Fatty acids of bovine milk glycolipids and phospholipids and their specific distribution in the diacylglycerophospholipids

Milk lipids were fractionated by silicic acid column chromatography and preparative thinlayer chromatography (TLC). Ceramide monohexoside (CMH), ceramide dihexoside (CDH), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl serine (PS), and sphingomyelin (Sph) were isolated, and the purity of each was checked by infrared spectroscopy and TLC. The diacylphospholipids were hydrolyzed with phospholipase A and the products separated by TLC. Fatty acid methyl esters were prepared from the various fractions and analyzed by gas chromatography. The glycolipids, CMH and CDH, and Sph contained large amounts of long-chain saturated fatty acids, especially C_22:0, C_23:0, and C_24:0, PE, PS, and PC contained C₁₀-C₂₂ normal and branched-chain saturated fatty acids, and C₁₅-C₂₀ unsaturated fatty acids (mainly monoenes). The distributions of saturated acids between the α′- and β-positions were respectively: PE, 46 and 11%; PS, 65 and 19%; and PC, 72 and 53%. PC was exceptional in that there was 10.8% myristic acid in the β-position and only 5.6% in the α′-position. PE and PS were similar in composition except that in PE oleic acid was evenly distributed, and in PS was largely in the β-position. In general, PC was much more saturated than PE or PS, and there was no overall pattern governing the specific distribution of the fatty acids in the three diacylphospholipids. Comparison with PC from other bovine tissues and from egg lecithin showed that fatty acids are located much less specifically in milk phospholipids than in PC from other sources. Presented at the AOCS Meeting, Houston, Texas, April, 1965.     

Lipid components of borage (Borago officinalis L.) seeds and their changes during germination

The changes in composition of total and neutral lipids (NL) as well as glycolipids (GL) and phospholipids (PL) of borage (Borago officinalis L.) seeds, germinated in the dark at 25^°C for 10 d, were studied. Total lipids constituted 34.0% of the dry matter of borage seeds. During germination, the content of total lipids was decreased by 95%. NL accounted for 95.7% of total lipids prior to germination and were composed of triacylglycerols (TAG; 99.1%), diacylglycerols (DAG; 0.06%), monoacylglycerols (MAG;0.02%), free fatty acids (FFA;0.91%), and sterols (0.02%). The content of TAG was significantly (P≤0.05) decreased, while that of other components, such as MAG and FFA, significantly (P≤0.05) increased during germination. However, the content of DAG did not change. GL and PL accounted for 2.0 and 2.3% of total lipids, respectively, and their contents significantly (P≤0.05) increased as germination proceeded. The thin layer chromatography-flame-ionization detection studies showed that phosphatidylcholine (PC; 69.7%) was the major PL present. The total content of phosphatidylserine (PS) and phosphatidylethanolamine (PE), which were coeluted, was 18.2%; phosphatidic acid (PA) was present at 11.2% of the total PL fraction. Lysophosphatidylcholine was detected at 0.9%. The proportion of PC, PS, and PE significantly (P≤0.05) decreased during germination, but that of PA increased (P≤0.05) markedly. The fatty acid composition of lipid fractions changed as germination proceeded. The predominant fatty acids of total lipids, NL, and GL were linoleic and linolenic acids, while those of PL were linoleic and palmitic acids. The present study demonstrated that the overall changes of lipids seen in borage seeds during germination agree well with results for other oilseeds. Changes in lipid compositions during germination result from the formation of tissues and metabolic interconversion of lipid classes. Rapid changes in lipid composition during seed germination may enhance the nutritional value of the sprouts.     

Fatty acid composition of late embryonic and early postnatal rat brain

The fatty acid (FA) composition and distribution in a variety of phospholipids (PL) and neutral lipids (NL) at two discrete stages during the embryonic rat brain development were investigated. Over 96% of the FA were acylated into fetal brain PL at embryonic day 17 after the peak of neuronal proliferation and at embryonic day 20, one day prior to delivery. Phosphatidylcholine constituted approximately 60% of the total PL pool, phosphatidylethanolamine (PE) 30%, phosphatidylserine (PS) 6%, and phosphatidylinositol (PI) 4%. The diacylglycerols and triacylglycerols constituted 1–2% of the fetal brain lipids. α-Linolenic acid (18∶3n−3) and linoleic acid (18∶2n−6) were found in very low amounts in all fetal brain PL and NL. The percentage of the n−6 polyunsaturated FA, consisting of arachidonic acid (AA), 22∶4n−6 and 22∶5n−6, remained unchanged in all the fractions, except in Pl, in which the proportion of AA increased. The concentration of docosahexaenoic acid (DHA) increased with age in all the fractions, with the bulk of accumulation accounted for by its increase in PE and, to a lesser extent, in PS. This finding suggests a “DHA accretion spurt” during the last three days of pregnancy.     

Lipid composition of erythrocytes and thrombocytes of a subantarctic seabird, the king penguin

Phospholipid (PL) compositions and fatty acid (FA) patterns of PL were determined in the erythrocytes and blood thrombocytes of a seabird, the king penguin, living in the subantarctic area and feeding on prey rich in n−3 polyunsaturated FA. Results were compared between birds in three different physiological states (breeding and molting adults, chicks) to those reported for other birds. In erythrocytes, the ratios of cholesterol to PL and of sphingomyelin to phosphatidylcholine (PC) were lower than in other birds. The PL distribution was similar to those previously reported in the hen and pigeon. In contrast to other birds, cardiolipin levels were unexpectedly high (4%). Very long chain n−3 FA were abundant (13–27%) in phosphatidylethanolamine (PE), phosphatidylserine and PC, probably in relation to the natural diet of these birds. Among n−3 FA, 22∶6n−3 was the most abundant in all PL (2−20%), whereas the highest levels of arachidonic acid were observed in PE (14%). In thrombocytes, the PL distribution and FA composition of the main PL (PC, PE) differed from those of erythrocytes, and in particular, levels of n−3 FA (9–12%) were 1.5–2 times lower. The highest levels of arachidonic acid were found in phosphatidylinositol (24%). The lipid profile of penguin erythrocytes could contribute to the efficiency of blood circulation and oxygen delivery in microvascular beds, thus favoring diving capacity of these animals. Our observations do not support the hypothesis of a common origin of avian thrombocytes and erythrocytes.     

Composition and metabolism of fatty acids in phospholipids of density-separated red cells of rats

Fatty acid compositions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and the rates of fatty acid esterification to these phospholipids (PL) were measured in intact rat red cell populations of different ages separated by density gradient centrifugation in order to clarify changes in membrane lipids of red blood cells during in vivo aging. Fatty acid compositions of PC and PE altered progressively as red cells became denser. Changes in unsaturated fatty acids occurred predominantly at the 2-position of PC and PE and those in saturated fatty acids at both positions. The esterification rates of 5 major fatty acids decreased as red cells became denser and those of oleic acid, linoleic acid and arachidonic acid to both PC and PE of fraction I cells (oldest cells) were 37–51% those of fraction IV cells (youngest cells). Reduction in the rates of fatty acid esterification appeared to occur in the course of red cell maturation because reticulocyte-enriched cell fractions showed 4.5–14.5 times higher rates of linoleic acid and arachidonic acid esterifications to PC and PE.     

Natural and accelerated docosahexaenoic acid accumulation in the prenatal rat brain

The quantity and distribution of docosahexaenoic acid (DHA) in major brain phospholipids (PL) was examined in the fetal rat brain before birth, using thin-layer and capillary column gas chromatography. A rapid increment of DHA content of about 187 μg/g brain/day was observed between 17 to 20 days gestation, as opposed to 39.3±2.9 μg/g brain/day prior to that. Single intraamniotic injections of 5 μL ethyl-docosahexaenoate (Et-DHA) 12 μM, 4.25 mg) administered to 17-day-old fetuses were used to examine the uptake of DHA into brain PL. Three days following injection, the amount of n-3 polyunsaturated fatty acids increased by 28% compared to ethyl-oleate (Et-Ole) injected fetuses. Compared to the n-6 fatty acid family, the relative amount of DHA increased in the phosphoatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine (PC) lipids by 15 (P=0.02), 13 and 14%, respectively. A major increase in the pool size of phosphatidylinositol and PS (110 and 50.3%, respectively), and a decrease in PC (8.2%) were observed 3 d after Et-DHA as compared to Et-Ole administration. The data suggest that a single intraamniotic administration of Et-DHA can modulate membrane PL content and alter PUFA composition.     

Separation and analysis of phospholipids in different foods with a light-scattering detector

A method for the separation of phospholipids (PL) from total lipids by solid-phase extraction (SPE) with reversephase C₈ cartridges is described. The method was validated with a standard mixture of PL and applied to natural food matrixes, such as egg, chicken meat, salami, and ripened cheese. The recovery of PL ranged between 93 and 99.7% and was evaluated by an organic phosphorus spectrophotometric determination. The egg powder PL fraction obtained by SPE contained about 20% (w/w) nonpolar PL material when 100–150 mg of lipids were loaded onto the cartridge. Higher percentages of nonphospholipid components (30–43%) were obtained when the amount of lipids loaded was below or above the 100–150 mg range. The purified PL fractions were analyzed by high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector. Good HPLC performance was observed even with low-purity SPE fractions (43% nonphospholipid material).     

The enzymatic hydrolysis of triglyceride-phospholipid mixtures in an organic solvent

The abilities of four commercially available lipolytic enzymes [three immobilized lipases—Lipozyme IM-20, SP-435 (Novo Biolabs, Danbury, CT), and AY-30/Celite (Amano Enzyme Co., Ltd., Troy, VA)—and a nonimmobilized Amano phospholipase B preparation] to hydrolyze mixtures of triacylglycerols (TG) and phospholipids (PL) were determined. All of the lipases hydrolyzed both types of substrates in water, with maximum rates of TG hydrolysis exceeding those of PL hydrolysis by between 20- and 200-fold. The phospholipase B preparation was inactive against both TG and PL in water. All the enzymes showed some activity against lipids in hexane. The amount of activity was sharply dependent on the amount of water added to the reaction. Lipozyme IM-20 and AY-30/Celite hydrolyzed both TG and PL in hexane. Their estimated initial activities were between 10- and 100-fold lower than those in water. Complete hydrolysis of the TG (measured as the hydrolysis of at least one ester bond in each molecule) was achieved, whereas only 40–60% of the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed. Lipase SP-435 was inactive against TG in hexane but hydrolyzed PC at a rate comparable to that seen in water, and it achieved complete hydrolysis of this substrate. Amano phospholipase B was inactive against TG in hexane but completely hydrolyzed the PC. The abilities of the enzymes to hydrolyze the TG, PC, and PE components of soybean soapstock, a by-product of edible oil production, were also examined. Lipozyme IM-20 hydrolyzed all the TG and a fraction of the PL in soapstock. SP-435 and AY-30/Celite were active only on soapstock that had been acidified prior to being dissolved in hexane. SP-435 displayed significant activity only toward PE under these conditions, whereas AY-30/Celite was active only toward TG. Phospholipase B was inactive against soapstock in hexane. The identity of the acid used in the acidification of soapstock affected the degree of hydrolysis by AY-30/Celite, with nitric and hydrochloric acids giving the best activity.     

Lecithins in oil-continuous emulsions. Fat crystal wetting and interfacial tension

Lecithin is a powerful emulsifier widely used in foods, feeds and pharmaceuticals. Several analytical methods have been proposed to characterize lecithins, but they are often inadequate to determine the industrial functionality. The purpose of this study was to find a relationship between the interfacial properties of lecithins (adsorption to oil/water and fat crystal/oil/water interfaces), phospholipid composition and functionality. Results show that all lecithins adsorb to fat crystals at the triglyceride oil/water interface, making their surface more polar (observed as an increase in the contact angle measured through the oil at the interface: fat crystal/oil/water). This adsorption process is quick (less than five minutes) for relatively polar lecithins, such as soybean phosphatidylcholine (PC), and results in highly polar surfaces (contact angle ∼180°). Less polar lecithins give slow adsorption (some hours) and less polar crystals (contact angle ≤90°). The adsorption of different lecithins to the oil/water interface, observed as a decrease in interfacial tension, follows the adsorption pattern to the fat crystals. We found a relation between high-fat crystal polarity and poor lecithin functionality in margarine (margarines spatters during frying), and also between high-fat crystal polarity and a high polar to nonpolar phospholipids [Σ(PI + PA + LPC)/ΣPE; PI, phosphatidylinositol; PA, phosphatidic acid; LPC, lysoPC, PE, phosphatidylethanolamine] ratio in lecithin. The correlations might bevia aggregation properties of lecithin in the oil. We found also that monoolein shifted the adsorption kinetics of lecithin (soybean PC) to fat crystals and the hydrophilicity of adsorbed layers probably due to formation of mixed aggregates between monoolein and soybean PC.     

Properties of diacylglycerol kinase purified from bovine brain

A nearly homogeneous but somewhat unstable diacylglycerol kinase (ca. MW 72,000 daltons) was purified from bovine brain by modification of the procedure of Kanoh et al. (Kanoh, H., Kondoh, H., and Ono, T. [1983]J. Biol. Chem. 258, 1767–1774). The purification consisted of four steps (brain cytosol isolation and successive chromatography on DEAE-cellulose, Sephadex G-25 for desalting and ATP-agarose) carried out in buffers stabilized with EDTA, ATP and dithiothreitol (DTT). Specific activities, determined within 4 hr of purification, ranged from 908–1857 nmol ATP incorporated/min/mg protein, with the variation reflecting the instability. Optimal activities required deoxycholate (0.1%), one of the phosphoglycerides [phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylserine (PS)] (0.025–0.25 mM), ATP (5 mM, apparent K_m=0.57 mM), 1,2-dioleoyl-rac-glycerol (5 mM, apparent K_m=1 mM) and Mg²⁺ (10 mM, apparent K_m=2.2 mM). Phosphatidylinositol (PI) was slightly less effective than PC, PE or PS and noninhibitory in combination with PC, PE or PS. Relative to PC phosphatidic acid (PA) (52%), sphingomyelin (48%), lyso-PC (1.5%) and lyso-PI (28.6%) were less effective activators. The sulfhydryl reagents,p-chloromercuribenzoic acid (PCMB) (1.0 mM),N-ethylmaleimide (NEM) (1.0 and 2.0 mM) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) (1.0 mM), showed strong inhibition of activity which was prevented by 0.5 mM DTT. In contrast to other reports, this purified enzyme showed no monoacylglycerol kinase activity. Comparison of diacylglycerols of varying fatty acid composition indicated that the enzyme showed a preference for substrates with at least one unsaturated fatty acid, particularly in the 2-position. With saturated fatty acids the order of preference was C₁₀ and C₁₂>C₁₄>C₁₆>C₁₈. Such a pattern indicates that the enzyme shows little selectivity that favors the generation of particular molecular species of PA.     

Effect of FA chain length on normal- and reversed-phase HPLC of phospholipids

Forty-seven saturated synthetic diacyl PA, PC, PE, PG, and PS and five unsaturated diacyl phospholipids (PL) underwent normal- and reversed-phase (RP) HPLC with isocratic isopropanol/hexane/water (5∶4∶1) and methanol/chloroform/acetonitrile/water (79.5∶9∶8∶3.5) mobile phases, respectively. For normal-phase HPLC, capacity factors (k′ _i ) decrease with chain length (n) of the two identical PL FA residues, whereas the opposite occurs with RP (C₁₈)-HPLC. Plots of In k′ _i vs. n for individual PL classes are in general curved, violating the linear free-energy relationship. For PL of the same n but with different head groups, k′ _i with normal-phase HPLC varies as PE<PG<PA<PS<PC, except when n≥16, when the order is PE<PS≈PA≈PG<PC. For RP-HPLC, the order of k′ _i values is PG<_A≈PS≤PC≈PE until n≥16, when it is PA≈PG<PS≪PC≈PE. With normal-phase HPLC, k′ _i values of PL with unsaturated FA of n=18 are ordered as PE<PA<PC. Increasing degrees of unsaturation lead to increasing k′ _i .     

Lipid analyses of isolated surface membranes ofLeishmania Donovani promastigotes

Constituent lipids of surface membranes (SM) isolated fromLeishmania donovani promastigotes were analyzed and compared with those obtained from whole cells and an isolated kinetoplast-mitochondrion fraction (KM). On a dry weight basis, the total extractable lipids constituted ≈47%, 12% and 24% of the SM, cells and KM, respectively. The total lipids of SM, cells and KM all were composed of ≈70% phospholipids (PL), 20–25% neutral lipids and 5–10% glycolipids. Sterols and diglycerides composed 60% and 30%, respectively, of the various neutral lipid fractions. Several mannose- and galactose-containing glycolipids were fractionated but not identified. The glycolipid fractions from cells and SM had demonstrable antigenic activities with rabbit anti-SM sera. Striking quantitative differences were apparent between the PL profiles of the 3 cellular components examined. The PL of SM, whole cells and KM, respectively, were composed of: 15%, 51% and 24% phosphatidylcholine; 37%, 13% and 11% phosphatidylethanolamine (PE); 18%, 10% and 14% phosphatidylinositol; 10%, 1% and 4% phosphatidylserine and traces of cardiolipin, phosphatidylglycerol and phosphatidic acid. An unknown PL containing sphingosine, choline and vicinal hydroxyl groups but no free amino moieties made up ≈19% of the PL of SM and whole cells, but it constituted ≈27% of the PL of KM. The PL side chain constituents of whole cells and SM were composed mainly of longchain fatty acids (C18–20). Further, over 50% of the PE of SM was in the alkyl and alK-1-enyl ether forms. These SM properties might contribute to the organism's resistance to digestion in the hydrolytic environs of both its insect vector and mammalian hosts.     

On the specificity of a phospholipase A2 purified from the 106,000×g pellet of bovine brain

Assessment has been made of the specificity of a purified phospholipase A₂ from the 106,000×g pellet (microsomal fraction) of bovine grey matter which shows strong activity toward phosphatidylinositol (PI). In the first series of experiments involving the utilization as substrates of PI with different¹⁴C- or³H-labeled fatty acids in the 2-position, the purified phospholipase A₂ most readily removed 16∶0 palmitic acid, followed by 18∶0 stearic acid, 18∶1 oleic acid and 20∶4 arachidonic acid. In the second series of experiments, the purified phospholipase A₂ showed preferential action toward PI (100%) compared to phosphatidylcholine (PC, 62.5%), phosphatidic acid (PA, 32.6%), phosphatidylethanolamine (PE, 25.1%) and phosphatidylserine (PS, 21.5%), where each phosphoglyceride was labeled in the 2-position with [1-¹⁴C] oleic acid. In the third series of experiments, fatty acids were shown to cause inhibition of action of the purified phospholipase A₂ on 1-acyl, 2-[1-¹⁴C] oleoyl PI in the order 20∶4>18∶1>18∶0>16∶0 which is the reverse order to that just noted. In the final series of experiments, the addition of the phosphoglycerides PC, PE, PS and PA in amounts of 5 or 10 μM caused either no inhibition (PE, 2%), slight inhibition (PC, 15%) or reasonably significant inhibition (PA, 20% and PS, 40%) of action of the purified phospholipase A₂ on 1-acyl, 2-[1-¹⁴C]-oleoyl PI. The pattern of specificity observed for the purified phospholipase A₂ combined with its microsomal location are the expected properties of a phospholipase A₂ that might function in a deacylation-reacylation cycle for modifying the fatty acid distribution in PI.     

Enzymatic method of increasing phosphatidylcholine content of lecithin

An enzymatic method was established to increase the phosphatidylcholine (PC) contents of soybean and egg lecithins. Other phospholipids of lecithin were phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidic acid (PA). Seven preparations of phospholipase D (PLD), PLD-1 to PLD-6 ofStreptomyces origin and PLD-7 of cabbage origin, were tested for their ability to increase PC by transphosphatidylation in the presence of choline chloride (CC). The reactions were carried out at 30 C in a biphasic system that consisted of an aqueous phase containing PLD along with a buffer (optimum pH) having desired concentration of CC and Ca²⁺ and an ethyl acetate phase containing lecithin phospholipids. Intermitttent samples were extracted and analyzed by HPLC. Four of six PLD’s ofStreptomyces origin showed good transphosphatidylation (increase of PC contents of soybean lecithin from approximately 35% to 60–70% on a phospholipid basis) at 2.5 M CC, but the other two microbial PLD’s completely hydrolyzed the phospholipids to PA. Cabbage PLD-7 showed poor transphosphatidylation. PLD-3 gave the highest PC contents (70%) at 1.75 M CC. One hundred percent transphosphatidylation of pure PE to PC was achieved with PLD-3. PI was inert to the attack of most PLD preparations examined with the exception that PLD-3 hydrolyzed PI significantly. Purified PI could not be transphosphatidylized to PC; 100% PA was formed. Soybean lecithin containing about 80% PC and purified egg yolk lecithin with 75% PC could be converted to products having 95% PC and almost 100% PC, respectively, by PLD-3 at 1.75M CC. Studies on Enzymatic Conversion of Phospholipids (v)     

New findings in the fatty acid composition of individual platelet phospholipids in man after dietary fish oil supplementation

Nine healthy male volunteers were given 15 Max EPA fish oil capsules providing 2.67 g of eicosapentaenoic acid (EPA, 20∶5ω3) and 1.72 g of docosahexaenoic acid (DHA, 22∶6ω3) daily for 3 wk. Measurements were taken at baseline, at the end of the fish-oil period, and at 2 and 6 wk postsupplementation. The effect of fish oil on plasma lipids and the fatty acid composition of individual platelet phospholipids was studied. In general, the proportions of 20∶5ω3 and 22∶6ω3 in platelet phosphoglycerides were substantially increased mainly at the expense of arachidonic acid (AA, 20∶4ω6). A large and significant increase in the relative EPA content of phosphatidylcholine (PC) (P<0.001) and phosphatidylethanolamine (PE) (P<0.001) was noted at the end of the 3 wk supplementation. We have also shown for the first time a small but significant (P<0.001) incorporation of EPA in phosphatidylserine (PS). Incorporation of DHA was also detected in PC, PE and PS, whereas the relative AA content of these phospholipids was significantly reduced. Fish oil supplementation led to a significant increase of 22∶5ω3 in PS and decreases of 20∶3ω6 in PC and 22∶4ω6 in PE. Postsupplementation measurements showed a gradual return of all fatty acids to baseline levels. The fatty acid composition of the phosphatidylinositol (PI) fraction remained unchanged throughout the trial period. We conclude that in humans ω3 fatty acids are incorporated into platelet membrane phospholipid subclasses with a high degree of specificity.     

Rapid separation of the major phospholipid classes on a single aminopropyl cartridge

A rapid method for the separation of the individual phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) by a single solid-phase extraction was developed. PC, PE, PS and PI were sequentially eluted from aminopropyl bonded silica with acetonitrile/n-propanol (2∶1, vol/vol), methanol, isopropanol/methanolic HCl (4∶1, vol/vol) and methanol/methanolic HCl (9∶1, vol/vol). Standard recoveries were over 95% for PC and PE and over 85% for PS and PI with undistorted fatty acid composition. The separation of complex lipid mixtures on aminopropyl minicolumns can be refined to the level of individual phospholipid classes.     

Light-scattering detection of phospholipids resolved by HPLC

An improved method for the analysis of phospholipids by normal-phase HPLC is described. Addition of methanol and acetonitrile to a gradient based on 2-propanol/hexane/water promoted a rapid separation of major classes of bovine surfactant phospholipids (PL) by using a conventional silica column. The use of an ELSD permitted an accurate analysis of a mixture of PL. Calibration curves were linear within the range of 5–40 μg with detection limits below 1 μg for PE and PC, and CV ranged from 0.6 to 9.6%. PL present in surfactant homogenates were separated by a solid-phase extraction (SPE) procedure before HPLC analysis. This methodology gave a recovery of 95% and combined SPE-HPLC and quantification of biological PL within a 30-min run. The use of ELSD detection of the eluted compounds was precise, linear, and sensitive.     

Molecular species composition of the major diacyl glycerophospholipids from muscle,liver, retina and brain of cod (Gadus morhua)

The molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) from white muscle, liver, retina and brain of cod (Gadus morhua) were determined by high-performance liquid chromatography of the respective 1,2-diacylglycerol 3,5-dinitrobenzoyl derivatives. A minimum of 69 diacyl species was identified. In muscle and liver saturated fatty acid/polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid/PUFA molecular species were predominant, particularly 16∶0/20∶5 and 16∶0/22∶6 in PC, 16∶0/22∶6 and 18∶1/22∶6 in PE and 18∶0/22∶6 and 18∶1/22∶6 in PS. Didocosahexaenoyl species were major components of PC, PE and PS from retina, comprising 29.3, 71.8 and 59.7% of the respective totals. Didocosahexaenoyl species were also abundant in PE and PS from brain, accounting for 13.8 and 24.0% of the totals, respectively. DiPUFA species were important in muscle, totalling 21.2% in PC and 38.3% in PE. PC from all tissues had the largest amounts of species containing only saturated or monounsaturated fatty acids, accounting for 59.8% of PC from brain, including 12.8% of 18∶1/24∶1 plus 24∶1/18∶1.     

High-Performance Thin-Layer Chromatography-Densitometry-Tandem ESI-MS to Evaluate Phospholipid Content in Exosomes of Cancer Cells

The question of whether exosome lipids can be considered as potential cancer biomarkers faces our current limited knowledge of their composition. This is due to the difficulty in isolating pure exosomes, the variability of the biological sources from which they are extracted, and the uncertainty of the methods for lipid characterization. Here, we present a procedure to isolate exosomes and obtain a deep, repeatable, and rapid phospholipid (PL) composition of their lipid extracts, from embryonic murine fibroblasts (NIH-3T3 cell line) and none (B16-F1) and high (B16-F10) metastatic murine skin melanoma cells. The analytical method is based on High Performance Thin-Layer Chromatography with Ultraviolet and fluorescence densitometry and coupled to Electrospray (ESI)-tandem Mass Spectrometry (MS). Under the conditions described in this work, separation and determination of PL classes, (sphingomyelins, SM; phosphatidylcholines, PC; phosphatidylserines, PS; and phosphatidylethanolamines, PE) were achieved, expressed as µg PL/100 µg exosome protein, obtained by bicinchoninic acid assay (BCA). A detailed structural characterization of molecular species of each PL class was performed by simultaneous positive and negative ESI-MS and MS/MS directly from the chromatographic plate, thanks to an elution-based interface.     

Lipidic characterization of full-grown amphibian oocytes and their plasma membrane-enriched fractions

The content and composition of neutral lipids and phosphoglycerides from full-grown prophase-arrestedBufo arenarum Hensel oocytes and from their ghost preparations were studied. The ghosts obtained are highly enriched in plasma membrane as suggested by the activity of 5′-nucleotidase, a marker enzyme, and the level of typical membrane components such as sphingomyelin, phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid. In whole oocytes, triacylglyceride (TAG) comprises about 60% of the total lipids followed by phosphatidylcholine (PC), cholesterol, and phosphatidylethanolamine (PE). TAG and diacylglycerides have a similar unsaturation index. PC and PE account for about 80% of the phosphoglycerides in the whole oocyte and in their plasma membrane-enriched fractions. Arachidonic acid (20∶4n−6), 18∶0, and 16∶0 make up about 80 mol% of the total fatty acids in Pl in whole oocytes and ghost fractions. The unsaturation index in PS is higher in intact oocytes than in ghost preparations, probably owing to the significant amount of 20∶4n−6 which comprises 23 mol% of the total fatty acids in whole oocytes. The fatty acid profile in phosphatidic acid from whole oocytes is rather different from that in ghosts. Sphingomyelin contains mainly saturated and monounsaturated fatty acids, 24∶1 being the principal very long chain unsaturated fatty acid in both oocytes and ghosts.