The BUME method: a novel automated chloroform-free 96-well total lipid extraction method for blood plasma

Abstract

Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10–100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources. Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10–100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources. butanol and methanol cholesteryl ester ceramide diacylglycerol diisopropyl ether free cholesterol glucosylceramide lactosylceramide lyso-PA lysophosphatidylcholine methyl-tert-butyl ether neutral loss phosphatidic acid phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol precursor ion scanning phosphatidylserine sphingomyelin triacylglycerol Lipids belong to a class of molecules with an immense structural diversity and function (1Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill Jr, A.H. Murphy R.C. Raetz C.R. Russell D.W. Seyama Y. Shaw W. et al.A comprehensive classification system for lipids.J. Lipid Res. 2005; 46: 839-861Abstract Full Text Full Text PDF PubMed Scopus (1127) Google Scholar). In addition to their important role in conserving membrane integrity, they participate in many intracellular processes, such as signal transduction, apoptosis, and membrane trafficking. As a consequence, disturbances in lipid metabolism have been implicated in several diverse diseases, such as type 2 diabetes, Alzheimer disease, (2Hartmann T. Kuchenbecker J. Grimm M.O. Alzheimer's disease: