期刊
ANALYTICAL CHEMISTRY
卷 81, 期 12, 页码 5023-5032出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac900545q
关键词
-
资金
- Foundation for Science and Technology, Portugal [FCT/PTDC/SAU-BEB-66896/2006, FCT/PTDC/QUI-68017/2006]
- CIMAGO, University of Coimbra [14/06]
The aim of this work was to investigate the effects of cell handling and storage on cell integrity and H-1 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 H-1 HRMAS spectra of-the three cell types are compared and discussed.
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