Analytical Approaches Toward Successful Human Cell Metabolome Studies by NMR Spectroscopy (original) (raw)
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Artefacts in 1 H NMR-based metabolomic studies on cell cultures
Object Metabolomic studies on cultured cells involve assays of cell extracts and culture medium, both of which are often performed by 1 H NMR. Cell culture is nowadays performed in plastic dishes or flasks, and the extraction of metabolites from the cells is typically performed with perchloric acid, methanol–chloroform, or acetonitrile, ideally while the cells are still adherent to the culture dish. We conducted this investigation to identify contaminants from cell culture plasticware in metabolomic studies. Materials and methods Human diploid fibroblasts (IMR90) (n = 6), HeLa cells (n = 6), and transformed astrocytes with HIF-1 knockout (Astro-KO) (n = 6) were cultured. Cells were seeded in 100 mm Petri dishes with 10 ml complete growth medium (Dulbecco's minimum essential medium) containing 10 % foetal bovine serum (FBS). Cell cultures were incubated at 37 °C in 5 % CO 2 for approximately 3 days. Metabolites were extracted by use of a perchloric acid procedure. 1 H NMR spectroscopy was used for metabolite analysis. ''Null sample'' (i.e. cell-free) experiments were performed by either rinsing dishes with medium or incubating the medium in Petri dishes from five different manufacturers for 72 h and then by performing a dummy ''extraction'' of each Petri dish by the perchloric acid, methanol–chloroform, or acetonitrile procedures. Principal components analysis was used for classification of samples and to determine the contaminants arising from plasticware. Results We found that even brief rinsing of cell culture plasticware with culture medium elutes artefactual chemicals , the 1 H NMR signals of which could confound assays of acetate, succinate, and glycolate. Incubation of culture medium in cell-culture dishes for 72 h (as in a typical cell-culture experiment) followed by perchloric extraction in the dishes enhanced elution of the artefacts. These artefacts were present, but somewhat less pronounced, in the 1 H NMR spectra of null samples extracted with methanol and acetonitrile. Ethanol, lactate, alanine, fructose, and fuma-rate signals that appear in the 1 H NMR spectrum of the unused (pure) medium originate from FBS. Conclusions Plastic Petri dishes from five different manufacturers gave rise to essentially identical artefactual peaks. Use of a pH indicator to assist neutralisation introduced still more artefactual signals in the aromatic region, as well as methanol and ethanol signals. Methanol and acetonitrile extracts also contained artefacts arising from the plasticware, although the amounts were less than in the perchloric acid extracts. Finally, we provide suggestions for minimizing these artefacts. The best practice would be to run a ''null'' extraction with every batch of cellular metabolomics experiments to test for contamination and to provide a ''background'' spectrum.
Journal of Biomedicine and Biotechnology, 2011
HRMAS NMR is considered a valuable technique to obtain detailed metabolic profile of unprocessed tissues. To properly interpret the HRMAS metabolomic results, detailed information of the actual state of the sample inside the rotor is needed. MRM (Magnetic Resonance Microscopy) was applied for obtaining structural and spatially localized metabolic information of the samples inside the HRMAS rotors. The tissue was observed stuck to the rotor wall under the effect of HRMAS spinning. MRM spectroscopy showed a transference of metabolites from the tissue to the medium. The sample shape and the metabolite transfer after HRMAS indicated that tissue had undergone alterations and it can not be strictly considered as intact. This must be considered when HRMAS is used for metabolic tissue characterization, and it is expected to be highly dependent on the manipulation of the sample. The localized spectroscopic information of MRM reveals the biochemical compartmentalization on tissue samples hidd...
Strategy for choosing extraction procedures for NMR-based metabolomic analysis of mammalian cells
Analytical and Bioanalytical Chemistry, 2011
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NMR in Biomedicine, 2008
We report principal component analysis (PCA) of 1 H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and 1 H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumour origin with different chemotherapy drug resistance patterns. For whole cells, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene mobile lipid 1 H resonance intensities, which correlate with our knowledge of the level of resistance displayed by the different cells. Similarly, in the 1 H NMR spectra of the aqueous and lipophilic extracts, significant quantitative differences in the metabolite distributions were apparent, which are consistent with the PCA results.
Influence of Drying Method on NMR-Based Metabolic Profiling of Human Cell Lines
Metabolites
Metabolic profiling of cell line and tissue extracts involves sample processing that includes a drying step prior to re-dissolving the cell or tissue extracts in a buffer for analysis by GC/LC-MS or NMR. Two of the most commonly used drying techniques are centrifugal evaporation under vacuum (SpeedVac) and lyophilization. Here, NMR spectroscopy was used to determine how the metabolic profiles of hydrophilic extracts of three human pancreatic cancer cell lines, MiaPaCa-2, Panc-1 and AsPC-1, were influenced by the choice of drying technique. In each of the three cell lines, 40–50 metabolites were identified as having statistically significant differences in abundance in redissolved extract samples depending on the drying technique used during sample preparation. In addition to these differences, some metabolites were only present in the lyophilized samples, for example, n-methyl-α-aminoisobutyric acid, n-methylnicotimamide, sarcosine and 3-hydroxyisovaleric acid, whereas some metaboli...
PeerJ, 2016
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 use1H 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 me...
Metabolites, 2016
According to World Health Organization (WHO) estimates, cancer is responsible for more deaths than all coronary heart disease or stroke worldwide, serving as a major public health threat around the world. High resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS) has demonstrated its usefulness in the identification of cancer metabolic markers with the potential to improve diagnosis and prognosis for the oncology clinic, due partially to its ability to preserve tissue architecture for subsequent histological and molecular pathology analysis. Capable of the quantification of individual metabolites, ratios of metabolites, and entire metabolomic profiles, HRMAS MRS is one of the major techniques now used in cancer metabolomic research. This article reviews and discusses literature reports of HRMAS MRS studies of cancer metabolomics published between 2010 and 2015 according to anatomical origins, including brain, breast, prostate, lung, gastrointestinal, and neur...
NMR Spectroscopy for Metabolomics Research
2019
Over the past two decades, nuclear magnetic resonance (NMR) has emerged as one of the three principal analytical techniques used in metabolomics (the other two being gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled with single-stage mass spectrometry (LC-MS)). The relative ease of sample preparation, the ability to quantify metabolite levels, the high level of experimental reproducibility, and the inherently nondestructive nature of NMR spectroscopy have made it the preferred platform for long-term or large-scale clinical metabolomic studies. These advantages, however, are often outweighed by the fact that most other analytical techniques, including both LC-MS and GC-MS, are inherently more sensitive than NMR, with lower limits of detection typically being 10 to 100 times better. This review is intended to introduce readers to the field of NMR-based metabolomics and to highlight both the advantages and disadvantages of NMR spectroscopy for metabolomic studies. It will also explore some of the unique strengths of NMR-based metabolomics, particularly with regard to isotope selection/detection, mixture deconvolution via 2D spectroscopy, automation, and the ability to noninvasively analyze native tissue specimens. Finally, this review will highlight a number of emerging NMR techniques and technologies that are being used to strengthen its utility and overcome its inherent limitations in metabolomic applications.
1 H HR-MAS NMR and S180 Cells: Metabolite Assignment and Evaluation of Pulse Sequence
Journal of the Brazilian Chemical Society, 2014
Ressonância magnética nuclear de 1 H de alta resolução com giro no ângulo mágico (HR-MAS NMR) é uma técnica empregada na avaliação de células e tecidos intactos. Entretanto, parâmetros bem estabelecidos de NMR são cruciais para a obtenção de resultados confiáveis. A fim de discutir as principais etapas envolvidas na otimização das análises de HR-MAS NMR, este artigo avaliou diferentes sequências de pulsos e parâmetros de NMR usando células de sarcoma 180 (S180). O completo assinalamento dos metabólitos de S180 é também apresentado para auxiliar estudos futuros. High resolution magic angle spinning 1 H nuclear magnetic resonance spectroscopy (HR-MAS NMR) is a useful technique for evaluation of intact cells and tissues. However, optimal NMR parameters are crucial in obtaining reliable results. To identify the key steps for the optimization of HR-MAS NMR parameters, we assessed different pulse sequences and NMR parameters using sarcoma 180 (S180) cells. A complete assignment of the metabolites of S180 is given to assist future studies.