Duarte Iola F, Marques Joana, Ladeirinha Ana F, Rocha Cláudia, Lamego Inês, Calheiros Rita, Silva Tânia M, Marques M Paula M, Melo Joana B, Carreira Isabel M, Gil Ana M
CICECO-Departmento de Química, Campus Universitário de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal.
Anal Chem. 2009 Jun 15;81(12):5023-32. doi: 10.1021/ac900545q.
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.
这项工作的目的是研究细胞处理和储存对细胞完整性以及氢-1高分辨率魔角旋转（HRMAS）核磁共振谱的影响。我们考虑了三种不同的细胞类型（肺肿瘤细胞、羊膜细胞和MG-63骨肉瘤细胞），以便识别样本依赖性效应。新鲜细胞和冷冻保存在低温保存溶液中的细胞的完整性约为70%-80%，前者显示出更高的膜降解，可能是酶促降解，如磷酸胆碱（PC）和/或甘油磷酸胆碱（GPC）增加所示。发现无保护冷冻（无论是逐步冷冻还是速冻）会使细胞完全裂解，类似于机械细胞裂解。除了增强代谢物的可见性外，与完整细胞相比，裂解细胞显示出不同的脂质谱，胆碱、PC和GPC增加，磷脂酰胆碱（PTC）减少。因此，细胞裂解对细胞脂质组成有显著影响，使得处理的可重复性成为脂质分析中的一个重要问题。发现样品旋转会破坏5%-25%的细胞，具体取决于细胞类型，并且HRMAS被证明优于悬浮液或上清液的溶液态核磁共振，能提供更多关于脂质的信息，并且对较小代谢物具有可比的分辨率。与裂解细胞相比，弛豫和扩散编辑的核磁共振实验对完整细胞提供的信息有限。对三种细胞类型的氢-1 HRMAS谱进行了比较和讨论。
