Soybean phospholipids

Cmde oils generally contain phospholipids that are removed during the degum-ming stage of refining as a crude mixture (lecithin). This valuable product is the basis of the phospholipid industry, and phospholipids are used extensively in food products, in animal feeds, and in industrial processes. The major members are phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols and are accompanied by smaller proportions of other phospholipids. Soybean oil (3.2%), rapeseed oil (2.5%), and sunflower seed oil (1.5%) contain the proportions of total phospholipids indicated in parentheses and are the main sources of commercial lecithins, especially soya lecithin. Palm oil contains little or no phospholipids (7-9). 

Phospholipids Soybean lecithin Egg lecithin Diolelyl phosphatidyl choline Distearoyl phosphatidyl glycerol PEGylated phospholipids Dimyristoyl phosphotidyl choline All routes... 

Other Reactions of Phospholipids. The unsaturated fatty acid groups in soybean lecithin can be halogenated. Acetic anhydride combined with the amino group of phosphatidylethanolamine forms acetylated compounds. PhosphoHpids form addition compounds with salts of heavy metals. Phosphatidylethanolamine and phosphatidjhnositol have affinities for calcium and magnesium ions that are related to interaction with their polar groups. 

The presence of impurities like free fatty acids in egg or soybean phosphatidylcholine, or in the (semi)synthetic phosphatidylcholines (e.g., DMPC, DPPC, DSPC) can be detected by monitoring the electrophoretic behavior of liposome dispersions of these phospholipids in aqueous media with low ionic strength a negative charge will be found on these liposomes when free fatty acids are present in the bilayers. 

An important characteristic of mammalian 15-LOX is its capacity to oxidize the esters of unsaturated acid in biological membranes and plasma lipoproteins without their hydrolysis to free acids. Jung et al. [19] found that human leukocyte 15-LOX oxidized phosphatidylcholine at carbon-15 of the AA moiety. Soybean and rabbit reticulocyte 15-LOXs were also active while human leukocyte 5-LOX, rat basophilic leukemia cell 5-LOX, and rabbit platelet 12-LOX were inactive. It was suggested that the oxygenation of phospholipid is a unique property of 15-LOX. However, Murray and Brash [20] showed that rabbit reticulocyte... 

Finally, besides conventional liposomes that are made from natural (e.g., egg yolk and soybean) or synthetic phospholipids, novel liposomes called archaeosomes that are prepared from the polar ether lipids extracted from various archaeobacteria proved also interesting for the design of vaccines as peptide antigen carriers (71) and as powerful self-adjuvanting vaccine delivery vesicles that promote both humoral and cell-mediated immunity (72). Related to this, one can mention that pseudopeptides, which are less prone to proteolysis when conjugated to liposomes, were also competent in triggering a humoral immune response (73). 

Phospholipid classes (see Section V.B.3) may be separated from one another by HPLC-UVD, measuring at 210 nm. Oxidized phospholipids, including hydroperoxide and hydroxy derivatives, can be detected by the same method measuring the signal at 234 nm. This was applied for assessment of the oxidation of phosphohpids in human erythrocite membranes catalyzed by soybean lipoxydase in the presence of various modifiers ... 

Instead of measuring the absorbance of the solution immediately after incubation, it is possible to determine the colored product 169 by HPLC-FLD (A.ex =515 nm, /. = 553 nm). This procedure was applied for determination of MDA after oxidation of phospholipids catalyzed by soybean lipoxygenase464. Peroxidation in the extracellular liquid of the brain cortex of rats was evaluated in vivo by microdialysis followed by reaction... 

Collectively, as found in nature, phospholipids are usually mixtures of different acyl groupings and terminal head groups. These mixtures can be extracted using solvents from, for example, plants (e.g., soybeans) or eggs and constitute the material usually known in commerce ay lecithin. Unfortunately there has been a tendency to call dipalmitoylphosphatidycholine, PC, one of the major constituents of most natural mixtures, lecithin, and it may be better to avoid applying this name to a purified derivative. 

Table 8 Phospholipids of Soybean Lecithin Distribution (By % Weight) of Fatty Acids...

Dispersed systems, such as emulsions, have also been employed to achieve high drug loading for parenteral administration. Emulsions generally consist of a vegetable oil (e.g., soybean), a phospholipid surfactant (e.g., lecithin), and glycerol added for isotonicity. The surfactant (emulsifier) is necessary to provide a barrier to agglomeration of the emulsion droplets. Unlike micellar solutions that are thermodynamically stable,... 

The unsaturated phospholipid from soybean lecithin also shows a similar effect, while the unsaturated phospholipids from red blood cell membranes, although showing a slight effect of cholesterol interaction, still show a prominent polymethylene peak in the high resolution spectrum. 

The phosphoric acid esters of diacyl glycerides, phospholipids, are important constituents of cellular membranes. Lecithins (phosphatidyl cholines) from egg white or soybeans are often added to foods as emulsifying agents or to modify flow characteristics and viscosity. Phospholipids have very low vapor pressures and decompose at elevated temperatures. The strategy for analysis involves preliminary isolation of the class, for example by TLC, followed by enzymatic hydrolysis, derivatization of the hydrolysis products, and then GC of the volatile derivatives. A number of phospholipases are known which are highly specific for particular positions on phospholipids. Phospholipase A2, usually isolated from snake venom, selectively hydrolyzes the 2-acyl ester linkage. The positions of attack for phospholipases A, C, and D are summarized on Figure 9.7 (24). Appropriate use of phospholipases followed by GC can thus be used to determine the composition of phospholipids. 

As an example of an asymmetric membrane integrated protein, the ATP synthetase complex (ATPase from Rhodospirillum Rubrum) was incorporated in liposomes of the polymerizable sulfolipid (22)24). The protein consists of a hydrophobic membrane integrated part (F0) and a water soluble moiety (Ft) carrying the catalytic site of the enzyme. The isolated ATP synthetase complex is almost completely inactive. Activity is substantially increased in the presence of a variety of amphiphiles, such as natural phospholipids and detergents. The presence of a bilayer structure is not a necessary condition for enhanced activity. Using soybean lecithin or diacetylenic sulfolipid (22) the maximal enzymatic activity is obtained at 500 lipid molecules/enzyme molecule. With soybean lecithin, the ATPase activity is increased 8-fold compared to a 5-fold increase in the presence of (22). There is a remarkable difference in ATPase activity depending on the liposome preparation technique (Fig. 41). If ATPase is incorporated in-... 

The value of antioxidant protection by way of natural food sources has been pointed out in the literature with considerable frequency. Among the components of soy flour known to have some antioxidant properties are isoflavones and phospholipids. Amino acids and peptides in soybean flour also possess some antioxidant activity. There also may be some antioxidant impact from aromatic amines and sulfhydryl compounds. 

Brash, A.R., Ingram, C.D., and Harris, T.M. 1987. Analysis of a specific oxygenation reaction of soybean lipoxygenase-1 with fatty acids esterified in phospholipids. Biochemistry 26 5465-5471. 

From a technical standpoint, phospholipids (e.g., from soybean) are composed mainly of lecithin, cephalin, or phosphatidylinositol. These complex mixtures (2-3% in soybean oil) are hydrated during the degumming step, removed, and dried. These products are sold as commercial lecithin used in margarines, confections, and shortenings where a fat-soluble emulsifier is required. 

In commercial formulations, phospholipids are not available as pure products. Mostly they are obtained as a by-product of the process of refining vegetable oils, during the so-called degum-ming step (3,4), from which a liquid-to-pasty product is obtained that is referred to as lecithin. Lecithin contains about 65% phospholipids plus about 30% residual neutral lipids and minor amounts of glycolipids. For historical reasons, most commercially available lecithins are derived from soybean oil, but lecithins of other oils could be used as well. From this discussion it follows that the determination and quantification of phospholipids is of importance both to control how efficiently the phospholipids have been removed from vegetable oils and to control the quality of the lecithin. For this purpose it is important to know not only the total amount of phospholipids but also the amount of the different types of phospholipids present, because it is well known that the functional properties of the various phospholipids differ widely (2-8). 

The separation of phospholipids by micellar electrokinetic capillary electrophoresis (MEKC) has been described (17-19). In this technique, solutes are separated based on their distribution between a mobile (usually aqueous) and a pseudostationary (micellar) phase. Szucs et al. found that the major soybean phospholipids were fully resolved in only 7 minutes using deox ycholic acid for micelle formation in combination with 30% n-propanol at 50°C (18). However, quantification of the separated compounds remains troublesome. This is due first of all to the fact that only UV detection can be used, thus making the response highly dependent on the degree of unsaturation of the phospholipids. Besides, the comparison of peak areas in MEKC is more complicated than in HPLC, because all compounds are moving with different velocities. 

Glycerophospholipids contain a glycerol skeleton to which two fatty acids are esterified saturated fatty acids occupy mostly sn-position 1, whereas unsaturated fatty acids are mainly present on sn-position 2. The third hydroxyl is linked to a phosphate group to which an organic base is mostly esterified (Fig. 1). The most important components of soybean lecithin are phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Phosphatidic acid (PA) may become important due to the presence of phospholipase D this enzyme slowly converts PC into PA in vegetable lecithins. Phosphatidylserine (PS), phosphatidylglycerol (PG), and lyso-phosphatidylcholine (LPC) are known as minor components lysophospholipids contain only one acyl group per molecule. Besides, ether phospholipids occur in which one or both fatty acyl... 

Olsson et al. (69), as well as Arnoldsson and Kaufmann (70), further improved the mobile-phase composition. Experimental design revealed that besides THF, n-butanol as well as ammonium acetate were appropriate modifiers too. The contribution of THF and n-butanol was kept constant throughout the whole solvent program and was about 5% for each solvent each mobile-phase mixture also contained 180 mg of ammonium acetate per liter. As a typical example, the chromatogram of the ILPS-proposed mixed soybean phospholipids standard, which is also referred to as the Spectralipid SN standard mixture, is shown in Fig. 5. 

Abidi et al. reported the separation of the major soybean PLs within 25 min at a flow rate of 1 ml /min on a beta-cyclodextrin-bonded silica using isocratic elution with mobile phases containing hexane, 2-propanol, ethanol, and 5 mM tetramethyl ammonium phosphate (TMAP) in water (pH 6.3) at a ratio of 35/32.7/26.8/5.5 (82). The latter seemed to be critical to improve resolution and enhance peak symmetry. The elution order appeared to follow the order of increasing phospholipid polarity with increasing retention times PE < PC < PI < PA. However, UV detection at 208 nm was required due to the presence of TMAP. Hence, the method is only qualitative. 

Recently, Mounts et al. studied the effect of genetic modification on the content and composition of tocopherols, sterols, and phospholipids in soybean oil (28). Although, in general, there was little impact on the phospholipid classes, a higher PA content was found in some crude oil samples. Compositional variations in molecular species indicated that genetic modification of soybeans affected the phospholipids at the molecular level. 

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