Bligh and Dyer procedure

Incorporation of Radioactive Precursors into Lipids. The cultures of surface-adhering cells were exposed for 16 h to either 400 pCi of Hp/C- syo (final specific activity, 50 yCi/ ymol) or 10 pCi of / %/, galactose ( the rate of incorporation was linear during this period). After 16 hr. the radioactive medium was removed and the cultures were washed four times with 0.9 NaCl. The cells were removed from the surface with a rubber policeman and suspended in physiological saline. Lipids were extracted by Bligh and Dyer procedure (26) and analyzed for various lipids according to Neskovic, et al. (27)... 

Lipids are commonly characterized by their solubility properties, i.e., they can be extracted by the Folch or Bligh and Dyer procedures. The next stage in the analytical procedure for mixtures involves... 

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.

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 20.9. 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.

Procedures for isolation and measurement of lipids in foods include exhaustive Soxhlet extraction with hexane or petroleum ether (AOAC, 1995 see Basic Protocol 1), chloro-form/methanol (Hanson and Olley, 1963 Ambrose, 1969), chloroform/methanol/water (Folch et al., 1957 Bligh and Dyer, 1959 see Basic Protocol 2 and Alternate Protocol 2), acid digestion followed by extraction (see Basic Protocol 4), or, for starchy material, extraction with n-propanol-water (e.g., Vasanthan and Hoover, 1992 see Basic Protocol 3). Each method has its own advantages and disadvantages and successful measurement of lipid content is often dictated by the type of sample and extraction medium employed. Commercial extraction and preparation of edible oils are explained in the literature (Williams, 1997). 

Prior to phospholipid analysis, it is imperative to extract the lipids from their matrix and free them of any nonlipid contaminants. Phospholipids are generally contained within the lipid fraction, which may be recovered by the traditional Bligh and Dyer or Folch extraction procedure (9,22). In any phospholipid extraction method it is recommended to include a rather polar solvent in addition to a solvent with high solubility for lipids. The former is needed to break down lipid-protein complexes that prevent the extraction of the lipids in the organic phase. Traditionally, mixtures of chloroform and methanol (especially 2 1, v/v) have been recommended. These are washed with water or aqueous saline to remove nonlipid contaminants. Comparing the recovery of phospholipids, Shaikh found that the neutral phospholipids PC, PE, SPH as well as DPG were nearly quantitatively extracted by all solvent systems studied (Table 1), although Bligh and Dyer, in which the lower phase was removed only once, was somewhat worse (23). 

However, Shaikh demonstrated that the aforementioned traditional methods are inappropriate to recover completely lysophospholipids as well as acidic phospholipids classical Folch gave 85-90% recovery of LPC and LPE, whereas Bligh and Dyer yielded only 75-80% recovery. Extraction with a mixture of chloroform and methanol, on the other hand, provided nearly complete recovery of acidic and lysophospholipids, but up to 15% losses were observed during subsequent washing, according to Folch. These losses could be circumvented by purification of the crude extract on Sephadex G-25, but this column chromatographic procedure is quite time-consuming. 

Extraction of fat by supercritical carbon dioxide was investigated as an important option for minimizing the expanded use of frequently flammable and carcinogenic solvents in food analysis. Unfortunately, the presence of moisture in foods has an adverse effect on the quantitative extraction of fat by supercritical fluid extraction (SEE). Hence, samples have to be lyophilized first. The total fat content of freeze-dried meat and oilseed samples was found to be comparable to values derived from Soxhlet-extracted samples (26). Besides, only small amounts of residual lipids could be recovered by an additional extraction of the SFE-extracted matrix by the Bligh and Dyer solvent extraction procedure. As far as the minor constituents are concerned, it was found that the extraction recovery ranged from 99% for PC to 88% for PA. Hence, Snyder et al. concluded that SFE can be used as a rapid, automated method to obtain total fat, including total phospholipids, from foods (26). 

Representative samples of the pre-treated material were analysed for water content for each herb using a Dean and Stark equipment and also for content of "oily" material (essential oils + natural oils + waxes) using the method of Bligh and Dyer (10). Samples were also subjected to a sieving procedure and examined microscopically to determine the range of sizes and shapes present. Samples of the residue material were analysed for water content. 

Extraction Exhaustive tissue extraction procedures are usually employed to isolate glycerolipids from biological matrices. Quantitative results can be achieved using the method of Bligh and Dyer (1959). 

Lipid Extration Procedures. Lipid was extracted from raw sludge with chloroform and methanol according to the method of Bligh and Dyer (22). Prior to use as a substrate, the lipid extract was dispersed in 2% Tween 80 by sonicating for 30 minutes at 60 °C. 

Gas Chromatography. Using the procedure described by Bligh and Dyer (14), tissue lipids were extracted with a... 

The most common mixture is that of chloroform and methanol. A typical procedure is that of Bligh and Dyer (1959) where a one-phase methanol system (chloroform-methanol-water, 1 2 0.8, by vol.) rapidly extracts lipids from most tissues and the extract is then diluted with 1 vol. each of chloroform and methanol to yield a two-phase mixture. The upper (aqueous) phase contains water-soluble contaminants while the lower phase contains lipids. Water-soluble contaminants can also be removed from solutions of lipids in organic solvents by passage through Sephadex columns (Rouser et ai, 1967). After rotary evaporation of the solvent, traces of water may remain with the lipid. These can be removed by azeotropic distillation with benzene. 

The Bligh and Dyer method is satisfactory for most animal tissues. Most simple and complex lipids can be extracted by this procedure. Kates (1972) and Christie (1982) detail practical points. For quantitative extraction of the polyphosphoinositides (and other very polar lipids) it is advisable to use high concentrations of salts (Garbus et al., 1963) or to add acid to the upper phase (Wells and Dittmer, 1965). The two phases can also be washed with fresh upper or lower phases, as appropriate, to ensure both quantitative extraction and removal of water-soluble contaminants. 

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.

The Bligh and Dyer method [25] is a simple adaptation of the Folch, Lees, and Stanley procedure, developed to minimize solvent consumption. At first, this method was adopted to extract phospholipids from a fish muscle tissue it is more generally intended for the extraction of large samples, with a high proportion of endogenous water (typically. 

The most widespread and effective solvent system for this purpose is the Bligh-Dyer procedure, which was mentioned earlier in Chapter 2. A typical protocol that can be employed for total lipid extraction follows. 

The experimental procedure below describes the uptake of ciprofloxacin into sphingomyelin (SPM)/Chol LUVs. Drug delivery vehicles prepared from SPM/Chol often exhibit greater efficacy than those prepared from DSPC/Chol (13). Included is a description of the Bligh-Dyer extraction procedure (78), which involves partitioning the lipid and water-soluble drug into organic solvent and aqueous layers, respectively. This is necessary because lipid interferes with the ciprofloxacin assay. 

The initial mixture and each time point are then assayed for ciprofloxacin and lipid. Lipid can be quantified using the phosphate assay (64,65) or by liquid scintillation counting. Ciprofloxacin is quantified by an absorbance assay following removal of drug from lipid by a Bligh-Dyer extraction procedure (78) (see below). The percent uptake is determined as described in the section Remote Loading of Doxorubicin into DSPC/Cholesterol (55 45) Large Unilamellar Vesicle. ... 

Figure 6.5 Representative two-dimensional mass spectrometric analyses of sphingomyelin molecular species in the alkaline-treated lipid extracts of mouse spinal cord in the positive-ion mode in the presence of LiOH. Lipid extracts from spinal cord of mice at 48 days of age were prepared by using a Bligh-Dyer extraction procedure as previously described [27]. A part of each lipid extract was treated with lithium methoxide as described previously [28] and the residual lipid extract was reconstituted with 50 pL of 1 1 CHClj/MeOH per milligram of original tissue protein. Each of the reconstituted lipid extracts was diluted 10 times prior to addition of a small amount of LiOH (10 pmol LiOH/pL). Positive-ion ESI mass spectrum in the full MS scan mode was acquired in the mass range from m/z 600 to 900 from the diluted lipid extract of mouse spinal cord, which displayed very low abundance ions corresponding to lithiated SM species. Neutral loss scans (NLS) as indicated were also acquired from the diluted lipid extract in the mass range. All scans were displayed after normalization to the base peak in individual scan. The ion at m/z 653.6 corresponds to the selected internal standard for quantitative analysis of SM species.

---