Choline containing metabolites during cell transfection: an insight into magnetic resonance spectroscopy detectable changes

Griffin, Julian L.; Mann, Christopher; Scott, James A.; Shoulders, Carol C.; Nicholson, Jeremy K.

doi:10.1016/s0014-5793(01)03175-1

Cited by 91 publications

(64 citation statements)

References 20 publications

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“…Even under MAS conditions used here, spinning speeds were too slow to remove the effects of the large dipolar couplings produced between lipids in cell membranes (35); thus, 1 H NMR-detected CHO are likely to be non-membrane bound. Previously, we have detected increases in CHO after cell membrane disruption in cultured hepatocytes, with the increased choline-containing metabolite resonance arising from membrane phosphocholine being redistributed to the cytosol (36). It is conceivable that during the end stage of PCD, the CHO resonance intensity is probably associated with cell membrane disruption and leakage of choline-containing metabolites into the cytosol and extracellular fluid.…”

Section: Discussionmentioning

confidence: 86%

Metabolite Changes in BT4C Rat Gliomas Undergoing Ganciclovir-Thymidine Kinase Gene Therapy-induced Programmed Cell Death as Studied by 1H NMR Spectroscopy in Vivo, ex Vivo, and in Vitro

Lehtimäki

Valonen

Griffin

et al. 2003

Journal of Biological Chemistry

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Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1 H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1 H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%. Meanwhile, both saturated and unsaturated 1 H NMR visible lipids increased. HR-MAS 1 H NMR spectroscopy of the tumor samples at 14.1 tesla demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo. The assignment of a doublet at 7.95 ppm to UDP was confirmed by spiking experiments of tumor extracts in conjunction with 1 H and 31 P NMR spectroscopy. HRMAS also resolved the choline-containing peak at 3.2 ppm in vivo into resonances from choline (3.20 ppm), phosphocholine (3.22 ppm), glycerophosphocholine (3.24 ppm), and taurine (3.26 ppm). These resonances were uncorrelated with temporal progression through programmed cell death. Our results show that 1 H NMR-detected lipids and some of the small molecular weight metabolites respond to gene therapy. However, the choline-containing compounds are unaffected by severe decline in cell density. The latter observation supports the idea that triacylglycerols, rather than membrane phospholipids, are the key components of 1 H NMR visible lipids, and it also casts doubt on the validity of resonance of choline-containing compounds as a diagnostic marker of programmed cell death in vivo. Programmed cell death (PCD)1 involves a cascade of biochemical processes in an ATP-dependent manner, and the process is associated with substantial morphological alterations in the cell interior before phagocytosis (1-3). Recent evidence from cell culture studies points to a number of characteristic metabolic perturbations appearing in the early phase of PCD. These include affections of intermediary metabolism, such as accumulation of glycolytic intermediates fructose 1,6-bisphosphate, dihydroxy acetone phosphate, and glycerol-3-phosphate because of inhibition of glyceraldehyde-3-phosphate dehydrogenase (4), retention of CDP-choline (5) as a result of inhibition of CDP-choline:1,2-diacylglycerol choline phosphotransferase (6), and collapse of NAD(H) levels (5). The severe decline in NAD(H), leading to inhibition of glycolysis, may result from activation of poly(ADP-ribose) polymerase in apoptotic cells (5). Inhibition of CDP-choline:1,2-diacylglycerol choline phospotransferase leads to cessation of phosphatidylcholine biosynthesis (6), and it has been proposed that this might be one of the mechanisms explaining accumulation of 1 H NMR lipids into cells undergoing apoptosis (7,8). I...

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Section: Discussionmentioning

confidence: 86%

Metabolite Changes in BT4C Rat Gliomas Undergoing Ganciclovir-Thymidine Kinase Gene Therapy-induced Programmed Cell Death as Studied by 1H NMR Spectroscopy in Vivo, ex Vivo, and in Vitro

Lehtimäki

Valonen

Griffin

et al. 2003

Journal of Biological Chemistry

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“…All of these procedures produced cells which showed total staining with Trypan blue, indicating total membrane disruption. Interestingly, the results obtained for samples C1 and C2 (Figure 1) showed that unprotected freezing, including snapfreezing (a method used previously in handling intact cells for NMR analysis 22,24 ), compromises cell integrity.…”

Section: Resultsmentioning

confidence: 96%

“…An increase in PC due to cell membrane disruption has been reported previously. 24 In sample A1, this may arise because of the activity of degrading enzymes during the 2-3 h refrigeration period between harvesting and NMR analysis, showing how sensitive cell membranes are to sample handling. Interestingly, choline content (evaluated by the area of its headgroup signal divided by the total spectral area) is similar between samples A1 and B for all cell types (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

Analytical Approaches toward Successful Human Cell Metabolome Studies by NMR Spectroscopy

et al. 2009

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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...

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“…To this end, brain tumor proton MR spectroscopy studies (Fig. 1) consistently demonstrate: (1) reduced or absent n-acetylaspartate (NAA) and total creatine (tCr) attributed to edema and necrosis; (2) increased Cho-containing compounds possibly due to cell membrane disruption (Griffin et al, 2001) and altered phospholipid metabolism (Aboagye and Bhujwalla, 1999;Podo, 1999;Ackerstaff et al, 2001); and (3) increased lactate due to metabolic acidosis (Bruhn et al, 1989;Alger et al, 1990;Arnold et al, 1990;Segebarth et al, 1990;Luyten et al, 1991;Nelson et al, 1997a;Wald et al, 1997;Aboagye et al, 1998). Reduced NAA is expected in glial tumors, since NAA is primarily localized to neurons.…”

Section: Biological Aspects Of Selected Metabolites Detected By Protomentioning

confidence: 94%

Pediatric Brain Tumors: Magnetic Resonance Spectroscopic Imaging

Tzika¹,

Astrakas²,

Zarifi³

2011

Diagnostic Techniques and Surgical Management of Brain Tumors

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Choline containing metabolites during cell transfection: an insight into magnetic resonance spectroscopy detectable changes

Cited by 91 publications

References 20 publications

Metabolite Changes in BT4C Rat Gliomas Undergoing Ganciclovir-Thymidine Kinase Gene Therapy-induced Programmed Cell Death as Studied by 1H NMR Spectroscopy in Vivo, ex Vivo, and in Vitro

Metabolite Changes in BT4C Rat Gliomas Undergoing Ganciclovir-Thymidine Kinase Gene Therapy-induced Programmed Cell Death as Studied by 1H NMR Spectroscopy in Vivo, ex Vivo, and in Vitro

Analytical Approaches toward Successful Human Cell Metabolome Studies by NMR Spectroscopy

Pediatric Brain Tumors: Magnetic Resonance Spectroscopic Imaging

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