A comparison of cell and tissue extraction techniques using high-resolution 1H-NMR spectroscopy.
Analysis of brain metabolites by a wide range of analytical techniques is typically achieved using biochemical extraction methodologies that require either two separate samples or two separate extraction steps to prepare both aqueous and organic metabolite fractions. However there are a number of brain pathologies in which both aqueous metabolite and lipid changes occur so that a simultaneous extraction of both fractions would be valuable. The methanol-chloroform (M/C) technique enables extraction of both aqueous metabolites and lipids simultaneously. It is already well established for lipid extraction of cells and tissue but its efficiency and reproducibility for extraction of aqueous metabolites is unknown. Therefore, we compared the aqueous metabolite yield and the reproducibility of the M/C method to the commonly used perchloric acid (PCA) method, using 1H-NMR spectroscopy of adult rat brain and purified rat astrocyte culture extracts. The results indicate that M/C is a superior technique for aqueous metabolite extraction from both brain tissue and cells when compared to the PCA method. The M/C extraction technique enables the simultaneous extraction of both lipids and aqueous metabolites from a single sample using small solvent-volumes, making it well suited for NMR investigations of both tissues and cells.
Le Belle
J E
JE
Unit of Biophysics, Institute of Child Health, UCL Medical School, London WC1N 1EH, UK. jl289@cam.ac.uk
Harris
N G
NG
Williams
S R
SR
Bhakoo
K K
KK
D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002478:Cells, Cultured; D002725:Chloroform; D008055:Lipids / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D000432:Methanol; D009419:Nerve Tissue Proteins / Q000032:analysis; D010472:Perchlorates; D010713:Phosphatidylcholines / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D014867:Water
D008055:Lipids; D009419:Nerve Tissue Proteins; D010472:Perchlorates; D010713:Phosphatidylcholines; D014867:Water; D002725:Chloroform; D000432:Methanol