Extraction of lipids

Although extraction of lipids from membranes can be induced in atomic force apparatus (Leckband et al., 1994) and biomembrane force probe (Evans et al., 1991) experiments, spontaneous dissociation of a lipid from a membrane occurs very rarely because it involves an energy barrier of about 20 kcal/mol (Cevc and Marsh, 1987). However, lipids are known to be extracted from membranes by various enzymes. One such enzyme is phospholipase A2 (PLA2), which complexes with membrane surfaces, destabilizes a phospholipid, extracts it from the membrane, and catalyzes the hydrolysis reaction of the srir2-acyl chain of the lipid, producing lysophospholipids and fatty acids (Slotboom et al., 1982 Dennis, 1983 Jain et al., 1995). SMD simulations were employed to investigate the extraction of a lipid molecule from a DLPE monolayer by human synovial PLA2 (see Eig. 6b), and to compare this process to the extraction of a lipid from a lipid monolayer into the aqueous phase (Stepaniants et al., 1997). 

Stepaniants et al., 1997] Stepaniants, S., Izrailev, S., and Schulten, K. Extraction of lipids from phospholipid membranes by steered molecular dynamics. J. Mol. Model. 3 (1997) 473-475... 

After the extraction of lipid and nonlipid components from the leaves of mandarin orange Citrus reticulata, the lipid fraction was further separated by PTLC to determine different lipid classes that affect the chemical deterrence of C. reticulata to the leaf cutting ecat Acromyrmex octopinosus. These lipids seem to be less attractive to the ants [81a]. The metabolism of palmitate in the peripheral nerves of normal and Trembler mice was studied, and the polar lipid fraction purified by PTLC was used to determine the fatty acid composition. It was found that the fatty acid composition of the polar fraction was abnormal, correlating with the decreased overall palmitate elongation and severely decreased synthesis of saturated long-chain fatty acids (in mutant nerves) [81b]. 

Fig. 3.4 Permeability profiles for (a) warfarin (acid), (b) propranolol (base) and (c) morphine (ampholyte) based on a BBB PAMPA model (plON) composed of animal brain extract of lipids. The data (unpublished) were analyzed with the pCEL-X program (plON), with the refined parameters indicated in the three frames. In all three cases, there was evidence for the permeation of charged...

Supercritical fluid extraction (SFE) is a technique in which a supercritical fluid [formed when the critical temperature Tf) and critical pressure Pf) for the fluid are exceeded simultaneously] is used as an extraction solvent instead of an organic solvent. By far the most common choice of a supercritical fluid is carbon dioxide (CO2) because CO2 has a low critical temperature (re = 31.1 °C), is inexpensive, and is safe." SFE has the advantage of lower viscosity and improved diffusion coefficients relative to traditional organic solvents. Also, if supercritical CO2 is used as the extraction solvent, the solvent (CO2) can easily be removed by bringing the extract to atmospheric pressure. Supercritical CO2 itself is a very nonpolar solvent that may not have broad applicability as an extraction solvent. To overcome this problem, modifiers such as methanol can be used to increase the polarity of the SFE extraction solvent. Another problem associated with SFE using CO2 is the co-extraction of lipids and other nonpolar interferents. To overcome this problem, a combination of SFE with SPE can be used. Stolker et al." provided a review of several SFE/SPE methods described in the literature. 

Bernardo-Gil G, Oneto C, Antunes P, Rodrigues MF and Empis JM. 2001. Extraction of lipids from cherry seed oil using supercritical carbon dioxide. EurFood Res Technol 212(2) 170—174. 

Analytical problems associated with the extraction of lipid from tissue with organic solvents are ... 

Ylinen et al. [53] developed an ion-pair extraction procedure employing tetrabutylamonium (TBA) counter ions for determination of PFOA in plasma and urine in combination with gas chromatography (GC) and flame ionisation detection (FID). Later on, Hansen et al. [35] improved the sensitivity of the ion-pair extraction approach using methyl tertiary butyl ether (MTBE) and by the inclusion of a filtration step to remove solids from the extract making it amenable to liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) determination. Ion-pair extraction procedure has been the basis of several procedures for biota [49,54-58] and food samples [50,59,60]. However, this method has shown to have some limitations, such as (1) co-extraction of lipids and other matrix constituents and the absence of a clean-up step to overcome the effects of matrix compounds and (2) the wide variety of recoveries observed, typically ranging. 

Lipids can be identified and quantified using thin-layer chromatography (TEC) and gas chromatography (GC) (Galliard, 1968). Extraction of lipids is achieved by homogenizing potato tubers with isopropanol in a blender, followed by a series of filtrations and extractions with chloroform-methanol (2 1). Chloroform is removed by rotary evaporation and the residue is redissolved in benzene-ethanol (4 1). This extract is passed through a DEAE-cellulose column, and the fractions collected are subjected to TEC on 250 p,m layers of silica gel G, using three solvent systems. Fatty acid methyl esters for GC analysis are prepared by transmethylation of the parent lipids, or by diazomethane treatment of the free fatty acids released by acid... 

Four aspects of research involving the use of SFE for the improvement of quality of muscle food products are briefly discussed. These include supercritical CO2 extraction of lipids fi om fresh ground beef and from dried muscle foods the extraction and separation of lipid and cholesterol from beef tallow supercritical CO2 extraction of flavor volatiles from beef and pork lipids for use as additives in synthetic meat flavors and identification and quantitation of flavor volatiles extracted with SC-CO2. 

Most studies of SC-CO2 quantitative extraction of lipids from muscle foods have been limited to dried materials with moisture levels below 10%. King et aL (26), Froning (27) and Hardardottir and Kinsella (28) were able to quantitatively remove lipid from muscle foods and data for Aese studies are shown in Table I. This table also includes data from study of dried pork. Maximum removal of lipid from dried beef and luncheon meat was obtained at 300-345 bar/45-80 C (26, 27) and at 275 bar/80 C for trout (28). 

Ethanol as a Co>Solvent with SC>C02 for Extracting Cholesterol from Beef Tallow. Figure 4 contains data for comparing the extraction of lipid and cholesterol with 5% ethanol as a co-solvent (entrainer) compared to extracting with SC-CO2 alone. 

High extraction pressure (>300 bar) is the predominant factor for increasing the extractability of lipid from muscle foods, dthough greater quantities of lipid are extracted at higher temperatures (50-80 C) compared to lower temperatures such as 35 or 40°C... 

Extraction of lipids from muscle foods is more predictable if the food has less than 10% moisture. Higb recoveries of glycerides can be obtained from dry meat products by extracting with SC-CO2 above 300 bar, but cholesterol is extracted less effectively. Cholesterol is extracted from beef tallow more efficiently at lower pressures than at higher pressures (<300 bar). 

Dried muscle foods, supercritical CO extraction of lipids, 121,122r... 

The spectra of dry films of intact ghosts prepared by lysis in 20 millios-molal phosphate buffer (21) and of ghost protein prepared by cold butyl alcohol extraction (51) are shown in Figure 6. In both cases the amide I band occurs at 1651 cm. 1 and shows no shoulder near 1630 cm. 1, characteristic of fl structure. The amide II band is also unaffected by removal of lipid and occurs at 1540 cm."1. As expected, extraction of lipid results in removal of the band at 1737 cm. 1 assigned to lipid ester carbonyl stretch and a decrease of the band at about 1455 cm."1 arising from methylene and methyl bending. 

N. Radin, in Methods in Enzymology, Vol. 72, J. M. Lowenstein, Editor (1981), Academic Press (New York), pp. 5-7. Extraction of lipids with hexane-isopropanol. 

D Holme and H Peck, Analytical Biochemtstiy, 3rd ed (1998), Addison Wi li Longman (New York), pp 406-442 Structure, function, and analysis of lipids C Matthews, K van Holde, and K Ahern, Biochemistry, 3rd ed (2000), Benjamin/ Cummings (San Francisco), pp 315-357 Lipid structure and function G Patton, S Cann, H Brunengraber, and J. Lowenstein, in Methods in Enzymology, Vol 72, J Lowenstein, Editor (1981), Academic Press (New York), pp 8-20 Separation of fatty acid methyl esters by gas chromatography on capillary columns N Radin, in Methods in Enzymology, Vol 72, J M Lowenstein, Editor (1981), Academic Press (New York), pp 5-7 Extraction of lipids with hexane-isopropanol L Stryer, Biochemistry, 4th ed (1995), W H Freeman (New York), pp 263-270, 603-606 Lipid structure and function... 

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

Basic Protocol 4 Extraction of Lipids Requiring Acid Digestion Dl.l. 7... 

In addition, to release lipids from source material, such as those in starch, fish meal, or milk, it might be necessary to treat the sample with an acid prior to lipid extraction (see Basic Protocol 4). In the case of milk, addition of ammonium hydroxide is necessary to dissolve casein prior to lipid extraction, which will release the lipids from its surrounding matrix (e.g., from the film surrounding the fat globules in milk). Furthermore, in certain cases, it is necessary to predry the sample in order to allow efficient and complete extraction of lipids. Particle size reduction is another factor that may improve lipid extraction efficacy. 

The Soxhlet extraction procedure is a semicontinuous process, which allows the buildup of the solvent in the extraction chamber for 5 to 20 min. The solvent surrounds the sample and is then siphoned back into the boiling flask (Fig. D 1.1.1). Multiextractor units are available for extraction of lipids from several different samples or replicate runs of the same material. The procedure provides a soaking effect and does not permit channeling. The fact that polar and bound lipids are not removed is a drawback to the procedure (see Background Information). 

BASIC CHLOROFORM/METHANOLAVATER EXTRACTION OF LIPIDS FROM... 

Extraction of lipid from small amounts (<1 g) of sample, such as that of rotifers, artemia, and larvae, may be achieved using an alternate procedure. In this approach, homogenization of sample is achieved using a vortex due to the delicate nature of tissues involved and the ease of their disintegration. The recovered lipids, similar to that in the original protocol (see Basic Protocol 2), may be used for further analysis. 

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