Polar lipids phosphatidylethanolamine

Materials. Egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS) were obtained from Avanti Polar Lipids Inc. (Birmingham, AL) and cholesterol was from Sigma (St. Louis, MO). Ganglioside GMj, bovine, was obtained from Calbiochem (San Diego, CA). Diethylenetriamine pentaacetic acid distearylamide complex (DPTA-SA) was synthesized according to ref. 17 and nlIn-DTPA-SA was prepared as described (7). This lipophilic radiolabel is not transferred to the serum components from liposomes (unpublished data), nor is it rapidly metabolized in vivo (7). The synthesis of N-(glutaryl)phosphatidylethanolamine(NGPE) has been described (18). Dipalmitoyl deoxyfluorouridine(dpFUdR) was synthesized as described (24). 

Global lipid profiling methods typically cover molecular lipid species from the major classes of lipids such as cholesteryl esters, ceramides, mono-(MG), di- (DG), and TGs, and membrane PLs, for example, sphingomyelins (SMs), PCs, phosphatidylethanolamines (PEs), PSs, and lysophospholipids. In targeted lipid analysis, specific lipid classes that are poorly covered by the global profiling methods are usually analyzed. These lipids include steroids, sterols, bile acids, fatty acids, signaling lipids such as eicosanoids, and ceramides, as well as polar lipids and inositol lipids. 

Phosphatidylcholine, phosphatidylethanolamine and sphingomyelin are the major polar lipids found in bovine milk and are present in similar proportions in the total phospholipids, about 25 to 35% (Table 1.11). Phosphatidylserine and phosphatidylinositol are present at lower levels, about 3 to 8%. There are also significant amounts of ceramides, as glucoceramide (GluCer, monohexose) and lactoceramide (LacCer, dihexose), 3 to 6%. Table 1.11 shows five sets of analytical data, four of which are relatively... 

Polar lipids. Polar lipids, mainly phospholipids, are present in fats and oils, and these originate primary as components of cell membranes and serve biological functions in the cells. Among phosphohpids present are phosphotidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). In general, saturated fatty acids are present at the sn-1 and unsaturated fatty acids at the sn-2 positions of phosphohpid molecules. 

Walnuts contain about 65% lipids, however, considerable differences exist among varieties (range 52-70%, w/w) (1,40). Walnuts also contain 15.8% protein, 13.7% carbohydrate, 4.1% water, and 1.8% ash (w/w) (1). The fatty acid composition of walnut oil is unique compared with other tree nut oils for two reasons walnut oil contains predominantly linoleic acid (49-63%) and a considerable amount of ot-linolenic acid (8-15.5%). Other fatty acids present include oleic acid (13.8-26.1%), palmitic acid (6.7-8.7%), and stearic acid (1.4—2.5%) (Table 5) (40). The tocopherol content of walnut oil varies among different cultivars and extraction procedures and ranges between 268 mg/kg and 436 mg/kg. The predominant tocol isomer is y-tocopherol (>90%), followed by a-tocopherol (6%), and then (3- and 8-tocopherols (41). Nonpolar lipids have been shown to constitute 96.9% of total lipids in walnut oil, whereas polar lipids account for 3.1%. The polar lipid fraction consisted of 73.4% sphingolipids (ceramides and galactosylcera-mides) and 26.6% phospholipids (predominantly phosphatidylethanolamine) (42). Walnut oil contains approximately 1.8g/kg phytosterols (1), primarily p-sitosterol (85%), followed by A-5-avenasterol (7.3%), campesterol (4.6%), and, finally, cholesterol (1.1%) (42). 

As the shortest carbon chain members, the polar lipids in lecithins are traditionally depicted in the sn-3 triacylglycerol position. The major polar lipids and fatty acids in deoiled lecithins from various oilseed species are presented in Tables 10 and 11, respectively (44, 45). In the trade, the term lecithin sometimes is used interchangeably with phosphatidylcholine, and cephalin is interchanged with phosphatidylethanolamine. 

It is natural to classify lipids as polar or non-polar according to their interaction with water. Non-polar lipids, for example triglyceride oils, do not form aqueous phases, whereas polar lipids do. Except for cholesterol, membrane-forming lipids form aqueous phases and have polar head groups. Within membranes there are also trace amounts of lipids in membranes that do not interact with water, for example diacylglycerols. The structural formulae of two common membrane lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are shown above. 

Rhodamine-phosphatidylethanolamine (Rh-PE) (Avanti Polar Lipids), stored at -80°C. 

L-a-Dioleoyl Phosphatidylethanolamine (DOPE), and 1,2-Dipalmitoyl-OT-Glycero-3-Phosphocholine (DPPC) were purchased from Avanti Polar Lipids. The chol-PEGjj was obtained in one step from the reaction of cholesteryl chloroformate and a-amino-co-methoxy-PEG. The luciferase encoding gene was obtained as reported (22). Picogreen was purchased from Molecular Probes, USA, Baftlomycin from Sigma, France, the BCA kit from Pierce and the luciferase kit from Promega. 

The membrane constituents are lipids (phospholipids, glycosphingolipids, and cholesterol Figure 10-5), carbohydrates, and proteins. The ratio of protein lipid carbohydrate on a weight basis varies considerably from membrane to membrane. For example, the human erythrocyte membrane has a ratio of about 49 43 8, whereas myelin has a ratio of 18 79 3. The composition of the normal human erythrocyte membrane is shown in Table 10-2. All membrane lipids are amphipathic (i.e., polar lipids). The polar heads of the phospholipids may be neutral, anionic, or dipolar. The surface of the membrane bears a net negative charge. The distribution of lipid constituents in the bilayer is asymmetrical. For example, in the erythrocyte membrane, phosphatidylethanolamine and phosphatidylserine are located primarily in the internal monolayer, whereas phosphatidylcholine and sphingomyelin are located in the external monolayer. 

A typical fish polar lipid fraction contains about 60% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), and several percent phosphatidylserine and sphingomyelin, while the remainder is composed of other minor phospholipids, hi fish, the phospholipid PC PE ratio is generally 2-3 1. The PC PE ratio in phospholipids of bream, a freshwater fish (total lipid content of 4 to 6% w/w), was as low as 1.3 to 1.9 1 (Kolakowska et al., 1993, 2000a). A PC PE ratio of approximately 2 1 was recorded in phospholipids of the light and dark muscle of trout (Ingemansson et al., 1991). Various cod tissues were found to differ in their phospholipid composition. The PC PE ratios of white and dark muscle of the cod were 3.5 1 and 2.7 1, respectively (Lie and Lambertsen, 1991b). Invertebrate phospholipids contain less PC than those from vertebrates. As shown by a study conducted over two annual... 

The lipid classes of hazelnut oil include triacylglycerols (TAG) as nonpolar lipids (98.4%) and glucolipids (1.4%) and phosphoUpids (<0.2%) [phosphatidylcholine (PC) and phosphatidylinositol (PI)] as polar lipids [116]. The 18 lo)9 is dominant in the nonpolar lipid class (TAG), whereas 16 0, 18 0, and 18 2o)6 are most predominant in the polar Upid class in hazelnut oil. Recently, Alasalvar et al. [117] examined lipid class composition of Tombul hazelnut oil and found that it contained 98.8% of nonpolar and 1.2% of polar constituents. The main nonpolar lipid class in hazelnut oil is TAG, contributing nearly 100% to the total amount PC, phosphatidylethanolamine (PE), and PI are main polar Upids, contributing 56.4%, 30.8%, and 11.7% to the total polar lipids, respectively. Similar results were reported by Parcerisa et al. [105]. More recently, Miraliakbari and Shahidi [216] examined the lipid classes in tree nut oils that included TAG, sterols and sterol esters, phospholipids, and sphingoUpids. Hazelnut oil contained TAG, sterols, sterol esters, phosphatidylserine (PS), PC, PI, phosphatidic acid, and sphingoUpids. 

Phospholipids can be separated from other polar lipids by normal-phase LC. Thus, on isocratic elution with acetonitrile-methanol-sulfuric acid (135 5 0.2), phosphatidylinositol elutes first followed by monogalactosyldiacylglycerol, N-acylphosphatidyle-thanolamine, N-acyl-lysophosphatidylethanolamine, phosphatidylethanolamine, and digalactosyldiacyl-glycerols. 

For the group separation of polar lipids, the packing material should be as inert as possible. Otherwise lipids such as phosphatidylethanolamine (PE) are eluted as tailing peaks. However, when there is some residual adsorptive activity from surface silanol groups on the packing these can be partially deactivated when the mobile phase contains an additive that has a deactivating ability, for example methanol. Class separation of moderately polar lipids, under subcritical conditions, was obtained on columns packed with diol-modified silica and a mobile phase modified with 19 mol% methanol (Fig. 2.5). Also, lipids with greater polarity, such as PE, could be... 

Chloroform solutions of dioleoylphosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), dioleoylphosphatidic acid (DOPA), and phosphatidylinositol (PI) from soybean are purchased from Avanti Polar Lipids (Alabaster, AL). Phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-biphosphate (PIP2), and cytidine 5 -diphosphate-diacylglycerol (CDP-DAG) are obtained from Sigma (St. Louis, MO). Cholesterol is purchased from Nacalai... 

NAPE has been reported from a variety of species. The highest concentration described was 70% of the total polar lipids of soybean seeds radiolabeled with acetate or pyruvate [4]. We found that NAPE comprised about 7.2% of the total microsomal phospholipid by weight, and when [ C]ethanolamine was introduced to a variety of tissues, as much as 14% of the label could be found in NAPE, with the majority of the remainder in phosphatidylethanolamine [5]. 

No evidence of exposed polar lipid groups could be obtained in several membranes, including mitochondria, by Weiss et al. (1968). Yet, two of the methods used were able to estimate phosphotidy-lethanolamine in the presence of phosphatidylcholine in bilayers. Since mitochondrial membrane lipids include a large proportion of these phospholipids, little, if any phosphatidylethanolamine would be exposed in this membrane (Papahadjopoulos and Weiss, 1969). This observation would appear to rule out the membrane model of Green et al. (1967) and that of Glaser et al. (1970). 

Chromatographic Separation of Lipids A mixture of lipids is applied to a silica gel column, and the column is then washed with increasingly polar solvents. The mixture consists of phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, cholesteryl palmitate (a sterol ester), sphingomyelin, palmitate, -re-tetradecanol, triacylglycerol, and cholesterol. In what order do you expect the lipids to elute from the column Explain your reasoning. 

Answer Because silica gel is polar, the most hydrophobic lipids elute first, the most hydrophilic last. The neutral lipids elute first cholesteryl palmitate and triacylglycerol. Cholesterol and %-tetradecanol, neutral but somewhat more polar, elute next. The neutral phospholipids phosphatidylcholine and phosphatidylethanolamine follow. Sphingomyelin, neutral but slightly more polar, elutes after the neutral phospholipids. The negatively charged phosphatidylserine and palmitate elute last—phosphatidylserine first because it is larger and has a lower charge-to-mass ratio. 

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