Simultaneous extraction of cellular lipids and water‐soluble metabolites: Evaluation by NMR spectroscopy

Tyagi, Rakesh K.; Azrad, Anat; Degani, Hadassa; Salomon, Yoram

doi:10.1002/mrm.1910350210

Cited by 131 publications

(119 citation statements)

References 21 publications

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“…One to 2 × 10 8 control and treated cells were extracted using a modification of the dual-phase extraction method (Ronen et al, 1990;Tyagi et al, 1996). Briefly, a pellet of saline-rinsed L1210 cells or detached SW620 cells was vortexed in 10 ml of ice-cold methanol, followed by 10 ml of ice-cold chloroform and 10 ml ice-cold deionized water.…”

Section: Magnetic Resonance Spectroscopymentioning

confidence: 99%

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis

Ronen¹,

Di-Stefano

McCoy

et al. 1999

Br J Cancer

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Summary Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31 P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line-or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31 P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects.

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Section: Magnetic Resonance Spectroscopymentioning

confidence: 99%

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis

Ronen¹,

Di-Stefano

McCoy

et al. 1999

Br J Cancer

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“…Tissue-targeted metabonomic analyses have been carried out on brain tissue [11][12][13], liver tissue [14][15][16], and kidney tissue [17][18][19]. Metabolite extraction is a critical step in tissue metabonomic study, and the chloroform-methanol-water solvent system is considered the preferred method in several reports [20][21][22][23]. The latest relevant study was conducted by Wu et al [24], in which fish liver was used to optimize the extraction strategy of the preferred solvent system for NMR-and FT-ICR MS-based metabonomic studies.…”

Section: Introductionmentioning

confidence: 99%

An optimized procedure for metabonomic analysis of rat liver tissue using gas chromatography/time-of-flight mass spectrometry

Qiu

Chen

et al. 2010

Journal of Pharmaceutical and Biomedical Analysis

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Abstract:In this paper, we present a tissue metabonomic method with an optimized extraction procedure followed by instrumental analysis with gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) and spectral data analysis with multivariate statistics. Metabolite extractions were carried out using three solvents: chloroform, methanol, and water, with design of experiment (DOE) theory and multivariate statistical analysis. A two-step metabolite extraction procedure was optimized using a mixed solvent of chloroform-methanol-water (1:2:1, v/v/v) and then followed by methanol alone. This approach was subsequently validated using standard compounds and liver tissues. Calibration curves were obtained in the range of 0.50-125.0 μg/mL for standards and 0.02-0.25 g/mL acceptable for liver tissue samples. For most of the metabolites investigated, relative standard deviations (RSD) were below 10% within a day (reproducibility) and below 15% within a week (stability). Rat liver tissues of carbon tetrachloride-induced acute liver injury models (n = 10) and healthy control rats (n = 10) were analyzed which demonstrated the applicability of the developed procedure for the tissue metabonomic study.

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“…The plates were washed twice with saline at room temperature and ice cold methanol was added (4 ml/plate or 150 µl/well). The plates were kept on ice and then subjected to dual-phase extraction [22], as described below. Each experiment was performed at least three times with triplicate samples ; the results of representative experiments are presented.…”

Section: Methodsmentioning

confidence: 99%

“…Cell extracts were obtained by the dual-phase extraction [22]. This method, which is a salt-free chloroform/methanol extraction, quantitatively allows recovery of both the water-soluble and lipid components from the same cell sample at a physiological pH.…”

Section: Methodsmentioning

confidence: 99%

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Stimulation of fructose 1,6‐bisphosphate production in melanoma cells by α‐melanocyte‐stimulating hormone

Tyagi

Azrad

Degani

et al. 1998

European Journal of Biochemistry

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In a 31 P-NMR spectroscopic study of cultured M2R mouse melanoma cells, we previously demonstrated the acute stimulation of three peaks in the phosphomonoester region of the spectrum by [Ahx 4 , DPhe 7 ]A-melanotropin (concomitant with an increase in cellular adenosine 3′,5′-phosphate (cAMP) and a decrease in ATP [Degani, H., DeJordy, J. O. & Salomon, Y. (1991) Proc. Natl Acad. Sci. USA 88, 1506Ϫ 1510. Chemical identification of these metabolites was performed in this study using 32 P metabolic labeling and polyethyleneimine-cellulose thin layer chromatography in combination with 31 P-NMR and 13C-NMR spectroscopic methods. Two of the stimulated signals were identified as P 1 and P 6 of fructose 1,6-bisphosphate (FruP 2 ) and their mode of regulation by A-melanotropin was examined. The FruP 2 response to A-melanotropin coincided in time and dose with a rise in cAMP and a decrease in levels of ATP, while elevation of cAMP by forskolin alone did not increase FruP 2. The stimulatory effect of A-melanotropin was not associated with a change in the overall rate of glycolysis, suggesting that FruP 2 levels were not rate limiting in this process. The data suggest the presence of a previously unknown response of M2R melanoma cells to A-melanotropin, which coincides in time with enhanced cAMP accumulation but is not mediated by cAMP and may relate to the control of FruP 2 in a non glycolytic context.Keywords : cAMP ; fructose 1,6-bisphosphate; melanoma ; A-melanotropin; NMR.A-melanotropin controls the synthesis of melanin in melanocytes by cAMP-mediated regulation of the levels and activity of tyrosinase, the rate-limiting enzyme in melanin synthesis [1Ϫ 4]. The cAMP response of M2R mouse melanoma cells to Amelanotropin has been previously studied in our laboratory [5Ϫ 7]. Stimulation of the cells by A-melanotropin and a low synergistic dose of forskolin, in the presence of isobutylmethylxanthine (iBuMeXan), resulted in an unusually high cAMP response; nearly 50% of the cellular [ 3 H]adenine was converted into [ 3 H]cAMP [5]. This phenomenon was further delineated by 31 P-NMR spectroscopy of cultured M2R cells, which showed that A-melanotropin stimulation under these conditions indeed elevated cellular cAMP to sub-millimolar concentrations coinciCorrespondence to Y. Salomon,

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Simultaneous extraction of cellular lipids and water‐soluble metabolites: Evaluation by NMR spectroscopy

Cited by 131 publications

References 21 publications

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis

An optimized procedure for metabonomic analysis of rat liver tissue using gas chromatography/time-of-flight mass spectrometry

Stimulation of fructose 1,6‐bisphosphate production in melanoma cells by α‐melanocyte‐stimulating hormone

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