Skip to main content
SpringerLink
Log in

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

  • Methods
  • Published:
Lipids

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

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

References

  1. Ledesma MD, Martin MG, Dotti CG (2012) Lipid changes in the aged brain: effect on synaptic function and neuronal survival. Prog Lipid Res 51:23–35

    Article  PubMed  CAS  Google Scholar 

  2. Conde C, Martinez M, Ballabriga A (1974) Some chemical aspects of human brain development. I. Neutral glycosphingolipids, sulfatides, and sphingomyelin. Pediatr Res 8:89–92

    Article  PubMed  CAS  Google Scholar 

  3. Cheng D, Jenner AM, Shui G, Cheong WF, Mitchell TW, Nealon JR, Kim WS, McCann H, Wenk MR, Halliday GM, Garner B (2011) Lipid pathway alterations in Parkinson’s disease primary visual cortex. PLoS ONE 6:e17299

    Article  PubMed  CAS  Google Scholar 

  4. Fabelo N, Martín V, Santpere G, Marín R, Torrent L, Ferrer I, Díaz M (2011) Severe alterations in lipid composition of frontal cortex lipid rafts from Parkinson’s disease and incidental Parkinson’s disease. Mol Med 17:1107–1118

    Article  PubMed  CAS  Google Scholar 

  5. Hejazi L, Wong JWH, Cheng D, Proschogo N, Ebrahimi D, Garner B, Don AS (2011) Mass and relative elution time profiling: two-dimensional analysis of sphingolipids in Alzheimer’s disease brains. Biochem J 438:165–175

    Article  PubMed  CAS  Google Scholar 

  6. Garner B (2010) Lipids and Alzheimer’s disease. Biochim Biophys Acta Mol Cell Biol Lipids 1801:747–749

    Article  CAS  Google Scholar 

  7. Karasinska JM, Hayden MR (2011) Cholesterol metabolism in Huntington disease. Nat Rev Neurol 7:561–572

    Article  PubMed  CAS  Google Scholar 

  8. Schwarz E, Prabakaran S, Whitfield P, Major H, Leweke FM, Koethe D, McKenna P, Bahn S (2008) High throughput lipidomic profiling of schizophrenia and bipolar disorder brain tissue reveals alterations of free fatty acids, phosphatidylcholines, and ceramides. J Proteome Res 7:4266–4277

    Article  PubMed  CAS  Google Scholar 

  9. Eisenstein M (2011) Genetics: finding risk factors. Nature 475:S20–S22

    Article  PubMed  CAS  Google Scholar 

  10. Bertram L, Tanzi RE (2012) The genetics of Alzheimer’s disease. Prog Mol Biol Transl Sci 107:79–100

    Article  PubMed  CAS  Google Scholar 

  11. Fonteh AN, Harrington RJ, Huhmer AF, Biringer RG, Riggins JN, Harrington MG (2006) Identification of disease markers in human cerebrospinal fluid using lipidomic and proteomic methods. Dis Markers 22:39–64

    PubMed  CAS  Google Scholar 

  12. Blanksby SJ, Mitchell TW (2010) Advances in mass spectrometry for lipidomics. Annu Rev Anal Chem 3:433–465

    Article  CAS  Google Scholar 

  13. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509

    PubMed  CAS  Google Scholar 

  14. Cequier-Sánchez E, Rodríguez C, Ravelo AG, Zárate R (2008) Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. J Agric Food Chem 56:4297–4303

    Article  PubMed  Google Scholar 

  15. Carlson LA (1985) Extraction of lipids from human whole serum and lipoproteins and from rat liver tissue with methylene chloride-methanol: a comparison with extraction with chloroform-methanol. Clin Chim Acta 149:89–93

    Article  PubMed  CAS  Google Scholar 

  16. Fraser T, Tayler H, Love S (2008) Low-temperature improved-throughput method for analysis of brain fatty acids and assessment of their post-mortem stability. J Neurosci Methods 169:135–140

    Article  PubMed  CAS  Google Scholar 

  17. Fraser T, Tayler H, Love S (2010) Fatty acid composition of frontal, temporal and parietal neocortex in the normal human brain and in Alzheimer’s disease. Neurochem Res 35:503–513

    Article  PubMed  CAS  Google Scholar 

  18. Römisch-Margl W, Prehn C, Bogumil R, Röhring C, Suhre K, Adamski J (2012) Procedure for tissue sample preparation and metabolite extraction for high-throughput targeted metabolomics. Metabolomics 8:133–142

    Article  Google Scholar 

  19. Wu H, Southam AD, Hines A, Viant MR (2008) High-throughput tissue extraction protocol for NMR- and MS-based metabolomics. Anal Biochem 372:204–212

    Article  PubMed  CAS  Google Scholar 

  20. Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lipid Res 49:1137–1146

    Article  PubMed  CAS  Google Scholar 

  21. Kosicek M, Kirsch S, Bene R, Trkanjec Z, Titlic M, Bindila L, Peter-Katalinic J, Hecimovic S (2010) Nano-HPLC–MS analysis of phospholipids in cerebrospinal fluid of Alzheimer’s disease patients—a pilot study. Anal Bioanal Chem 398:2929–2937

    Article  PubMed  CAS  Google Scholar 

  22. Kosicek M, Zetterberg H, Andreasen N, Peter-Katalinic J, Hecimovic S (2012) Elevated cerebrospinal fluid sphingomyelin levels in prodromal Alzheimer’s disease. Neurosci Lett 516:302–305

    Article  PubMed  CAS  Google Scholar 

  23. Bligh EG, Dyer WJ (1959) A Rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917

    Article  PubMed  CAS  Google Scholar 

  24. Graessler J, Schwudke D, Schwarz PEH, Herzog R, Shevchenko A, Bornstein SR (2009) Top-down lipidomics reveals ether lipid deficiency in blood plasma of hypertensive patients. PLoS ONE 4:e6261

    Article  PubMed  Google Scholar 

  25. Ki Ichihara, Yoneda K, Takahashi A, Hoshino N, Matsuda M (2011) Improved methods for the fatty acid analysis of blood lipid classes. Lipids 46:297–306

    Article  Google Scholar 

  26. Schuhmann K, Almeida R, Baumert M, Herzog R, Bornstein SR, Shevchenko A (2012) Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes. J Mass Spectrom 47:96–104

    Article  PubMed  CAS  Google Scholar 

  27. Deeley JM, Mitchell TW, Wei X, Korth J, Nealon JR, Blanksby SJ, Truscott RJW (2008) Human lens lipids differ markedly from those of commonly used experimental animals. Biochim Biophys Acta Mol Cell Biol Lipids 1781:288–298

    Article  CAS  Google Scholar 

  28. Saville JT, Zhao Z, Willcox MDP, Blanksby SJ, Mitchell TW (2010) Detection and quantification of tear phospholipids and cholesterol in contact lens deposits: the effect of contact lens material and lens care solution. Invest Ophthalmol Visual Sci 51:2843–2851

    Article  Google Scholar 

  29. Ejsing CS, Duchoslav E, Sampaio J, Simons K, Bonner R, Thiele C, Ekroos K, Shevchenko A (2006) Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning. Anal Chem 78:6202–6214

    Article  PubMed  CAS  Google Scholar 

  30. Zemski Berry K, Murphy R (2004) Electrospray ionization tandem mass spectrometry of glycerophosphoethanolamine plasmalogen phospholipids. J Am Soc Mass Spectrometry 15:1499–1508

    Article  CAS  Google Scholar 

  31. Mitchell TW, Buffenstein R, Hulbert AJ (2007) Membrane phospholipid composition may contribute to exceptional longevity of the naked mole-rat (Heterocephalus glaber): a comparative study using shotgun lipidomics. Exp Gerontol 42:1053–1062

    Article  PubMed  CAS  Google Scholar 

  32. Ginsberg L, Rafique S, Xuereb JH, Rapoport SI, Gershfeld NL (1995) Disease and anatomic specificity of ethanolamine plasmalogen deficiency in Alzheimer’s disease brain. Brain Res 698:223–226

    Article  PubMed  CAS  Google Scholar 

  33. Han X, Holtzman DM, McKeel DW, Kelley J, Morris JC (2002) Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer’s disease: potential role in disease pathogenesis. J Neurochem 82:809–818

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Adult brain tissue was received from the Sydney Brain Bank at Neuroscience Research Australia which is supported by the National Health and Medical Research Council of Australia, the University of New South Wales and Neuroscience Research Australia. This research was supported by a project grant (APP1008307) from the Australian National Health and Medical Research Council (NHMRC) awarded to GMH and BG. TWM and BG are supported by Australian Research Council Future Fellowships (FT110100249 and FT0991986, respectively). GMH is supported by an NHMRC Principal Research Fellowship (630434).

Conflict of interest

All authors disclose that there are no conflicts of interest.

Author information

Authors and Affiliations

  1. Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, 2522, Australia

    Sarah K. Abbott, Andrew M. Jenner, Todd W. Mitchell, Simon H. J. Brown & Brett Garner

  2. School of Biological Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia

    Sarah K. Abbott, Andrew M. Jenner & Brett Garner

  3. School of Health Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia

    Todd W. Mitchell & Simon H. J. Brown

  4. Neuroscience Research Australia and the University of New South Wales, Sydney, NSW, 2031, Australia

    Glenda M. Halliday

Authors
  1. Sarah K. Abbott
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew M. Jenner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Todd W. Mitchell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Simon H. J. Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Glenda M. Halliday
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Brett Garner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sarah K. Abbott.

About this article

Cite this article

Abbott, S.K., Jenner, A.M., Mitchell, T.W. et al. An Improved High-Throughput Lipid Extraction Method for the Analysis of Human Brain Lipids. Lipids 48, 307–318 (2013). https://doi.org/10.1007/s11745-013-3760-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-013-3760-z

Keywords

Search

Navigation