An improved high-throughput lipid extraction method for the analysis of human brain lipids.
We have developed a protocol suitable for high-throughput lipidomic analysis of human brain samples. The traditional Folch extraction (using chloroform and glass-glass homogenization) was compared to a high-throughput method combining methyl-tert-butyl ether (MTBE) extraction with mechanical homogenization utilizing ceramic beads. This high-throughput method significantly reduced sample handling time and increased efficiency compared to glass-glass homogenizing. Furthermore, replacing chloroform with MTBE is safer (less carcinogenic/toxic), with lipids dissolving in the upper phase, allowing for easier pipetting and the potential for automation (i.e., robotics). Both methods were applied to the analysis of human occipital cortex. Lipid species (including ceramides, sphingomyelins, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserines) were analyzed via electrospray ionization mass spectrometry and sterol species were analyzed using gas chromatography mass spectrometry. No differences in lipid species composition were evident when the lipid extraction protocols were compared, indicating that MTBE extraction with mechanical bead homogenization provides an improved method for the lipidomic profiling of human brain tissue.
Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia. sarahmac@uow.edu.au
Sarah K
SK
Abbott
Andrew M
AM
Jenner
Todd W
TW
Mitchell
Simon H J
SH
Brown
Glenda M
GM
Halliday
Brett
B
Garner
D000369:Aged, 80 and over; D001923:Brain Chemistry; D005591:Chemical Fractionation / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry / Q000191:economics / Q000379:methods; D057166:High-Throughput Screening Assays / Q000191:economics / Q000379:methods*; D006801:Humans; D008055:Lipids / Q000032:analysis / Q000302:isolation & purification*; D008297:Male; D008738:Methyl Ethers / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000191:economics / Q000379:methods; D013997:Time Factors
D008055:Lipids; D008738:Methyl Ethers; C043243:methyl tert-butyl ether