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 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.
Duarte
Iola F
IF
CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal.
Marques
Joana
J
Ladeirinha
Ana F
AF
Rocha
Cláudia
C
Lamego
Inês
I
Calheiros
Rita
R
Silva
Tânia M
TM
Marques
M Paula M
MP
Melo
Joana B
JB
Carreira
Isabel M
IM
Gil
Ana M
AM
D002478:Cells, Cultured; D006801:Humans; D008055:Lipids / Q000032:analysis / Q000737:chemistry; D055442:Metabolome; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010713:Phosphatidylcholines / Q000032:analysis; D010767:Phosphorylcholine / Q000032:analysis
D008055:Lipids; D010713:Phosphatidylcholines; D010767:Phosphorylcholine