title: Analytical approaches toward successful human cell metabolome studies by NMR spectroscopy.
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 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.
authors.affiliation: CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal.
authors.forename: Iola F
authors.initials: IF
authors.lastname: Duarte
authors.forename: Joana
authors.initials: J
authors.lastname: Marques
authors.forename: Ana F
authors.initials: AF
authors.lastname: Ladeirinha
authors.forename: Cláudia
authors.initials: C
authors.lastname: Rocha
authors.forename: Inês
authors.initials: I
authors.lastname: Lamego
authors.forename: Rita
authors.initials: R
authors.lastname: Calheiros
authors.forename: Tânia M
authors.initials: TM
authors.lastname: Silva
authors.forename: M Paula M
authors.initials: MP
authors.lastname: Marques
authors.forename: Joana B
authors.initials: JB
authors.lastname: Melo
authors.forename: Isabel M
authors.initials: IM
authors.lastname: Carreira
authors.forename: Ana M
authors.initials: AM
authors.lastname: Gil
mesh_terms: 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
chemical_list: D008055:Lipids; D010713:Phosphatidylcholines; D010767:Phosphorylcholine