Bligh and Dyer method

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

Hanson, S.W.F. and Olley, J. 1963. Application of the Bligh and Dyer method for lipid extraction to tissue homogenates. Biochem. J. 83 101-105. 

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. 

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.)...

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. 

Fatty acids can be extracted either as FFAs or as BFAs. FFAs are obtained by the hydrolysis of BFAs. Liquid-liquid extraction has been successfully applied in the isolation of several classes of lipids, or of single lipids from complex mixtures. Popular extraction methods for lipids are the Folch extraction technique, or the Bligh and Dyer method. Solid-phase extraction (SPE), and solid-phase microextraction (SPME), are also available as simple and economical time- and solvent-efficient sample preparation methods. Prefractionation can be performed using SPE silica or aminopropyl-silica columns. 

Moisture content determination was done according to the AO AC method (1990). Total lipids (TL) were quantified by the Bligh and Dyer method (1959) and classified into polar lipid (PL) and nonpolar lipids (NL). NL analysis was carried out according to the method of Ohshima and Ackman (1991), PL were analysed using the method of Rat-nayake and Ackman (1985). TBA values of minced fish meat were measured using the method of Sinnhuber and Yu (1972). 

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. 

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.

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... 

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). 

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). 

Currently, there is no doubt that the most widely used method for extraction of tissue lipids is that of Bligh and Dyer (1959). Basically, this is a modification of the Folch method and employs a careful calculation of the amount of sample (tissue) water such that the overall mixture will have a final composition of chloroform-methanol-water of 1 2 0.8 (v/v). Thus, a singlephase extract can be obtained and extraction completed very rapidly, even within minutes. Recovery of the lipid in a chloroform-rich phase can be achieved by addition of equal volumes of chloroform (under certain conditions) and water to produce a two-phase system. The lower (CHC13) phase is subsequently washed with a methanol-water (1 0.9, v/v) mixture to allow removal of a substantial amount of the nonlipid contaminant with little or no problems with interfacial fluff formation or emulsions. However, even though this is a highly efficient method, it is still advisable that one take steps... 

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. 

To promote good lipid accumulation in the cells, the culture was transferred to a nitrogen-absent medium. The rate of depletion of soluble potassium nitrate in culture medium was determined with Hitachi HPLC L6000. And the fluorescent intensity of the Nile Red-dyed microalgal cells was measured by Hitachi fluorescent spectrophotometer UV-2000. Stainability with Nile Red was determined as fluorescent intensity at 575 nm per cell density. After the cultivation, cells were harvested by centrifugation, washed twice with distilled water and lyophilized. Cellular lipid was extracted by the method of Bligh and Dyer [5] and determined gravimetrically. 

The DNA may then be extracted by the method of Bligh and Dyer (113). The sample is mixed with 1 part chloroform and 2.1 parts methanol, forming a monophase upon vortex mixing. 

Estimation of total lipids and phospholipids The total lipid content was estimated according to Tsuda et al.(23) and purified according to Bligh and Dyer(24). Phospholipids were separated by thin layer chromatography (TLC) method(25). The phospholipids were quantified by estimating the phosphorous content(26). 

The first hurtle is to reproducibly extract lipids from a matrix. The most common lipids extraction methods are those of Bligh and Dyer [42] and Eolch [43]. Recent analysis of these two methods has shown that the Eolch method tends to have a greater total recovery of lipid [44]. A variety of other solvent mixtures has been compared and may offer fewer hazards with similar recoveries [45]. These extraction methods are designed to recover the principal lipid classes, but may not be as useful for recovery of lipids that have unique charge characteristics. For example, fatty acids, phosphatidic acids, and lyso-phosphatidic acids usually require acidic solvents to facilitate recovery from an aqueous solution while neutral lipids may not be sufficiently soluble in an organic solvent [46]. Other complexities include solvent manipulations required to extract more polar lipids like the phosphatidylinositol phosphates. 

Iverson, S. J., Lang, S. L., and Cooper, M. H. 2001. Comparison of the Bligh and Dyer and Folch methods for total lipid determination in a broad range of marine tissue,... 

Lipids were extracted from the PSII preparations according to Bligh and Dyer (11). The extracted lipids were subjected to reverse-phase HPLC in order to remove Triton X-100 from the lipid extract. The resultant lipid extract was separated into lipid classes by silica gel HPLC according to the method of Demandre et al (12). The lipid classes in the eluted fractions were identified by comparing the retention times in the HPLC with those of authentic lipid classes, and the identification was confirmed by TLC. The separated lipid classes were subjected to methanolysis. The resultant methyl esters were analyzed by gas-liquid chromatography and gas chromatography-mass spectrometry. 

The Bligh and Dyer technique is based on the homogenization of wet foods with chloroform and methanol. Further addition of chloroform and water leads to the separation of the phases, with the lipid contained in the chloroform layer. The AO AC details a similar extraction method based on chloroform-methanol extraction, the advantage of these methods being that no heating stage is involved and the lipid extract can then be analyzed to determine the individual fatty acids. 

Legumes contain predominantly unsaturated fatty acids there are exceptions to this, with certain legumes containing appreciable proportions of saturated fats. In order to determine accurately the fatty acid profile of legumes, the fat portion must be extracted without heating chloroform-methanol extraction methods are therefore widely used, e.g., those of Bligh and Dyer, Karow. 

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