Document Detail

Analytical approaches toward successful human cell metabolome studies by NMR spectroscopy.
MedLine Citation:
PMID:  19462963     Owner:  NLM     Status:  MEDLINE    
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 approximately 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.
Iola F Duarte; Joana Marques; Ana F Ladeirinha; Cláudia Rocha; Inês Lamego; Rita Calheiros; Tânia M Silva; M Paula M Marques; Joana B Melo; Isabel M Carreira; Ana M Gil
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical chemistry     Volume:  81     ISSN:  1520-6882     ISO Abbreviation:  Anal. Chem.     Publication Date:  2009 Jun 
Date Detail:
Created Date:  2009-06-12     Completed Date:  2009-08-14     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5023-32     Citation Subset:  IM    
CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal.
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MeSH Terms
Cells, Cultured
Lipids / analysis,  chemistry
Nuclear Magnetic Resonance, Biomolecular / methods*
Phosphatidylcholines / analysis
Phosphorylcholine / analysis
Reg. No./Substance:
0/Lipids; 0/Phosphatidylcholines; 107-73-3/Phosphorylcholine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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