Analytical Approaches toward Successful Human Cell Metabolome Studies by NMR Spectroscopy
authors Duarte, IF; Marques, J; Ladeirinha, AF; Rocha, C; Lamego, I; Calheiros, R; Silva, TM; Marques, MPM; Melo, JB; Carreira, IM; Gil, AM
nationality International
journal ANALYTICAL CHEMISTRY
keywords MAGNETIC-RESONANCE-SPECTROSCOPY; SPINNING H-1-NMR SPECTROSCOPY; HIGH-RESOLUTION; CANCER-CELLS; HRMAS-NMR; IN-VITRO; TUMOR; SPHEROIDS; MARKERS; LINES
abstract The aim of this work was to investigate the effects of cell handling and storage on cell integrity and (1)H high resolution magic angle spinning (HRMAS) NMR spectra. Three different cell types have been considered (lung tumoral, amniocytes, and MG-63 osteosarcoma cells) in order for sample-dependent effects to be identified. Cell integrity of fresh cells and cells frozen in cryopreservative solution was similar to 70-80%, with the former showing higher membrane degradation, probably enzymatic, as indicated by increased phosphocholine (PC) and/or glycerophosphocholine (GPC). Unprotected freezing (either gradual or snap-freezing) was found to lyse cells completely, similar to mechanical. cell lysis. Besides enhanced metabolites visibility, lysed cells showed a different lipid profile compared to intact cells, with increased choline, PC, and GPC and decreased phosphatidylcholine (PTC). Cell lysis has, therefore, a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid analysis. Sample spinning was found to disrupt 5-25% of cells, depending on cell type, and HRMAS was shown to be preferable to solution-state NMR of suspensions or supernatant, giving enhanced information on lipids and comparable resolution for smaller metabolites. Relaxation- and diffusion-edited NMR experiments gave limited information on intact cells, compared to lysed cells. The (1)H HRMAS spectra of-the three cell types are compared and discussed.
publisher AMER CHEMICAL SOC
issn 0003-2700
year published 2009
volume 81
issue 12
beginning page 5023
ending page 5032
digital object identifier (doi) 10.1021/ac900545q
web of science category Chemistry, Analytical
subject category Chemistry
unique article identifier WOS:000266969700045
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journal analysis (jcr 2017):
journal impact factor 6.042
5 year journal impact factor 6.035
category normalized journal impact factor percentile 95.679
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