Encyclopedia of Lipidomics

Living Edition
| Editors: Markus R. Wenk

Liquid Extraction: BUME

  • Lars LöfgrenEmail author
Living reference work entry
DOI: https://doi.org/10.1007/978-94-007-7864-1_98-1

Keywords

Lipid Class Liquid Extraction Total Lipid Extraction Folch Method Molecular Species Composition 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Synonyms

Definitions

BUME refers to the chloroform-free, total lipid extraction from biofluids and tissue samples using a mixture of 1-butanol and methanol (BUME mixture) followed by a two-phase separation step for purification of the upper lipid extract. The method was developed to simplify and speed-up lipid extractions and facilitate automation while maintaining lipid results comparable to the Folch method.

Introduction

An important part of the workflow of lipid analysis is the extraction procedure. This step extracts and purifies the lipids from the biological sample and removes substances such as proteins, carbohydrates, and other polar metabolites that may interfere with lipid quantification and impair the long-term performance of the analytical system. While there has been an impressive development of automated and fast analytical methods, the extraction procedure is still often performed manually with traditional methods. Two of the most commonly used methods over the years (Folch et al. 1957; Bligh and Dyer 1959) are based on a mixture of chloroform and methanol, which makes these methods highly efficient in extracting lipids with a wide range of polarity. However, there are several drawbacks with these methods in addition to the potential health risks from carcinogenic chloroform exposure. Lipids need to be retrieved from the lower lipid fraction after the two-phase separation. During aspiration of the lipid extract, the upper aqueous phase and the insoluble fraction that sits between the phases need to be penetrated. This may lead to contamination of the lipid extract. The risk of contamination and plugging of the tips used for lipid extract transfer from the lower phase increase when using automated and unsupervised protocols. Moreover, spontaneous phase separation is slow or inadequate and thus the need for a centrifugation step is difficult to overcome and the solvent-to-sample ratio requirement is high. Altogether, these features of the classical Folch method limit the opportunity to simplify and automate sample processing using standard 96-well robots.

To overcome the drawbacks of classical chloroform methods, development work was performed with the aim to:
  • Develop a new rapid and automated chloroform-free method for total lipid extraction from biofluids and tissue samples

  • Replace the need for the laborious chloroform-based method

  • Deliver lipid data fully compatible with data obtained from the Folch method – in less time using less resources

The BUME Method for Biofluids

The BUME method is the result of development work based on the automation perspective rather than replacing chloroform in current manual protocols. Based on the technical capabilities of a standard 96-format pipetting robot (i.e., Bravo, Agilent) a pilot protocol for 100 % automation was defined based on repeated solvent extraction steps, multiple mixing and transfer steps, built-in time for spontaneous phase separation without the need for centrifugation steps, and small volumes of solvents and buffers keeping total volumes within the range of the 1.2 ml 96-well format.

Nonchloroform solvents and solvent mixtures were then screened for high lipid recoveries, spontaneous phase separation, and full compatibility with automation. The result was the BUME method for biofluids (Löfgren et al. 2012) as out-lined in Fig. 1.
Fig. 1

Workflow for the BUME method for biofluids (Full details are given in Löfgren et al. (2012))

An extensive evaluation of the developed protocol was performed covering lipid recoveries and fatty acid species composition in a number of lipid classes from polar and hydrophobic lipids using one single robot protocol for the sample volume range of 10–100 μl of biofluid, with a fat content up to 10 % (Löfgren et al. 2012). Typical results for lipid recoveries from 25 μl of serum are given in Fig. 2.
Fig. 2

Typical lipid recoveries from blood serum using the automated BUME method for biofluids (Full details on the method performance in terms of lipid class recoveries and molecular species compositions are given in Löfgren et al. (2012))

The BUME Method for Tissues

The BUME method for biofluids was recently extended to and validated for tissue samples (Löfgren et al. 2016). Snap-frozen tissue (15–150 mg) is collected and stored in 2 ml homogenization tubes. 500 μl of BUME mixture (1-butanol:methanol 3:1) is added and automated homogenization of up to 24 frozen samples at a time in less than 60 s is performed, followed by a 5-min single-phase extraction. After the addition of 500 μl of heptane:ethyl acetate (3:1) and 500 μl of 1 % acetic acid a 5-min two-phase extraction is performed. Lipids are recovered from the upper phase by automated liquid handling using a standard 96-tip robot. A second two-phase extraction is performed using 500 μl of heptane:ethyl acetate (3:1). The workflow for the BUME method for tissues is illustrated in Fig. 3.
Fig. 3

Workflow for the BUME method for tissues (Full details are given in Löfgren et al. (2016))

Besides the benefit of being able to perform the complete process of sample collection, storage, homogenization, and extraction in the same small sample collection tube, the chosen solvent system results in a lipid-enriched upper phase. This enables the use of a pipetting robot for automatic transfer of lipid extract without the risk of contamination by the aqueous phase and insoluble fraction. The automated transfer also minimizes the risk of errors and relieves strain on neck and shoulders.

By using the developed method, 96 tissue samples can be extracted in 4 h moving sample preparation into the high-throughput workflows, which are fundamental in the field of lipidomics.

Validation of the method in terms of lipid class recoveries and molecular species compositions showed that the extraction recoveries for the investigated lipids, which included cholesterol, cholesteryl ester, triglyceride, diglyceride, phospholipids (PC, PE, PS, PA, PG, LPC), sphingomyelin, ceramide, dihydroceramides, and glucosylceramides were similar or better than for the Folch method. Typical lipid recoveries are illustrated in Fig. 4. Furthermore molecular species compositions were near identical for BUME and Folch methods (Löfgren et al. 2016).
Fig. 4

Typical lipid recoveries from liver tissue using the semi-automated BUME method for tissues (Full details on the method performance in terms of lipid class recoveries and molecular species compositions are given in Löfgren et al. (2016))

Conclusions

In conclusion, the new BUME methods for biofluids and tissue samples presented here are superior to the old, laborious, and toxic but still commonly used chloroform-based extraction methods. The use of the BUME mixture as the single-phase extraction solvent enables us to collect the sample at the site of the experiment, rapidly snap-freeze the sample, and perform both the automated homogenization and extraction in a single 2-ml homogenization tube with the tissue sample frozen at all time to prevent biochemical degradation of lipids until total lipid extraction is completed. Furthermore, the method shows high recovery for a wide range of lipids, and lipid species profiles are comparable with the gold standard Folch method.

Cross-References

References

  1. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37(8):911–7.CrossRefPubMedGoogle Scholar
  2. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226(1):497–509.PubMedGoogle Scholar
  3. Lofgren L, Stahlman M, Forsberg GB, Saarinen S, Nilsson R, Hansson GI. The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma. J Lipid Res. 2012;53(8):1690–700.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Lofgren L, Forsberg GB, Stahlman M. The BUME method: a new rapid and simple chloroform-free method for total lipid extraction of animal tissue. Sci Rep. 2016;6: article number 27688. doi:10.1038/srep27688.Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Department of Translational SciencesCardiovascular and Metabolic Diseases, AstraZeneca R&DGothenburgSweden