Simple lipids isolation

Structural variations of isolated cell membranes and even, in favourable cases, whole cells can be studied by DSC. In spite of the inherent difficulties, the available data confirm that, in essence, the results obtained from simple lipid-water systems and model membranes can be applied to understand the structure-function relationships of cell membranes. 

Some preliminary fractionation of lipid samples into simple lipid, glycolipid and phospholipid groups may then be desirable to facilitate their analysis or the isolation of single lipid classes on a small scale. The last objective can be accomplished by high-performance liquid, thin-layer or ion-exchange chromatography. 

The complexity of natural lipid extracts is such that it is rarely possible to claim that all the lipid classes of a sample can be separated in one operation. It is, therefore, often worthwhile to be able to isolate distinct simple lipid, phospholipid or glycolipid fractions for further analysis. For example, it is frequently easier technically to isolate small amounts of pure lipid classes preparatively by means of HPLC (or other methods), after a preliminary fractionation has been carried out. Unfortunately, no procedure appears yet to have been described that is satisfactory in all respects, although some useful methods are available provided that their limitations are recognised. 

Alcohols may be released from the esterified form by any of the hydrolytic or transesterification procedures described in Chapter 4. If a pure wax ester fraction is hydrolysed, the alcohols are obtained simply by solvent extraction of the alkaline solution. On the other hand, when other lipids are present, it is advisable to isolate them as a class by adsorption chromatography. TLC on layers of silica gel G with the elution system described for simple lipid separations in Chapter 2, i.e. with hexane-diethyl ether-formic acid (80 20 2 by volume) as the mobile phase, is usually used. With such a system, any secondary alcohols migrate ahead of primary alcohols, which in turn are slightly less polar than cholesterol diols migrate just in front of monoacylglycerols. If cholesterol is present in an extract, it may be necessary to re-run the plate in the same direction to obtain additional resolution and ensure that primary alcohols and cholesterol are fully separated. Procedures of this kind were utilised to isolate trace levels of fatty alcohols from animal tissues, for example [108,662,904]. When wax esters are transesterified, the methyl esters and free alcohols can be separated on a mini-column of... 

The ion channel receptors are relatively simple in functional terms because the primary response to receptor activation is generated by the ion channel which is an integral part of the protein. Therefore, no accessory proteins are needed to observe the response to nicotinic AChR activation and the full functioning of the receptor can be observed by isolating and purifying the protein biochemically and reconstituting the protein in an artificial lipid membrane. In contrast, the G-protein-coupled receptors require both G-proteins and those elements such as phospholipase-C illustrated in Fig. 3.1, in order to observe the response to receptor activation (in this case a rise in intracellular calcium concentration resulting from the action of IP3 on intracellular calcium stores). 

Kossjakow114 claims to have isolated a polysaccharide in this way, by a relatively simple procedure, but the reviewer and coworkers11 have been unable to obtain active material by this method. It does appear that the specific substances contain carbohydrate, lipid, and possibly polypeptide constituents similar perhaps to the Foreman antigen (see below) and that the linkages between these constituents are very fragile. 

By the second half of the nineteenth century German chemists had established a dominant position in analytical and synthetic organic chemistry. Various simple sugars and aminoacids were being isolated and characterized, as well as more complex plant products. Studies on the composition of blood and the properties of hemoglobin were also well under way. The composition of lipid-rich components and the order of the different units within complex macromolecules, such as proteins and nucleic acids, could not however be resolved by techniques then available. 

The heterogeneous class of compounds marked by solubility in so-called lipid solvents (acetone, hydrocarbons, ether, etc.) and relative insolubility in water, has traditionally been called lipids (3). This historical classification, based upon isolation procedures from natural products, is obviously too broad for simple generalizations since it includes triglycerides, fatty acids, phospholipids, sterols, sterol esters, bile acids, waxes, hydrocarbons, fatty ethers and hydrocarbons. For the purposes of this chapter, we will consider lipids to be fatty acids and their derivatives. 

Folch, J., Lees, M., and Sloane Stanley, G.H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226 497-509. 

In addition to simple model systems, more complex systems which are closer to actual foodstuffs have been used to investigate the formation of flavor chemicals in the Maillard reaction. Sixty-three volatile chemicals were isolated and identified from starch heated with glycine (4). When beef fat was used as a carbonyl compound precursor in a Maillard model system with glycine, 143 volatile chemicals were identified (6). These included fifteen n-alkanes, twelve n-alkenes, thirteen n-aldehydes, thirteen 2-ketones, twelve n-alcohols, and eleven n-alkylcyclohexanes. Recently, the effect of lipids and carbohydrates on the thermal generation of volatiles from commercial zein was studied (7). 

The metabolome refers to the entire collection of metabolites, including lipids, sugars, amino acids, and nucleosides within an organism [2]. Though there is still some debate as to the exact number of metabolites, the current estimate is 6,800 human metabolites [25]. Compared to large biopolymers consisting of thousands of atoms, such as proteins and nucleic acids, the structures of many metabolites seem simple. However, this simplicity masks the unique challenge associated with analysis of the metabolome due to the distinct physicochemical properties of different classes of metabolites. For example, the isolation and analysis protocols... 

The isolation of lipids from cells or tissues is not as simple and straightforward as one might desire, but is essentially an important adjunct to characterization of membranes (composition, lipid-to-protein ratio, structure proof, definition, new lipids, etc.). While this is recognized by many investigators in the field, it is difficult for the novice in this area to become aware of some of the potential problems in extraction procedures and the reasons for particular approaches. Thus it seems fitting at this point in time to comment on some of the nuances of the approaches used in isolation, purification, and identification of lipids present in cell membranes. These topics are subdivided into areas which are considered to be of major import to a successful consideration of the extraction procedure. 

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