Bligh—Dyer extraction modified

While there is no doubt that free lipids can facilitate the formation of hemozoin in model systems, their potential biological role must be placed in the appropriate context. The vast majority of these lipids are involved in cellular structures (organism membrane, organelles, etc.), not freely soluble in the cytoplasm. The methods of extraction modified from Bligh and Dyer [35] by Cohen [36] were designed to extract all of the available... 

A protocol on lipid extraction modified from the pioneering work of Folch et al. (1957) is included in this unit for convenience (see Support Protocol). The authors laboratory uses this protocol for virtually all kinds of samples, and have found it to be effective with reproducibly high recoveries. Another very popular lipid extraction technique is the Bligh and Dyer method, which uses a 1 1 (v/v) mixture of chloroform/methanol (Bligh and Dyer, 1959). For more information on lipid extraction, the discussion by Nelson (1991) on solvent selection and extraction efficiency is recommended. 

For the microbial assay, lipids were extracted using a single phase chloroform/methanol/water extraction after Bligh Dyer (1959) as modified by White et al. (1979) to include a phosphate buffer. The total lipid was fractionated into neutral lipid, glyco-lipid and polar lipid fractions by silicic acid column chromatography (SACC) (Parker et al., 1982 White et al., 1983) using the three solvents of increasing polarity (chloroform, acetone and methanol). The polar lipid fraction (PLFA) was... 

C.1.1 Modified Bligh and Dyers Method for Phospholipid Extraction... 

Lipid Extracts. Lipids were extracted from the hairless mouse stratum corneum using a modified method based on methods developed by Bligh and Dyer (37) and Elias and coworkers (17.20.21). 

Figure 2.4 MDMS of a chloroform extract of mouse retina prepared by a modified Bligh and Dyer method [25]. Direct-infusion ESI spectra were acquired directly from the diluted lipid extract (total Upid 50 pmol/pL) using a TSQ Quantum Ultra tandem quadrupole instrument. The first (x) dimension is represented in the top panel (negative-ion ESI-MS). The second dimension (y) is represented by successive neutral-loss (NL) and precmsor-ion (PI) scans. All traces were displayed after normalization to the base peak in each spectrum. Internal standard (IS) m n, acyl chain containing m carbons and n double bonds. (Modified with permission from Han, X. et al., 2005, Shotgun Lipidomics of Phosphoethanolamine-Containing Lipids in Biological Samples after One-Step in Situ Derivatization, /. Upid Res. 46 1548-60.)...

Figure 3.2 Mass spectral comparison of PC molecular species present in mouse heart lipid extracts acquired by either ESI or MALDI. Extracts of mouse myocardium were prepared by a modified Bligh and Dyer procedure and analyzed by ESI-MS in the presence of LiOH (a) or MALDI-MS utilizing 9-aminoacridine as matrix dissolved in isopropanol/acetonitrile (60/40, v/v) (b). IS denotes internal standard. Sun et al. [108], Reproduced with permission of the American Chemical Society.

Figure 4.9 Representative ESI-MS analysis of lipid classes resolved by intrasource separation. Lipid extracts from mouse liver samples were prepared by using a modified procedure of Bligh and Dyer [1]. MS analysis was performed with a TSQ Vantage triple-quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with an automated nanospray apparatus (i.e., TriVersa, Advion Bioscience Ltd., Ithaca, NY) and Xcalibur system software. Mass spectra were acqnired directly from the diluted hpid extract in the negative-ion mode (a), after addition of 50 nmol LiOH/mg of protein in the diluted lipid extract and analyzed in the negative-ion mode (h), or the identical hpid solution to that in (b) in the positive-ion mode (c). IS denotes internal standard PC, PE, PG, PI, PS, TAG, NEFA, and CL stand for phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidyUnositoL phosphatidylserine, triacylglycerol, nonesterified fatty acid, and doubly charged cardioUpin, respectively.

Lipid extraction is one of the key steps for the successful analyses of cellular lipidomes by ESI-MS, in general, and by approaches using direct infusion, in particular. Traditionally, lipid samples from biological sources are extracted using a mixture of chloroform and methanol based on the Folch method [17] or the modified method of Bligh and Dyer [18] or other solvent combinations [19, 20]. 

A Modified Bligh and Dyer Procedure for Lipidomics To succeed in lipidomic analysis with ESI-MS using direct infusion or with MALDI-MS, a key point is to have a lipid extract carrying only a minimal amount of inorganic salts. Although solid-phase extraction cartridges can be used to eliminate the salt contaminants, a careful solvent wash is recommended as routinely used in the author s laboratory [36, 38]. 

Figure 20.5. The effects of sanqile preparation on adduct ions of phosphatidylcholine mixture. The hpid mixture comprised of 2pmol/ jL each of 14 1-14 1 and 18 1-18 1 GPCho molecular species was extracted against 50 mM LiCl, (A) 50 mM ammonium acetate, (B) 50 mM NH4CI, (C) and tenfold diluted PBS (D) by a modified Bligh and Dyer method. Positive-ion ESI mass spectra were acquired using a TSQ Quantum Ultra ESI mass spectrometer (Thermo, San Jose, CA) at a flow rate of 4 pL/min.

Figure 20.9. Two-dimensional mass spectrometric analysis of GPIns molecular species in a lipid extract of mouse myocardium. The lipid extract of mouse myocardium was prepared by a modified Bligh and Dyer procedure as previously described. Each MS or MS/MS trace of the 2D ESI mass spectrum was acquired by sequentially programmed, customized scans operating under Xcalibur software. For negative-ion tandem mass spectrometry in the precursor-ion (PI) mode, the first quadrupole was scanned in the selected mass range and the second quadrupole was used as a coUision cell while the third quadrupole was fixed to monitor the ion of interest (i.e., either inositol phosphate, glycerophosphate, or a fatty acyl carboxylate fragmented from GPIns molecular species). All mass spectral traces were displayed after being normalized to the base peak in each individual trace.

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