Analytical Approaches toward Successful Human Cell Metabolome Studies by NMR Spectroscopy


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.



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Duarte, IF; Marques, J; Ladeirinha, AF; Rocha, C; Lamego, I; Calheiros, R; Silva, TM; Marques, MPM; Melo, JB; Carreira, IM; Gil, AM

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Funding is acknowledged from the Foundation for Science and Technology, Portugal, through the research projects FCT/PTDC/SAU-BEB-66896/2006 and FCT/PTDC/QUI-68017/2006, and also from CIMAGO, University of Coimbra (project 14/06). The authors also acknowledge Dr. Manfred Spraul from Bruker BioSpin, Germany, for access to a comprehensive compound database for NMR assignment. The authors thank the Associate Laboratory IBMC-INEB (Portugal) for having provided the MG-63 osteosarcoma cell line.

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