demonstrate that folinic acid,responsive seizures are due to ,-AASA dehydrogenase deficiency and mutations in the ALDH7A1 gene. Thus, folinic acid,responsive seizures are identical to the major form of pyridoxine-dependent epilepsy. We recommend consideration of treatment with both pyridoxine and folinic acid for patients with ,-AASA dehydrogenase deficiency, and consideration of a lysine restricted diet. The evaluation of patients with neonatal epileptic encephalopathy, as well as those with later-onset seizures, should include a measurement of ,-AASA in urine to identify this likely underdiagnosed and treatable disorder. Ann Neurol 2008 [source] Integrated Sampling Procedure for Metabolome AnalysisBIOTECHNOLOGY PROGRESS, Issue 5 2006Jochen Schaub Metabolome analysis, the analysis of large sets of intracellular metabolites, has become an important systems analysis method in biotechnological and pharmaceutical research. In metabolic engineering, the integration of metabolome data with fluxome and proteome data into large-scale mathematical models promises to foster rational strategies for strain and cell line improvement. However, the development of reproducible sampling procedures for quantitative analysis of intracellular metabolite concentrations represents a major challenge, accomplishing (i) fast transfer of sample, (ii) efficient quenching of metabolism, (iii) quantitative metabolite extraction, and (iv) optimum sample conditioning for subsequent quantitative analysis. In addressing these requirements, we propose an integrated sampling procedure. Simultaneous quenching and quantitative extraction of intracellular metabolites were realized by short-time exposure of cells to temperatures ,95 °C, where intracellular metabolites are released quantitatively. Based on these findings, we combined principles of heat transfer with knowledge on physiology, for example, turnover rates of energy metabolites, to develop an optimized sampling procedure based on a coiled single tube heat exchanger. As a result, this sampling procedure enables reliable and reproducible measurements through (i) the integration of three unit operations into a one unit operation, (ii) the avoidance of any alteration of the sample due to chemical reagents in quenching and extraction, and (iii) automation. A sampling frequency of 5 s,1 and an overall individual sample processing time faster than 30 s allow observing responses of intracellular metabolite concentrations to extracellular stimuli on a subsecond time scale. Recovery and reliability of the unit operations were analyzed. Impact of sample conditioning on subsequent IC-MS analysis of metabolites was examined as well. The integrated sampling procedure was validated through consistent results from steady-state metabolite analysis of Escherichia coli cultivated in a chemostat at D = 0.1 h,1. [source] Urinary F2 -isoprostane metabolite analysis: a step closer to obtaining a reliable measure of oxidative stress?CLINICAL & EXPERIMENTAL ALLERGY, Issue 3 2001Frank J. Kelly [source] | ||||||||||||||||