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 ∼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.Nuclear magnetic resonance (NMR) spectroscopy has been, in recent years, increasingly employed for the analysis of metabolic processes in biological systems because of its ability to provide rapid detection of many different metabolites present in complex systems such as biofluids, biological tissues, or cells. The analysis of the metabolome of biological systems provides important information on their biochemical phenotypes and on the metabolic changes occurring in response to external stimuli, e.g., drug exposure, disease onset, medication. 1,2The study of cellular metabolism using NMR has been successfully carried out with strong emphasis on cell extracts, either hydrophilic or lipophilic. For instance, acidic extracts, in the presence of ice-cold perchloric acid (PCA) or trichloroacetic acid (TCA), allow polar metabolites to be identified 3,4 as shown for PCA extracts of human colon adenocarcinoma cells 5 and human osteosarcoma cells 6,7 and TCA extracts of human rhabdomyosarcoma cells 8 and human lung cancer cells. 9 Other extraction methods have been used to identify aqueous and lipophilic metabolites, for instance in human colon carcinoma cells, 10 rat astrocyte cells, 11 human prostate cancer cells, 12 and human lung carcinoma cell lines. 13 In addition to the unavoidable selectivity of extraction methods, rendered useful only when the nature of the compounds of interest is known a priori, sample extraction may involve significant loss of particular cellular components, retained in the residual insoluble precipitate and not amenable to study by solution-st...

Aggregated Electronegative Low Density Lipoprotein in Human Plasma Shows a High Tendency toward Phospholipolysis and Particle Fusion

“…For this reason, the spectra at time 0 could not be acquired. The content of choline-containing phospholipids (3.170 -3.280 ppm) (27,28), ceramide (5.250 -5.350 ppm) (28), and bisallylic compounds (2.650 -5.850 ppm) (29) was measured by area integration of the corresponding resonance signal.…”

“…The spectra of the three subfractions remained almost unchanged after 24 h (monitored for a total time of 48 h; data not shown), except for the signal of the fatty acid-CH 3 in agLDL(Ϫ), which increased in intensity, probably because of particle decomposition, as was observed by TEM. 1 H NMR spectra also provides information on degradation of phospholipids by monitoring the intensity of the resonance signals corresponding to choline-containing phospholipids (3.170 -3.280 ppm) and ceramide (5.250 -5.350 ppm) (28). In addition, the progression of oxidation can be detected by monitoring the decrease of the signal corresponding to bisallylic groups of fatty acids (CH-CH 2 -CH, 2.650 -2.850 ppm) (27).…”

Nuclear Magnetic Resonance (NMR) Study of the Effect of Cisplatin on the Metabolic Profile of MG-63 Osteosarcoma Cells

“…13 In addition, many different anticancer drugs have been investigated through NMR analysis of other types of cultured cells, mostly in the form of cell suspensions [14][15][16][17] or extracts. [18][19][20][21][22] High resolution magic angle spinning (HRMAS) NMR spectroscopy, which is well established in tissue analysis, 23,24 has been shown to enable well-resolved spectra to be obtained for intact or lysed cells 25 and to provide valuable information on both lipid and small metabolite changes upon treatment with anticancer drugs of endometrial, 26 melanoma, 27 colon carcinoma, 28 hepatocarcinoma and osteosarcoma 13 cells. In the present study, 1 H HRMAS NMR spectroscopy was used to assess the changes in the intracellular metabolic profile of MG-63 human OS cells induced by the chemotherapy agent cisplatin (cis-diamminedichloroplatinum(II) or CDDP) at different times of exposure.…”

Processing of high resolution magic angle spinning spectra of breast cancer cells by the filter diagonalization method

“…In the case of in vitro or in vivo NMR studies of cellular metabolomics, the analysis is performed with intact cells, which can be a much more reliable process. 2 However, in this case the achieved NMR resolution is lower than that observed for the extracts, due to the restricted mobility of large molecules or assemblies and differences in the magnetic susceptibility of the sample. 3 To improve the spectral resolution of heterogeneous biological samples, the high resolution magic angle spinning (HR-MAS) technique has been used for more than a decade.…”

“…2 Besides, the extraction process is also laborious and time consuming. In the case of in vitro or in vivo NMR studies of cellular metabolomics, the analysis is performed with intact cells, which can be a much more reliable process.…”