Lipids

, Volume 48, Issue 3, pp 307–318 | Cite as

An Improved High-Throughput Lipid Extraction Method for the Analysis of Human Brain Lipids

  • Sarah K. Abbott
  • Andrew M. Jenner
  • Todd W. Mitchell
  • Simon H. J. Brown
  • Glenda M. Halliday
  • Brett Garner
Methods

Abstract

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.

Keywords

Lipidomics Human-brain Ceramide Sphingomyelin Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Sterol 

Abbreviations

BHT

Butylated hydroxytoluene

BSTFA

(N,O-bis(trimethylsilyl)trifluoroacetamide

CE

Collision energy

Cer

Ceramide

CerPCho

Sphingomyelin

ChoGpl

Choline glycerophospholipid

CV

Coefficient of variation

CXP

Collision cell exit potential

DP

Declustering potential

EP

Entrance potential

ESI–MS

Electrospray ionization mass spectrometry

EtnGpl

Ethanolamine glycerophospholipid

GC-MS

Gas chromatography mass spectrometry

MTBE

Methyl-tert-butyl ether

MRM

Multiple reaction monitoring

NL

Neutral loss

PI

Precursor ion

PtdSer

Phosphatidylserine

SGalCer

Sulfatide

TMCS

Trimethylchlorosilane

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Copyright information

© AOCS 2013

Authors and Affiliations

  • Sarah K. Abbott
    • 1
    • 2
  • Andrew M. Jenner
    • 1
    • 2
  • Todd W. Mitchell
    • 1
    • 3
  • Simon H. J. Brown
    • 1
    • 3
  • Glenda M. Halliday
    • 4
  • Brett Garner
    • 1
    • 2
  1. 1.Illawarra Health and Medical Research InstituteUniversity of WollongongWollongongAustralia
  2. 2.School of Biological SciencesUniversity of WollongongWollongongAustralia
  3. 3.School of Health SciencesUniversity of WollongongWollongongAustralia
  4. 4.Neuroscience Research Australia and the University of New South WalesSydneyAustralia

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