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Simultaneous extraction of cellular lipids and water‐soluble metabolites: Evaluation by NMR spectroscopy
Author(s) -
Tyagi Rakesh Kumar,
Azrad Anat,
Degani Hadassa,
Salomon Yoram
Publication year - 1996
Publication title -
magnetic resonance in medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.696
H-Index - 225
eISSN - 1522-2594
pISSN - 0740-3194
DOI - 10.1002/mrm.1910350210
Subject(s) - chloroform , chemistry , perchloric acid , extraction (chemistry) , chromatography , nuclear magnetic resonance spectroscopy , methanol , proton nmr , metabolite , solid phase extraction , biochemistry , organic chemistry
Abstract A method for simultaneous extraction of lipids and water‐soluble metabolites from a single cell sample was developed and optimized for NMR spectroscopy. Intermediary metabolites in cultured M2R mouse melanoma cells and changes therein in response to challenge with melanotropin were studied by 31 P and 13 C NMR. Cells were extracted with methanol, chloroform, and water (1:1:1, v/v/v). The contents of the chloroform and methanol‐water phases were separated and quantitatively recovered. The contents of the upper and lower phases compared well with the homologous fractions obtained by perchloric acid and Folch's lipid extraction methods. The pH of the extracts remained within the physiologic range, eliminating potential deleterious effect on cellular metabolites. The water phase contained minimal amounts of salts, making these extracts amenable to subsequent analytical procedures. Obtaining lipid‐ and water‐soluble metabolites from the same sample enables characterization of metabolic pathways that bridge the two cellular components in a quantitative manner.