PubMed

The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells. Cytotechnology. 2015 Sep 25; Authors: Wenner MI, Maker GL, Dawson LF, Drummond PD, Mullaney I Abstract Several studies of neuropathic pain have linked abnormal adrenergic signalling to the development and maintenance of pain, although the mechanisms underlying this are not yet fully understood. Metabolomic analysis is a technique that can be used to give a snapshot of biochemical status, and can aid in the identification of the mechanisms behind pathological changes identified in cells, tissues and biological fluids. This study aimed to use gas chromatography-mass spectrometry-based metabolomic profiling in combination with reverse transcriptase-polymerase chain reaction and immunocytochemistry to identify functional α1-adrenergic receptors on cultured N1E-115 mouse neuroblastoma cells. The study was able to confirm the presence of mRNA for the α1D subtype, as well as protein expression of the α1-adrenergic receptor. Furthermore, metabolomic data revealed changes to the metabolite profile of cells when exposed to adrenergic pharmacological intervention. Agonist treatment with phenylephrine hydrochloride (10 µM) resulted in altered levels of several metabolites including myo-inositol, glucose, fructose, alanine, leucine, phenylalanine, valine, and n-acetylglutamic acid. Many of the changes observed in N1E-115 cells by agonist treatment were modulated by additional antagonist treatment (prazosin hydrochloride, 100 µM). A number of these changes reflected what is known about the biochemistry of α1-adrenergic receptor activation. This preliminary study therefore demonstrates the potential of metabolomic profiling to confirm the presence of functional receptors on cultured cells. PMID: 26408527 [PubMed - as supplied by publisher]

Detection of Ophthalmic Acid in Serum from Acetaminophen-Induced Acute Liver Failure Patients Is More Frequent in Non-Survivors. PLoS One. 2015;10(9):e0139299 Authors: Kaur G, Leslie EM, Tillman H, Lee WM, Swanlund DP, Karvellas CJ, US Acute Liver Failure Study Group Abstract BACKGROUND/AIM: Acetaminophen (APAP) hepatotoxicity is related to the formation of N-acetyl-p-benzoquinone imine (NAPQI), which is detoxified through conjugation with reduced glutathione (GSH). Ophthalmic acid (OA) is an analogue of GSH in which cysteine is replaced with 2-aminobutyrate. Metabolomics studies of mice with APAP-induced acute liver failure (APAP-ALF) identified OA as a marker of oxidative stress and hepatic GSH consumption. The aim of the current study was to determine whether OA is detectable in APAP-ALF human patients either early (day 2) or late (day 4) and whether OA levels were associated with in-hospital survival in the absence of liver transplant. METHODS: Serum samples from 130 APAP-ALF patients (82 survivors, 48 non-survivors) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and correlated with clinical data from the United States Acute Liver Failure Study Group (US ALFSG) Registry (2004-2011). RESULTS: Survivors had significantly lower admission bilirubin (4.2 vs. 5.7 mg/dl) and lactate levels (3.3 vs. 6.5 μmol/l, p<0.05 for all). During the first 7 days of the study, survivors were less likely to require mechanical ventilation (55% vs. 88%), vasopressor support (9.8% vs. 67%) or renal replacement therapy (26% vs. 63%, p< 0.001 for all). Non-survivors were more likely to have detectable OA levels early (31% vs. 15%, p = 0.034) and late (27% vs. 11%, p = 0.02). However there were no significant differences in mean OA levels between non-survivors and survivors (early 0.48 vs. 0.36, late 0.43 vs. 0.37, P > 0.5 for all). CONCLUSION: OA was detectable more frequently in APAP-ALF non-survivors but mean OA levels were not associated with survival. The routine clinical administration of N-acetyl cysteine could replenish GSH levels and prevent OA production. PMID: 26407170 [PubMed - as supplied by publisher]

Biodiversity, Anti-Trypanosomal Activity Screening, and Metabolomic Profiling of Actinomycetes Isolated from Mediterranean Sponges. PLoS One. 2015;10(9):e0138528 Authors: Cheng C, MacIntyre L, Abdelmohsen UR, Horn H, Polymenakou PN, Edrada-Ebel R, Hentschel U Abstract Marine sponge-associated actinomycetes are considered as promising sources for the discovery of novel biologically active compounds. In the present study, a total of 64 actinomycetes were isolated from 12 different marine sponge species that had been collected offshore the islands of Milos and Crete, Greece, eastern Mediterranean. The isolates were affiliated to 23 genera representing 8 different suborders based on nearly full length 16S rRNA gene sequencing. Four putatively novel species belonging to genera Geodermatophilus, Microlunatus, Rhodococcus and Actinomycetospora were identified based on a 16S rRNA gene sequence similarity of < 98.5% to currently described strains. Eight actinomycete isolates showed bioactivities against Trypanosma brucei brucei TC221 with half maximal inhibitory concentration (IC50) values <20 μg/mL. Thirty four isolates from the Milos collection and 12 isolates from the Crete collection were subjected to metabolomic analysis using high resolution LC-MS and NMR for dereplication purposes. Two isolates belonging to the genera Streptomyces (SBT348) and Micromonospora (SBT687) were prioritized based on their distinct chemistry profiles as well as their anti-trypanosomal activities. These findings demonstrated the feasibility and efficacy of utilizing metabolomics tools to prioritize chemically unique strains from microorganism collections and further highlight sponges as rich source for novel and bioactive actinomycetes. PMID: 26407167 [PubMed - as supplied by publisher]

Putative identification of new p-coumaroyl glycoside flavonoids in grape by ultra-high performance liquid chromatography/high-resolution mass spectrometry. Rapid Commun Mass Spectrom. 2015 Feb 28;29(4):357-366 Authors: Panighel A, De Rosso M, Dalla Vedova A, Flamini R Abstract RATIONALE: Grape polyphenols are antioxidant compounds, markers in vine chemotaxonomy, and involved in color stabilization of red wines. Sugar acylation usually confers higher stability on glycoside derivatives and this effect is enhanced by an aromatic substituent such as p-coumaric acid. Until now, only p-coumaroyl anthocyanins have been found in grape. METHODS: A method of 'suspect screening analysis' by ultra-high-performance liquid chromatography/high-resolution mass spectrometry (UHPLC/QTOFMS) has recently been developed to study grape metabolomics. In the present study, this approach was used to identify new polyphenols in grape by accurate mass measurement, MS/MS fragmentation, and study of correlations between fragments observed and putative structures. RESULTS: Three putative p-coumaroyl flavonoids were identified in Raboso Piave grape extract: a dihydrokaempferide-3-O-p-coumaroylhexoside-like flavanone, isorhamnetin-3-O-p-coumaroylglucoside, and a chrysoeriol-p-coumaroylhexoside-like flavone. Accurate MS provided structural characterization of functional groups, and literature data indicates their probable position in the molecule. A fragmentation scheme is proposed for each compound. CONCLUSIONS: Compounds were identified by overlapping various analytical methods according to recommendations in the MS-based metabolomics literature. Stereochemistry and the definitive position of substituents in the molecule can only be confirmed by isolation and characterization or synthesis of each compound. These findings suggest addressing research of acylated polyphenol glycosides to other grape varieties. Copyright © 2015 John Wiley & Sons, Ltd. PMID: 26406348 [PubMed - as supplied by publisher]

Profiling of volatile compounds in APC(Min/+) mice blood by dynamic headspace extraction and gas chromatography/mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Sep 11;1003:35-40 Authors: Kakuta S, Nishiumi S, Yoshida M, Fukusaki E, Bamba T Abstract Various volatile compounds as well as hydrophilic compounds exist in the blood. For example, 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes have been reported as oxidized lipid-derived volatiles in blood. These specific volatiles have been associated with diseases; however, multi-volatile analyses have not been performed. In this study, volatile profiling of APC(Min/+) mouse plasma by dynamic headspace extraction was performed for multi-volatile analysis. In total, 19 volatiles were detected in the plasma of mice, based on information regarding oxidized lipid-derived volatile compounds, and eight of these compounds differed significantly between normal and diseased mice. 2-Methyl-2-butanol and benzyl alcohol were previously unreported in blood samples. Furthermore, 3,5,5-trimethyl-2(5H)-furanone was only detected in normal mice. 5-Methyl-3-hexanone and benzaldehyde have been detected in subjects with gastrointestinal diseases and lung cancer, respectively. Therefore, volatile profiling can be used to detect differences between samples and to identify compounds associated with diseases. PMID: 26406113 [PubMed - as supplied by publisher]

Metabolomics for genomics: the role of vitamin D in nonalcoholic fatty liver disease. J Gastrointestin Liver Dis. 2015 Sep;24(3):394-5 Authors: Crisan D, Radu C, Suciu A, Grigorescu MD, Stefanescu H, Romanciuc F, Socaciu C, Grigorescu M PMID: 26405717 [PubMed - in process]

Autocrine signaling of type 1 interferons in successful anticancer chemotherapy. Oncoimmunology. 2015 Aug;4(8):e988042 Authors: Vacchelli E, Sistigu A, Yamazaki T, Vitale I, Zitvogel L, Kroemer G Abstract Anthracycline-based chemotherapies are particularly effective if they succeed in reinstating immunosurveillance by the induction of immunogenic cell death (ICD) in the tumor. Recently, we discovered that ICD is coupled to the induction of type 1 interferons (IFNs-I) that act in an autocrine fashion on cancer cells, thereby increasing their immunogenic potential. PMID: 26405588 [PubMed - as supplied by publisher]

A majority of m6A residues are in the last exons, allowing the potential for 3' UTR regulation. Genes Dev. 2015 Sep 24; Authors: Ke S, Alemu EA, Mertens C, Gantman EC, Fak JJ, Mele A, Haripal B, Zucker-Scharff I, Moore MJ, Park CY, Vågbø CB, Kuśnierczyk A, Klungland A, Darnell JE, Darnell RB Abstract We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons. Moreover, m(6)A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m(6)A density peaks early in the 3' UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m(6)A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m(6)A modification in regulating proximal alternative polyA choice. PMID: 26404942 [PubMed - as supplied by publisher]

Investigation of Interspecies Interactions within Marine Micromonosporaceae Using an Improved Co-Culture Approach. Mar Drugs. 2015;13(10):6082-98 Authors: Adnani N, Vazquez-Rivera E, Adibhatla SN, Ellis GA, Braun DR, Bugni TS Abstract With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes. PMID: 26404321 [PubMed - in process]

MS-Based Metabolite Profiling of Aboveground and Root Components of Zingiber mioga and Officinale. Molecules. 2015;20(9):16170-16185 Authors: Han JS, Lee S, Kim HY, Lee CH Abstract 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. PMID: 26404226 [PubMed - as supplied by publisher]

In situ biomarker discovery and label-free molecular histopathological diagnosis of lung cancer by ambient mass spectrometry imaging. Sci Rep. 2015;5:14089 Authors: Li T, He J, Mao X, Bi Y, Luo Z, Guo C, Tang F, Xu X, Wang X, Wang M, Chen J, Abliz Z Abstract Sensitive and spatial exploration of the metabolism of tumors at the metabolome level is highly challenging. In this study, we developed an in situ metabolomics method based on ambient mass spectrometry imaging using air flow-assisted desorption electrospray ionization (AFADESI), which can spatially explore the alteration of global metabolites in tissues with high sensitivity. Using this method, we discovered potential histopathological diagnosis biomarkers (including lipids, amino acids, choline, peptides, and carnitine) from 52 postoperative lung cancer tissue samples and then subsequently used these biomarkers to generate images for rapid and label-free histopathological diagnosis. These biomarkers were validated with a sensitivity and a specificity of 93.5% and 100%, respectively. Moreover, a single imaging analysis of a cryosection that visualized all these biomarkers, taking tens of minutes, revealed the type and subtype of the cancer. This method could potentially be used as a molecular pathological tool for rapid clinical lung cancer diagnosis and immediate image-guided surgery. PMID: 26404114 [PubMed - as supplied by publisher]

Metabolomic analysis can detect the composition of pasta enriched with fibre after cooking. J Sci Food Agric. 2015 Sep 25; Authors: Beleggia R, Menga V, Platani C, Nigro F, Fragasso M, Fares C Abstract BACKGROUND: Several studies have demonstrated that metabolomics has a definite place in food quality, nutritional value, and safety issues. The aim of the present study was to determine and compare the metabolites in different pasta samples with fibre, and to investigate the modifications induced in these different kinds of pasta during cooking, using a gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach. RESULTS: Differences were seen for some of the amino acids, which were absent in control pasta, while were present both in the commercial available high-fibre pasta (A-C) and the enriched pasta (D-F). The highest content in reducing sugars was observed in enriched samples in comparison with high fibre - pasta. The presence of stigmasterol in sample enriched with wheat bran was relevant. Cooking decreased all of the metabolites: the high-fibre pasta (A-C) and Control showed losses of amino acids and tocopherols, while for sugars and organic acids, the decrease depended on the pasta sample. The enriched pasta samples (D-F) showed the same decreases with the exception of phytosterols, and in pasta with barley the decrease of SFAs was not significant as for tocopherols in pasta with oat. The PCA of the metabolites and the pasta discrimination was effective in the differentiation of the enriched pasta from the commercial pasta, both uncooked and cooked. CONCLUSIONS: The study has established that such metabolomic analyses provide useful tools in the evaluation of the changes in nutritional compounds in high-fibre and enriched pasta, both before and after cooking. PMID: 26403803 [PubMed - as supplied by publisher]

Evaluation of changes induced in rice metabolome by Cd and Cu exposure using LC-MS with XCMS and MCR-ALS data analysis strategies. Anal Bioanal Chem. 2015 Sep 24; Authors: Navarro-Reig M, Jaumot J, García-Reiriz A, Tauler R Abstract The comprehensive analysis of untargeted metabolomics data acquired using LC-MS is still a major challenge. Different data analysis tools have been developed in recent years such as XCMS (various forms (X) of chromatography mass spectrometry) and multivariate curve resolution alternating least squares (MCR-ALS)-based strategies. In this work, metabolites extracted from rice tissues cultivated in an environmental test chamber were subjected to untargeted full-scan LC-MS analysis, and the obtained data sets were analyzed using XCMS and MCR-ALS. These approaches were compared in the investigation of the effects of copper and cadmium exposure on rice tissue (roots and aerial parts) samples. Both methods give, as a result of their application, the whole set of resolved elution and spectra profiles of the extracted metabolites in control and metal-treated samples, as well as the values of their corresponding chromatographic peak areas. The effects caused by the two considered metals on rice samples were assessed by further chemometric analysis and statistical evaluation of these peak area values. Results showed that there was a statistically significant interaction between the considered factors (type of metal of treatment and tissue). Also, the discrimination of the samples according to both factors was possible. A tentative identification of the most discriminant metabolites (biomarkers) was assessed. It is finally concluded that both XCMS- and MCR-ALS-based strategies provided similar results in all the considered cases despite the completely different approaches used by these two methods in the chromatographic peak resolution and detection strategies. Finally, advantages and disadvantages of using these two methods are discussed. Graphical Abstract Summary of the workflow for untargeted metabolomics using the compared approaches. PMID: 26403240 [PubMed - as supplied by publisher]

Related Articles Metabolomic profiling in liver of adiponectin-knockout mice uncovers lysophospholipid metabolism as an important target of adiponectin action. Biochem J. 2015 Jul 1;469(1):71-82 Authors: Liu Y, Sen S, Wannaiampikul S, Palanivel R, Hoo RL, Isserlin R, Bader GD, Tungtrongchitr R, Deshaies Y, Xu A, Sweeney G Abstract Adiponectin mediates anti-diabetic effects via increasing hepatic insulin sensitivity and direct metabolic effects. In the present study, we conducted a comprehensive and unbiased metabolomic profiling of liver tissue from AdKO (adiponectin-knockout) mice, with and without adiponectin supplementation, fed on an HFD (high-fat diet) to derive insight into the mechanisms and consequences of insulin resistance. Hepatic lipid accumulation and insulin resistance induced by the HFD were reduced by adiponectin. The HFD significantly altered levels of 147 metabolites, and bioinformatic analysis indicated that one of the most striking changes was the profile of increased lysophospholipids. These changes were largely corrected by adiponectin, at least in part via direct regulation of PLA2 (phospholipase A2) as palmitate-induced PLA2 activation was attenuated by adiponectin in primary hepatocytes. Notable decreases in several glycerolipids after the HFD were reversed by adiponectin, which also corrected elevations in several diacyglycerol and ceramide species. Our data also indicate that stimulation of ω-oxidation of fatty acids by the HFD is enhanced by adiponectin. In conclusion, this metabolomic profiling approach in AdKO mice identified important targets of adiponectin action, including PLA2, to regulate lysophospholipid metabolism and ω-oxidation of fatty acids. PMID: 25915851 [PubMed - indexed for MEDLINE]

Related Articles Systems medicine, personalized health and therapy. Pharmacogenomics. 2015 Sep 24; Authors: Siest G, Auffray C, Taniguchi N, Ingelman-Sundberg M, Murray H, Visvikis-Siest S, Ansari M, Marc J, Jacobs P, Meyer U, Van Schaik RH, Müller MM, Wevers RA, Simmaco M, Kussmann M, Manolopoulos VG, Alizadeh BZ, Beastall G, Németh G Abstract The 7th Santorini Conference was held in Santorini, Greece, and brought together 200 participants from 40 countries in several continents, including Europe, USA but also Japan, Korea, Brazil and South Africa. The attendees had the opportunity to: listen to 60 oral presentations; participate in two lunch symposia; look at 103 posters, which were divided in two groups ('systems medicine and environment' and 'pharmacogenomics and cancer') and attend a dedicated exhibition with six companies. The meeting was organized by the Institut National de la Santé et de la Recherche Médicale (INSERM) U1122; IGE-PCV and by 'Biologie Prospective' with the collaboration of the European Society of Pharmacogenomics and Theranostics (ESPT), under the auspices of international organizations (e.g., International Federation of Clinical Chemistry and Laboratory medicine [IFCC], European Federation of Clinical Chemistry and Laboratory Medicine [EFLM], European Diagnostic Manufacturers Association [EDMA], Federation of European Pharmacological Societies [EPHAR], European Science Foundation [ESF]). The 3 days of the conference stimulated intensive discussions on systems biology and the influence of omics technologies on personalized health. Sixty speakers were invited or selected from early abstracts and gave presentations on the following topics: From systems biology to systems medicine/pharmacology; Omics/translating pharmacogenomics/proteomic biomarkers/metabolomics; Human nutrition and health/personalized medicine. We are summarizing here the main topics and presentations, according to the successive sessions. PMID: 26401575 [PubMed - as supplied by publisher]

Related Articles Biomarkers in Sporadic and Familial Alzheimer's Disease. J Alzheimers Dis. 2015 Jul 24;47(2):291-317 Authors: Lista S, O'Bryant SE, Blennow K, Dubois B, Hugon J, Zetterberg H, Hampel H Abstract Most forms of Alzheimer's disease (AD) are sporadic (sAD) or inherited in a non-Mendelian fashion, and less than 1% of cases are autosomal-dominant. Forms of sAD do not exhibit familial aggregation and are characterized by complex genetic and environmental interactions. Recently, the expansion of genomic methodologies, in association with substantially larger combined cohorts, has resulted in various genome-wide association studies that have identified several novel genetic associations of AD. Currently, the most effective methods for establishing the diagnosis of AD are defined by multi-modal pathways, starting with clinical and neuropsychological assessment, cerebrospinal fluid (CSF) analysis, and brain-imaging procedures, all of which have significant cost- and access-to-care barriers. Consequently, research efforts have focused on the development and validation of non-invasive and generalizable blood-based biomarkers. Among the modalities conceptualized by the systems biology paradigm and utilized in the "exploratory biomarker discovery arena", proteome analysis has received the most attention. However, metabolomics, lipidomics, transcriptomics, and epigenomics have recently become key modalities in the search for AD biomarkers. Interestingly, biomarker changes for familial AD (fAD), in many but not all cases, seem similar to those for sAD. The integration of neurogenetics with systems biology/physiology-based strategies and high-throughput technologies for molecular profiling is expected to help identify the causes, mechanisms, and biomarkers associated with the various forms of AD. Moreover, in order to hypothesize the dynamic trajectories of biomarkers through disease stages and elucidate the mechanisms of biomarker alterations, updated and more sophisticated theoretical models have been proposed for both sAD and fAD. PMID: 26401553 [PubMed - as supplied by publisher]

Related Articles Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells. J Proteomics Bioinform. 2015 Jun;8(6):126-132 Authors: Ulmer CZ, Yost RA, Chen J, Mathews CE, Garrett TJ Abstract Metabolomics is the comprehensive study of metabolism as it pertains to an organism or biological system. Lipidomics, a subset discipline of metabolomics, encompasses the study of cellular lipid functions: including pathways, networks, and interactions. The abundance of metabolites and lipids, along with their contribution to health and disease, makes metabolomics a valuable tool for biomarker research. Disease biomarker identification requires a reproducible, sensitive, and accurate analytical platform. Although transcriptomic and proteomic areas have well-established protocols for sample preparation and data processing, the metabolomics field is still developing comparable standardized conventions. Furthermore, of the few comparative LC-MS metabolomic studies that have been applied to mammalian cell cultures, most are targeted to adherent cell lines. The purpose of this work was to optimize a sample preparation workflow for the cellular metabolomic analysis of suspension-cultured mammalian cells using commercially available Jurkat T lymphocytes as a model system. The current investigation evaluated commonly used sample preparation techniques for reproducibility, accuracy, and applicability to untargeted biomarker discovery. Results show ammoniated cell rinsing solutions to be an effective means to remove extracellular components present in the media without causing ion suppression or affecting the integrity of the cellular membrane. Additionally, a novel workflow was designed to allow for the combined analysis of metabolites and lipids from mammalian suspension cells from a single cell pellet. The Folch lipid extraction protocol was coupled to an 80% MeOH metabolite isolation to ensure high extraction efficiency for phospholipids and triacylglycerides. While the workflow was tailored to cells in suspension, it could also be applied to adherent cell lines. PMID: 26401069 [PubMed - as supplied by publisher]

Related Articles Herring and Beef Meals Lead to Differences in Plasma 2-Aminoadipic Acid, β-Alanine, 4-Hydroxyproline, Cetoleic Acid, and Docosahexaenoic Acid Concentrations in Overweight Men. J Nutr. 2015 Sep 23; Authors: Ross AB, Svelander C, Undeland I, Pinto R, Sandberg AS Abstract BACKGROUND: Dietary guidelines generally recommend increasing fish intake and reducing red meat intake for better long-term health. Few studies have compared the metabolic differences between eating meat and fish. OBJECTIVE: The objective of this study was to determine whether there are differences in the postprandial plasma metabolic response to meals containing baked beef, baked herring, and pickled herring. METHODS: Seventeen overweight men (BMI 25-30 kg/m(2), 41-67 y of age) were included in a randomized crossover intervention study. Subjects ate baked herring-, pickled herring-, and baked beef-based meals in a randomized order and postprandial blood plasma samples were taken over 7 h. Plasma metabolomics was measured with the use of gas chromatography-mass spectrometry and area under the curve for detected metabolites was compared between meals. RESULTS: The plasma postprandial response of 2-aminoadipic acid, a suggested marker of diabetes risk, was 1.6 times higher after the beef meal than after the baked herring meal (P < 0.001). Plasma β-alanine and 4-hydroxyproline both increased markedly after beef intake compared with herring intake (16 and 3.4 times the response of baked herring, respectively; P < 0.001). Herring intake led to a greater plasma postprandial response from docosahexaenoic acid (DHA) and cetoleic acid vs. beef (17.6 and 150 times greater, respectively; P < 0.001), whereas hippuric acid and benzoic acid were elevated after pickled herring compared with baked herring (5.4 and 43 times higher; P < 0.001). CONCLUSIONS: These results in overweight men confirm that DHA and cetoleic acid reflect herring intake, whereas β-alanine and 4-hydroxyproline are potential biomarkers for beef intake. The greater postprandial rise in 2-aminoadipic acid after the beef meal, coupled to its proposed role in stimulating insulin secretion, may have importance in the context of red meat intake and increased diabetes risk. This trial was registered at clinicaltrials.gov as NCT02381613. PMID: 26400963 [PubMed - as supplied by publisher]

Related Articles Can we predict the intracellular metabolic state of a cell based on extracellular metabolite data? Mol Biosyst. 2015 Sep 24; Authors: Granucci N, Pinu FR, Han TL, Villas-Boas SG Abstract The analysis of extracellular metabolites presents many technical advantages over the analysis of intracellular compounds, which made this approach very popular in recent years as a high-throughput tool to assess the metabolic state of microbial cells. However, very little effort has been made to determine the actual relationship between intracellular and extracellular metabolite levels. The secretion of intracellular metabolites has been traditionally interpreted as a consequence of an intracellular metabolic overflow, which is based on the premise that for a metabolite to be secreted, it must be over-produced inside the cell. Therefore, we expect to find a secreted metabolite at increased levels inside the cells. Here we present a time-series metabolomics study of Saccharomyces cerevisiae growing on a glucose-limited chemostat with parallel measurements of intra- and extracellular metabolites. Although most of the extracellular metabolites were also detected in the intracellular samples and showed a typical metabolic overflow behaviour, we demonstrate that the secretion of many metabolites could not be explained by the metabolic overflow theory. PMID: 26400772 [PubMed - as supplied by publisher]

Related Articles Rhynchophorus ferrugineus attack affects a group of compounds rather than rearranging Phoenix canariensis metabolic pathways. J Integr Plant Biol. 2015 Sep 24; Authors: Giovino A, Martinelli F, Saia S Abstract The red palm weevil (RPW; Rhynchophorus ferrugineus) is spreading worldwide and severely harming many palm species. However, most studies on RPW focused on insect biology, and little information is available about the plant response to the attack. In the present experiment, we used metabolomics to study the alteration of the leaf metabolome of Phoenix canariensis at initial (1(st) stage) or advanced (2(nd) stage) attack by RPW compared with healthy (unattacked) plants. The leaf metabolome significantly varied among treatments. At the 1(st) stage of attack, plants showed a reprogramming of carbohydrate and organic acid metabolism; in contrast, peptides and lipid metabolic pathways underwent more changes during 2(nd) then 1(st) stage of attack. Enrichment metabolomics analysis indicated that RPW attack mostly affected a particular group of compounds rather than rearranging plant metabolic pathways. Some compounds selectively affected during 1(st) rather than 2(nd) stage (e.g. phenylalanine; tryptophan; cellobiose; xylose; quinate; xylonite; idonate; and iso-threonate; cellobiotol and arbutine) are upstream events in the phenylpropanoid, terpenoid and alkaloid biosynthesis. These compounds could be designated as potential markers of initial RPW attack. However, further investigation is needed to determine efficient early screening methods of RPW attack based on the concentrations of these molecules. PMID: 26399847 [PubMed - as supplied by publisher]