Lipid Extraction Methods

Important analytical considerations regarding lipid normalization In addition to the problems associated with normalization of contaminants to total lipid discussed above, the problem of lipid normalization is further compounded by differences in the efficiency of various lipid extraction methods. In short, different lipid classes are extracted by different solvents. For example, bluefish liver yielded total lipid levels of 25% when extracted using chloroform/methanol but only 8% when acetonitrile was the extracting solvent92. This reflects differences in the lipid class extracted by each solvent neutral lipids were preferentially extracted by non-polar solvents and more polar membrane lipids were extracted more efficiently by more... 

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. 

Yeasts have several difficulties for Upid extraction, including the presence of a thick cell wall that renders the yeast cells resistant to many solvents, as well as the possible presence of lipases in their cell extracts, and most of the neutral lipids are intracellularly stored in lipid bodies. However, lipid bodies also contain other lipophilic compounds, in particular aromatic compounds, which are difficult to remove during lipid purification (Ageitos et al. 2011). Table 7 shows the profile of TAG for some lipid extraction methods from L. starkeyi. 

Table 7 Lipid extraction methods from L. starkeyi...

Bligh, E.G. and E)yer, W.J. 1959 A rapid method of total lipid extraction and purification. Canadian Journal of Biochemical Physiology 37 911-917. 

Raith and Neubert [57] have developed a method for the profihng of human stratum comeum ceramides. The method enables the investigation of the role of ceramides in maintaining the barrier function of stratum comeum. TLC using automated multiple development was modified for semipreparative purposes. The fractionation of complex lipid extracts using this method ensured specific, sensitive, and... 

Recently, a quantitative electrospray ionization/mass spectrometry method (ESI/MS) has been developed to analyze the molecular profile, or hpidome of different lipid classes in very small samples. In this method, total lipid extracts from tissues or cultured cells can be directly analyzed. By manipulating the ionization method, the mass spectrographs of polar or even non-polar lipids can be obtained [8]. This method and the use of lipid arrays allow precise and quantitative identification of the lipid profile of a given tissue, and map functional changes that occur. 

One advance in the area of LLE is the use of solid supports that facilitate the partitioning of the analyte(s) of interest. LLE extraction methods involving nonpolar matrices often suffer from the formation of emulsions, and using the solid support is a possible solution. In one study, polychlorinated biphenyls, dioxins, and furans were extracted from the lipid fraction of human blood plasma [32], using diatomaceous earth as the solid support. Long glass columns (30 cm) were packed with several layers of Chem-Elut (a Varian product) and sodium chloride. The plasma samples were diluted with water and ethanol and passed over the columns. A mixture of isopropanol and hexane (2 3) was passed over the column and the LLE was performed. It can be concluded that the LLE with the solid support is easier to perform and can be applied to other lipid heavy matrices such as milk [32]. 

Treatment with hot organic solvents was the next step in the tissue fractionation, to remove lipid-phosphorous and breakdown lipid-protein interactions. In the Schneider procedure, nucleic acids were then extracted in hot dilute trichloroacetic or perchloric acid, leaving a protein residue with any phosphoprotein links still intact. This method was to become particularly useful when 3H thymidine became the preferred label for DNA in the early 1960s. For investigations where both RNA and DNA were to be examined the Schmidt-Thannhauser process was often chosen. Here the lipid-extracted material was hydrolyzed with dilute sodium hydroxide releasing RNA nucleotides and any hydroxyamino acid bound phosphorus. DNA could be precipitated from the extract but the presence in the alkaline hydrolysate of the highly labeled phosphate released from phosphoprotein complicated... 

A thin film of oil-like material was visible after 28 d on the exterior surfaces of the SPMD membrane. Analysis of this film indicated that the triolein impurities, oleic acid and methyl oleate, were the major constituents. This external lipid film (Petty et al., 1993) appeared to contain imbibed particulates. Although the film was removed from the SPMDs by solvent rinsing and analyzed separately, some lipid-mediated desorption of particle-associated PCBs and subsequent diffusion into the SPMD may have occurred prior to solvent-removal of the film. This observation suggests the potential for SPMD concentrations to reflect both vapor phase concentrations and to a lesser extent, lipid-extracted particulate-associated residues (see Section 3.9.2.). Unfortunately, concentrations of more chlorinated congeners in particulates collected on GFFs from the NIOSH method were often below quantitation limits, because only a small volume of air was sampled (1 m ) using this active method. 

This method is particularly suitable for lipid extraction of incubation medium, tissue homogenates or cell suspensions. 

Choudhury RB, Poddar MK. (1984) Andrographolide and Kahnegh (Andrographis paniculata) extract In vivo and in vitro effect on hepatic lipid peroxidation. Methods Find Exp Clin Pharmacol 6 481. 

Crude and three diethyl ether extracted, acetone treated, fractions were isolated from large-scale cultures of Gambierdiscus toxicus. Crude extracts at. 04 mg/ml inhibited the histamine contraction response in smooth muscle of the guinea pig ileum. Three semi-purified fractions at 5 ng/ml, effectively inhibited the guinea pig ileum preparation. Two of these fractions followed Michaelis-Menten kinetics for a competitive inhibition. The third fraction inhibited in a non-reversible manner. This study has established the presence of three lipid extracted toxins in toxicus, outlined a method for their assay in small quantities, and identified at least two of the effects of these toxic extracts in animals. 

NonenaL 2-decenal and 2-undecenal were concentrated appreciably in fraction FI of the 207 bar/50°C extract, whereas aldehydes associated with lipid oxidation such as pentanaL hexanal and octanal were not concentrated by the extraction method used, probably because these constituents continue to accumulate following extraction or because low volatiles are not extracted well by the SC-COj method used. 

Separation of crude lipid extracts into individual lipid classes is difficult and time-consuming. In some cases a crude separation of lipids can be attained by selective solvent extraction. For more extensive purification of lipids, the researcher must turn to chromatography. Chromatographic methods can both resolve a complex lipid mixture into the various classes of lipids and separate the individual components of a single class of lipids. 

Alternate Protocol 1 Goldfisch Method for Lipid Extraction Basic Protocol 2 Chloroform/MethanolAVater Extraction of Lipids from Dl.1.3... 

Lipid content (%) = mass of lipid extracted (g)/sample weight (g) x 100 GOLDFISCH METHOD FOR LIPID 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. 

Lee, C.M., Trevino, B., and Chaiyawat, M. 1996. A simple and rapid solvent extraction method for determining total lipids in fish tissue. JAOCS International 79 487-492. 

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