Liquid Extraction: BUME
KeywordsLipid Class Liquid Extraction Total Lipid Extraction Folch Method Molecular Species Composition
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.
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.
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.
The BUME Method for Tissues
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.
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.
- 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