Free lipid

It must be emphasized that the problem of unsubstituted hydroxyl groups is usually studied employing free lipid A prepared by treatment of LPS with acid. The demonstration of a free hydroxyl group at C-4 in monophosphated lipid A therefore does not exclude the possibility that, in LPS, a substituent, present in acid-labile linkage, could be bound to 0-4 of lipid A. This possibility has thus far been excluded for E. coli Re LPS which was analyzed by 1H- and 13C-n.m.r. and shown to contain an unsubstituted hydroxyl group at C-4 of GlcN(I) (34,110). 

Single crystals of free lipid A or LPS are as yet not available. Therefore, the most promising approach to obtain molecular models is to perform theoretical calculations. After the chemical structures of enterobacterial lipid A had been elucidated, this methodology was successfully applied with heptaacyl S. minnesota lipid A (220) and hexaacyl E. coli Re LPS (221). As an example, Fig. 13 shows the atomic model of the E. coli lipid A molecule, as calculated by Kastowsky et al. (221) using energy-minimization techniques. 

Fig. 14.—Phase diagram (227) of free lipid A.The phase diagram was established using S. minnesota Re LPS-derived lipid A.

The results from all biological assays performed showed that chemically synthesized E. coli lipid A (compound 506 or LA-15-PP) expresses, with similar doses, the same spectrum of endotoxic effects as bacterial (E. coli) free lipid A (5,234-237). Thus, lipid A constitutes the lethal, pyrogenic, leukopenic, and mediator-inducing, that is, the endotoxically essential region of LPS, its endotoxic properties being embedded in a molecule having the structure shown in Fig. 2. 

In the early seventies, Galanos et al. (241) showed that lipid A may be immunogenic, and acid-treated bacteria (exposing free lipid A on their sur-... 

As one increases the size of the cages, the cavity becomes sufficiently large to be used as a nanoreactor. In fact, nanometer size capsules or spheres made of lipid bilayers have been investigated for other applications including drug delivery. However, without modification, such vesicles are probably not suitable as reactors since the lipid bilayer is rather fluid, and the lipid molecules exchange with free lipids in solution. In order to use such vesicles as reactors, it is necessary to convert them into a robust... 

The foam-negative effects of lipids can also be counteracted with the addition of a lipid-binding protein, wheat puroindoline (PIN), to beer. The PIN may bind the residual free lipids in such a way that they can no longer destabilize the foam. (Adapted from Cooper et ah, 2002)... 

Gurtovenko, A.A., Vattulainen, I. Ion leakage through transient water pores in protein-free lipid membranes driven by transmembrane ionic charge imbalance. Biophys. J. 2007, 92,1878-90. 

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... 

Lipids in starchy foods may occur in the free as well as bound forms. The latter being either in the form of amylose inclusion complexes or linked via ionic or hydrogen bonding to the hydroxyl groups of the starch components. Free lipids are easily extractable at ambient temperatures, while use of nonalcoholic solvents for a prolonged period or disruption of the granular structure by acid hydrolysis (see Basic Protocol 4) may be required for the efficient extraction of bound lipids. While acid hydrolysis allows the release and quantitation of lipids, the procedure leads to destruction of the starch components therefore, the alcohol extraction system involving propanol and water would be most desirable in these cases. This system removes both nonpolar and polar lipids from samples. 

Lipid A. All R form lipopolysaccharides as well as free lipid A (obtained by mild acid cleavage of the KDO linkage) represent potent endotoxins, comparable in activity to complete lipopolysaccharides. This shows that lipid A represents the component of lipopolysaccharides which is responsible for its endotoxic properties. 

Of the lipid portions of bacterial macromolecular amphiphi-les, that of lipopolysaccharides is the structurally most complex. For its designation the term lipid A has been coined. More specifically, it was suggested that the lipid, as it is present in intact lipopolysaccharide, should be called lipid A, while the lipid in a separated form should be termed free lipid A (10,11). This nomenclature will be used throughout this paper. In the following, ways and methods will be described which have been used to elucidate the chemical structure of lipid A. The present discussion will deal in more detail with the elucidation of the structure of Salmonella lipid A. Relative to this structure, chemical features of other lipid A s will then be discussed. 

Total fatty acids were liberated by subjecting Salmonella minnesota Re lipopolysaccharide (or free lipid A) to acidic (4 N HC1, 5 h, 100°C) followed by alkaline (1 N NaOH, 1 h, 100°C) hydrolysis. After extraction (chloroform), the free fatty acids were converted into their methyl esters (diazomethane) and analysed by combined gas-liquid chromatography/mass spectrometry. Alternatively, the fatty acids of lipid A are transesterified by treatment of lipopolysaccharide with methanolic HC1 (2 N HC1 in water-free CHaOH, 18 h, 85°C). By these procedures the following fatty acids were identified (in approximate amounts relative to 2 moles glucosamine) dodecanoic (12 0, 1.1 mole), tetradecanoic (14 0, 0.8 mole), hexadecanoic (16 0, 0.9 mole), 2-hydroxytetradecanoic (2-OH-l4 0, 0.1 mole), and 3-hydroxytetradecanoic acid (3-OH-14 0, 4 moles). In total, therefore, approximately 7 moles of fatty acids are present per mole of lipid A backbone. The stereochemistry of the hydroxylated fatty acids was determined by gas-liquid chromatography of their diastereomeric methoxyacyl-L-phenylethylamide derivatives (24). It was found that 2-hydroxyte-tradecanoic acid possesses the-Ts), and the predominating 3-hydroxytetradecanoic acid the (R) configuration. 

When this method, in a slightly modified form, was applied to Salmonella free lipid A, mainly two compounds were liberated which were detected by gas-liquid chromatography and characterized by mass spectrometry. The spectrum of the first compound was identical with that of authentic 3-hydroxytetradecanoic acid, 3-0-acylated by dodecanoic acid (3-0-dodecanoyl-tetradecanoic acid methyl ester, 3-0(12 0)-14 0) (27). The mass spectrum of the second peak showed, inter alia, characteristic fragments at 496 (M ), 465 (M-31), 239, 240 and 241, thus corresponding to 3-0-hexadecanoyl-tetradecanoic acid methyl ester (3—0(16 0)—14 0). Therefore, the two components present in Salmonella lipid A in amide linkage and released by the above described procedure... 

Figure 4. Steps involved in the selective liberation ofamide-bound acyl groupsfrom free lipid A. (Reproduced with permission from Ref 27. Copyright 1982, Federation of European Biochemical Societies.)...

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