High-throughput lipid extraction for the analysis of human brain lipid
Instrument: Precellys® 24 Dual
Sample: Human occipital cortex
Protocol:
- Precellys kits: CK14_0.5mL (03961-1-203).
- Samples: 10mg pulverized brain aliquot (human occipital cortex) weighted directly into the 0.5mL tube.
- Buffer: 300μL ice-cold methanol containing internal standards.
- Run the Precellys 24 at 6000 rpm, 2x30 sec.
- Lipids were extracted using MTBE or chloroform and analyzed using electrospray ionization mass spectrometry (ESI-MS; for phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, ceramide and sphingomyelin) and sterol species analyzed using gas chromatography (GC-MS).
Results:
No differences in lipid species composition were evident when the protocols were compared. From these studies we conclude that the high-throughput Bead-MTBE protocol is equivalent to the traditional Glass-Chloroform protocol (Folch) for lipid extraction and quantification of glycerophospholipid, sphingolipid and sterol species in human brain tissue.
This high-throughput Bead-MTBE protocol improves upon traditional lipid extraction methods as it is safer (less carcinogenic/toxic) and much more efficient. The Bead-MTBE protocol is approximately four times quicker than Glass-Chloroform in the homogenization of 24 samples (i.e.1 vs. 4 h), with the additional benefit being that tissue aliquots can be weighted directly into the Precellys tubes prior to homogenization (thus reducing double-handling times). The lower density of MTBE further enhances the lipid extraction procedure (by dissolving lipids in the upper phase) and is also better for the potential incorporation of robotics to further streamline lipidomic studies.
