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LC/MS lipid profiling from human serum: a new method for global lipid extraction

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Abstract

Over the last decade, technological advances have improved the sensitivity and selectivity of LC/MS analyzers, providing very efficient tools for lipidomics research. In particular, the nine lipid classes that constitute 99 % of the human serum lipidome (sterols, cholesteryl esters, phosphocholines, phosphoethanolamines, sphingomyelins, triacylglycerols, fatty acids, lysophosphocholines, and diacylglycerols) can be easily detected. However, until today there has not been a unique technique for sample preparation that provides a satisfactory recovery for all of these nine classes together. In this work, we have developed and validated a new one-phase extraction (OPE) method that overcomes this limitation. This method was also compared with the gold standard lipid extraction methods such as Folch, Bligh & Dyer, and recently developed methods with methanol and methyl-tert-butyl ether. Results demonstrate that the mixture of methanol/chloroform/MTBE (MMC) provides a recovery very close to 100 % for all nine lipid classes of the human serum investigated. For this extraction method, 100 μL of human serum is incubated with 2 mL of the solvents mixture, then vortexed and centrifuged. For its simplicity of execution, rapidity, reproducibility, and the reduced volume of sample required, this method opens the door to the use of human serum lipid profiling for large-scale applications in scientific research and clinical trials.

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Abbreviations

BD:

Bligh & Dyer

FO:

Folch

MA:

Methanol:acetone method

MC:

Methanol:chloroform method

ME:

Methanol method

MIPA:

Methanol:isopropanol method

MMC:

Methanol:methyl-tert-butyl ether:chloroform method

OPE:

One-phase extraction

SD:

Standard deviation

TPE:

Two-phase extraction

References

  1. Wolf C, Quinn PJ (2008) Lipidomics: practical aspects and applications. Prog Lipid Res 47:15–36

    Article  CAS  Google Scholar 

  2. Wenk MR (2005) The emerging field of lipidomics. Nat Rev Drug Discov 4:594–610

    Article  CAS  Google Scholar 

  3. Feng L, Prestwich GD (2005) Functional lipidomics. Dekker-CRC, New York 1–329

  4. Yang K, Han X (2011) Accurate quantification of lipid species by electrospray ionization mass spectrometry meets a key challenge in lipidomics. Metabolites 1:21–40

    Article  CAS  Google Scholar 

  5. Han X, Holtzman DM, McKeel DW Jr, 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  CAS  Google Scholar 

  6. Mills GB, Moolenaar WH (2003) The emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer 3:582–591

    Article  CAS  Google Scholar 

  7. Zhao Z, Xiao Y, Elson P, Tan H, Plummer SJ, Berk M, Aung PP, Lavery IC, Achkar JP, Li L, Casey G, Xu Y (2007) Plasma lysophosphatidylcholine levels: potential biomarkers for colorectal cancer. J Clin Oncol 25:2696–2701

    Article  CAS  Google Scholar 

  8. Postle AD, Wilton DC, Hunt AN, Attard GS (2007) Probing phospholipid dynamics by electrospray ionization mass spectrometry. Prog Lipid Res 46:200–224

    Article  CAS  Google Scholar 

  9. 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  Google Scholar 

  10. Fernandez C, Sandin M, Sampaio JL, Almgren P, Narkiewicz K, Hoffman M, Hedner T, Wahlstrand B, Simons K, Shevchenko A, James P, Melander O (2013) Plasma lipid composition and risk of developing cardiovascular disease. PLoS ONE 8:e71846

    Article  CAS  Google Scholar 

  11. Rossouw JE (1994) The effects of lowering serum cholesterol on coronary heart disease risk. Med Clin N Am 78:181–195

    CAS  Google Scholar 

  12. Han X, Gross RW (2003) Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J Lipid Res 44:1071–1079

    Article  CAS  Google Scholar 

  13. Breuer HW (2001) Hypertriglyceridemia: a review of clinical relevance and treatment options: focus on cerivastatin. Curr Med Res Opin 17:60–73

    Article  CAS  Google Scholar 

  14. Pulfer M, Murphy RC (2003) Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev 22:332–364

    Article  CAS  Google Scholar 

  15. 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

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Retra K, Bleijerveld OB, van Gestel RA, Tielens AGM, van Hellemond JJ, Brouwers JF (2008) A simple and universal method for the separation and identification of phospholipid molecular species. Rapid Commun Mass Spectrom 22:1853–1862

    Article  CAS  Google Scholar 

  18. Reis A, Rudnitskaya A, Blackburn GJ, Fauzi NM, Pitt AR, Spickett CM (2013) A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL. J Lipid Res 54:1812–1824

    Article  CAS  Google Scholar 

  19. 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  CAS  Google Scholar 

  20. Löfgren L, Ståhlman M, Forsberg GB, Saarinen S, Nilsson R, Hansson GI (2012) The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma. J Lipid Res 53:1690–1700

    Article  Google Scholar 

  21. Quehenberger O, Armando AM, Brown AH, Milne SB, Myers DS, Merrill AH, Bandyopadhyay S, Jones KN, Kelly S, Shaner RL, Sullards CM, Wang E, Murphy RC, Barkley RM, Leiker TJ, Raetz CRH, Guan Z, Laird GM, Six DA, Russell DW, McDonald JG, Subramaniam S, Fahy E, Dennis EA (2010) Lipidomics reveals a remarkable diversity of lipids in human plasma. J Lipid Res 51:3299–3305

    Article  CAS  Google Scholar 

  22. Zhao Z, Xu Y (2010) An extremely simple method for extraction of lysophospholipids and phospholipids from blood samples. J Lipid Res 51:652–659

    Article  CAS  Google Scholar 

  23. Thompson M, Ellison SLR, Wood R (2002) Harmonized guidelines for single laboratory validation of methods of analysis. (IUPAC Technical Report). Pure Appl Chem 74:835–855

    Article  CAS  Google Scholar 

  24. Bird SS, Marur VR, Sniatynski MJ, Greenberg HK, Kristal BS (2011) Serum lipidomics profiling using LC-MS and high-energy collisional dissociation fragmentation: focus on triglyceride detection and characterization. Anal Chem 83:6648–6657

    Article  CAS  Google Scholar 

  25. Sud M, Fahy E, Cotter D, Brown A, Dennis EA, Glass CK, Merrill AH, Murphy RC, Raetz CR, Russell DW, Subramaniam S (2007) LMSD: LIPID MAPS structure database. Nucleic Acids Res 35:D527–D532

    Article  CAS  Google Scholar 

  26. Taguchi R, Houjou T, Nakanishi H, Yamazaki T, Ishida M, Imagawa M, Shimizu T (2005) Focused lipidomics by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 823:26–36

    Article  CAS  Google Scholar 

  27. Camera E, Ludovici M, Galante M, Sinagra J, Picardo M (2010) Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry. J Lipid Res 51:3377–3388

    Article  CAS  Google Scholar 

  28. Cruciani G, Crivori P, Carrupt PA, Testa B (2000) Molecular fields in quantitative structure-permeation relationships: the VolSurf approach. J Mol Struct THEOCHEM 503:17–30

    Article  CAS  Google Scholar 

  29. Milne S, Ivanova P, Forrester J, Brown HA (2006) Lipidomics: an analysis of cellular lipids by ESI-MS. Methods 39:92–103

    Article  CAS  Google Scholar 

  30. Liebisch G, Lieser B, Rathenberg J, Drobnik W, Schmitz G (2004) High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm. Biochim Biophys Acta 1686:108–117

    Article  CAS  Google Scholar 

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Acknowledgments

A.D.V. thanks Regione Umbria, this research project was partly supported by 2007–2013 ESF “Competitiveness and Employment objective” Umbrian Regional Operational  Programme (ROP), Avviso pubblico aiuti individuali per la realizzazione di progetti di ricerca, project “Correlazione tra impronta lipidica del sangue e incidenza delle malattie neurodegenerative. Sviluppo di un metodo non invasivo e poco costoso per abbattere i costi della sanità pubblica regionale”.

The authors thank the Centro Ospedaliero di Riabilitazione Intensiva (C.O.R.I.), Passignano sul Trasimeno (Perugia, Italy), for providing serum samples, and Molecular Discovery Ltd. (UK) for free software and financial support.

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Authors and Affiliations

  1. Department of Chemistry, Biology, and Biotechnologies, University of Perugia, Via Elce di Sotto 8, 06123, Perugia, Italy

    Roberto Maria Pellegrino, Alessandra Di Veroli, Aurora Valeri, Laura Goracci & Gabriele Cruciani

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  1. Roberto Maria Pellegrino
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  2. Alessandra Di Veroli
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  3. Aurora Valeri
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  4. Laura Goracci
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  5. Gabriele Cruciani
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Corresponding author

Correspondence to Gabriele Cruciani.

Additional information

Roberto Maria Pellegrino and Alessandra Di Veroli contributed equally to this work.

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Pellegrino, R.M., Di Veroli, A., Valeri, A. et al. LC/MS lipid profiling from human serum: a new method for global lipid extraction. Anal Bioanal Chem 406, 7937–7948 (2014). https://doi.org/10.1007/s00216-014-8255-0

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  • DOI: https://doi.org/10.1007/s00216-014-8255-0

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