Simultaneous extraction of cellular lipids and water-soluble metabolites: evaluation by NMR spectroscopy.
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 31P and 13C 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.
Tyagi
R K
RK
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Azrad
A
A
Degani
H
H
Salomon
Y
Y
D000818:Animals; D009682:Magnetic Resonance Spectroscopy; D008546:Melanoma, Experimental / Q000737:chemistry*; D051379:Mice; D010743:Phospholipids / Q000032:analysis*; D012995:Solubility; D014407:Tumor Cells, Cultured / Q000737:chemistry
D010743:Phospholipids