Determination of substrate preference in phosphatidylserine decarboxylation by liquid chromatography-electrospray ionization mass spectrometry

A method has been developed to determine the substrate preference in phosphatidylserine decarboxylation (PSD), the process by which phosphatidylserine is converted to phosphatidylethanolamine (PE) in the mitochondria. The in vitro assay utilized liposomes containing deuterium-labeled PS molecular species incubated with liver and brain cortex mitochondria, and the conversion of PS to the corresponding PE species was monitored by electrospray ionization mass spectrometry in conjunction with reversed-phase liquid chromatography. Employing this approach we were able to establish for the first time that there exists a substrate preference in PSD in liver (18:0,18:1 > or = 18:0,22:6 > 18:0,20:4-PS) and brain cortex (18:0,22:6 > 18:0,18:1 > 18:0,20:4-PS). The observed PSD molecular species preference, however, did not reflect the mitochondrial PE profile, suggesting that selectivity in other processes such as de novo PE synthesis, intracellular transport of phospholipid molecules, or remodeling by deacylation-reacylation may be important contributors in maintaining a specific lipid profile in mitochondria.     

Differential effects of n-3 fatty acid deficiency on phospholipid molecular species composition in the rat hippocampus

In this study, we have examined the effects of n-3 fatty acid deficient diets on the phospholipids (PL) molecular species composition in the hippocampus. Female rats were raised for two generations on diets containing linoleic acid (18:2n-6), with or without supplementation of alpha-linolenic acid (18:3n-3) or 18:3n-3 plus docosahexaenoic acid (22:6n-3). At 84 days of age, the hippocampal phospholipids were analyzed by reversed phase HPLC-electrospray ionization mass spectrometry. Depleting n-3 fatty acids from the diet led to a reduction of 22:6n-3 molecular species in phosphatidylcholine (PC), phosphatidylethanolamine (PE), PE-plasmalogens (PLE), and phosphatidylserine (PS) by 70-80%. In general, 22:6n-3 was replaced with 22:5n-6 but the replacement at the molecular species level did not always occur in a reciprocal manner, especially in PC and PLE. In PC, the 16:0,22:6n-3 species was replaced by 16:0,22:5n-6 and 18:0,22:5n-6. In PLE, substantial increases of both 22:5n-6 and 22:4n-6 species compensated for the decreases in 22:6n-3 species in n-3 fatty acid deficient groups. While the total PL content was not affected by n-3 deficiency, the relative distribution of PS decreased by 28% with a concomitant increase in PC.The observed decrease of 22:6n-3 species along with PS reduction may represent key biochemical changes underlying losses in brain-hippocampal function associated with n-3 deficiency.     

Effect of Docosahexaenoic Acid on the Synthesis of Phosphatidylserine in Rat Brain Microsomes and C6 Glioma Cells

Abstract: Docosahexaenoic acid (22:6n-3) is the major polyunsaturated fatty acid (PUFA) in the CNS and accumulates particularly in phosphatidylserine (PS). We have investigated the effect of the 22:6n-3 compositional status on the synthesis of PS. The fatty acid composition of brain microsomes from offspring of rats artificially reared on an n-3-deficient diet showed a dramatic reduction of 22:6n-3 content (1.7 ± 0.1%) when compared with control animals (15.0 ± 0.2%). The decrease was accompanied by an increase in docosapentaenoic acid (22:5n-6) content, which replaced the 22:6n-3 phospholipids with 22:5n-6 molecular species, as demonstrated using HPLC/electrospray mass spectrometry. The n-3 deficiency did not affect the total amount of polyunsaturated phospholipids in brain microsomes; however, it was associated with a decrease in the total polyunsaturated PS content and with increased levels of 1-stearoyl-2-docosapentanoyl (18:0/22:5n-6) species, particularly in phosphatidylcholine. Incorporation of [³H]serine into PS in rat brain microsomes from n-3-deficient animals was slightly but significantly less than that of the control animals. Similarly, C6 glioma cells cultured for 24 h in 22:6n-3-supplemented media (10–40 µ M ) showed a significant increase in the synthesis of [³H]PS when compared with unsupplemented cells. Our data show that neuronal and glial PS synthesis is sensitive to changes in the docosahexaenoate levels of phospholipids and suggest that 22:6n-3 may be a modulator of PS synthesis.     

Optimization of receptor-G protein coupling by bilayer lipid composition II: formation of metarhodopsin II-transducin complex.

The visual transduction system was used as a model to investigate the effects of membrane lipid composition on receptor-G protein coupling. Rhodopsin was reconstituted into large, unilamellar phospholipid vesicles with varying acyl chain unsaturation, with and without cholesterol. The association constant (K(a)) for metarhodopsin II (MII) and transducin (G(t)) binding was determined by monitoring MII-G(t) complex formation spectrophotometrically. At 20 degrees C, in pH 7.5 isotonic buffer, the strongest MII-G(t) binding was observed in 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC), whereas the weakest binding was in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) with 30 mol% cholesterol. Increasing acyl chain unsaturation from 18:0,18:1PC to 18:0,22:6PC resulted in a 3-fold increase in K(a). The inclusion of 30 mol% cholesterol in the membrane reduced K(a) in both 18:0,22:6PC and 18:0,18:1PC. These findings demonstrate that membrane compositions can alter the signaling cascade by changing protein-protein interactions occurring predominantly in the hydrophilic region of the proteins, external to the lipid bilayer. These findings, if extended to other members of the superfamily of G protein-coupled receptors, suggest that a loss in efficiency of receptor-G protein binding is a contributing factor to the loss of cognitive skills, odor and spatial discrimination, and visual function associated with n-3 fatty acid deficiency.     

Optimization of receptor-G protein coupling by bilayer lipid composition I: kinetics of rhodopsin-transducin binding.

The role of membrane composition in modulating the rate of G protein-receptor complex formation was examined using rhodopsin and transducin (G(t)) as a model system. Metarhodopsin II (MII) and MII-G(t) complex formation rates were measured, in the absence of GTP, via flash photolysis for rhodopsin reconstituted in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC) bilayers, with and without 30 mol% cholesterol. Variation in bilayer lipid composition altered the lifetime of MII-G(t) formation to a greater extent than the lifetime of MII. MII-G(t) formation was fastest in 18:0,22:6PC and slowest in 18:0,18:1PC/30 mol% cholesterol. At 37 degrees C and a G(t) to photolyzed rhodopsin ratio of 1:1 in 18:0,22:6PC bilayers, MII-G(t) formed with a lifetime of 0.6 +/- 0.06 ms, which was not significantly different from the lifetime for MII formation. Incorporation of 30 mol% cholesterol slowed the rate of MII-G(t) complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-G(t) formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-G(t) formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system.     

Alterations in hippocampal phospholipid profile by prenatal exposure to ethanol

It has been suggested that hippocampus-related cognitive processes are especially sensitive to ethanol. To provide an insight into the biochemical mechanisms underlying the hippocampus-related functional deficits associated with prenatal ethanol exposure, we investigated the effects of chronic ethanol exposure on the phospholipid profile in developing rat hippocampi. High-performance liquid chromatography/electrospray ionization-mass spectrometry analysis revealed that ethanol lowered the levels of total phosphatidylserine (PS) by 15-20% at all ages examined, primarily owing to the reduction in 1-stearoyl-2-docosahexaenoyl-PS (18:0,22:6n-3-PS) species. Ethanol exposure also led to a decrease in phosphatidylcholine (PC) and an increase in phosphatidylethanolamine (PE), but the total phospholipid content was not significantly changed. At the fatty acid level, ethanol exposure significantly decreased the 22:6n-3 content at postnatal days 0 and 21, with a slight increase in 22:5n-6, without changing the total fatty acid content significantly. In conclusion, ethanol depleted PS, especially 22:6-containing species, and PC from hippocampal membranes with concomitant increase in PE. Alteration of the phospholipid profile in the hippocampus resulting from exposure to ethanol during prenatal and developmental stages may have significant implications with respect to the cognitive dysfunction observed in fetal alcohol syndrome.     

Deficit of didocosahexaenoyl phospholipid in the eyes of larval sea bass fed an essential fatty acid deficient diet

Larval sea bass Dicentrarchus labrax of 27 days old were reared on Artemia enriched with Super Selco©, Tuna Orbital Oil or Yeast. The first diet is commonly used in mariculture for larval rearing, the second diet was designed to deliver an optimal docosahexaenoic acid (22: 6n-3) to eicosapentaenoic acid (20: 5n-3) ratio, and the third diet was deficient in docosahexaenoic acid (22: 6n-3). The eyes of these larvae were analysed after 28 days and the molecular species of the three main phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) determined. Eyes from larvae fed Artemia enriched with yeast showed large decreases in molecular species containing 22: 6n-3 compared to those supplemented with tuna orbital oil, most notably in 16: 0/22: 6n-3 PC which fell from 10.6 to 0.4%, in 22: 6n-3/22: 6n-3 and 18: 1/22: 6n-3 PE which fell from 29.6 to 0.3% and from 10.8 to 1.1% respectively, and in 22: 6n-3/22: 6n-3 PS which fell from 34.3 to 1.7%. Molecular species containing all other fatty acids, and especially 20: 5n-3, were elevated in eyes from the yeast-supplemented fish. In larvae fed Artemia enriched with Super Selco, amounts of eye 22: 6n-3/22: 6n-3 phospholipid were slightly lower in all three phospholipid classes compared to eyes from the tuna orbital oil-supplemented larvae. There was also a trend of decreased saturated fatty acid/22: 6n-3 and monounsaturated fatty acid/22: 6n-3 molecular species in all classes from the Super Selco-supplemented fish, the deficits being made up with molecular species containing 20: 5n-3 and 22: 5n-3. These results are discussed in relation to larval viability with particular respect to visual function.     

Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains

The partition coefficient Kp was measured for a headgroup-labeled phospholipid (12:0,12:0)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE (12-NBD-PE), equilibrated between LUV of a series of phosphatidylcholines (PC). Fluorescence resonance energy transfer between the 12-NBD-PE and a headgroup-rhodamine-labeled PE was used to find the equilibrium concentration of the 12-NBD-PE in the different LUV. Reliable equilibrium concentrations were obtained by monitoring the approach to equilibrium starting from a concentration below and from a concentration above the ultimate values. Using (16:0,18:1delta9)-PC as the reference lipid, Kp ranged from a high value of 1.65 favoring (16:0,18:1delta9)-PC over (16:1delta9,16:1delta9)-PC, to a low value of 0.90, favoring (22:1delta13,22:1delta13)-PC over (16:0,18:1delta9)-PC. The Kp values enabled calculation of the acyl chain contribution to the excess free energy of mixing for (12:0,12:0) acyl chains at infinite dilution in the L alpha phase of PC having acyl chains of (16:0,18:1delta9), (16:1delta9,16:1delta9), (18:1delta9,18:1delta9), (18:1delta6,18:1delta6), (20:1delta11,20:1delta11), and (22:1delta13,22:1delta13). (14:1delta9,14:1delta9)-PC was found to transfer so rapidly between LUV as to preclude reliable Kp measurement.     

Labeling of phosphatidylcholines of retina subcellular fractions by [1-14C]eicosatetraenoate (20:4(n-6)), docosapentaenoate (22:5(n-3)) and docosahexaenoate (22:6(n-3))

The labeling of molecular species of phosphatidylcholine (PC) has been studied in bovine retinas incubated for 2 h with (1-14C)-labeled (n-6) eicosatetraenoate (n-3) docosapentaenoate and (n-3) docosahexaenoate (20:4, 22:5 and 22:6, respectively) and in four subcellular fractions isolated after such incubations. Of the total radioactivity incorporated in PC, the following percentages of the above fatty acids, respectively, are found in its dipolyunsaturated species: 58, 56 and 53% in rod outer segments; 29, 41 and 49% in mitochondria; 24, 28 and 39% in microsomes; 12, 14 and 16% in postmicrosomal supernatants; 28, 36 and 58% in entire retinas. The remainder percentages are in tetra-, penta- and hexaenoic species of PC, respectively. The levels of pentaenoic species in the PCs of all fractions are similar, while tetraenes are lowest and hexaenes highest in photoreceptor membranes. Dipolyunsaturated species are highly concentrated in photoreceptor membranes, but are minor components of mitochondrial, microsomal and cytosolic PC. The specific radioactivities of tetraenoic, pentaenoic and hexaenoic PCs are decreasingly lower in the following order: postmicrosomal supernatants, microsomes, mitochondria, photoreceptor membranes. In contrast, the specific radioactivities of dipolyunsaturated PCs are higher in mitochondria and microsomes than in the other fractions, especially with 22:5 and 22:6. It is suggested that mitochondria as well as the endoplasmic reticulum could play a role in the synthesis and further modifications of dipolyunsaturated PCs before being supplied to photoreceptor membranes.     

The esterification and modification of n-3 and n-6 polyunsaturated fatty acids by hepatocytes and liver microsomes of turbot (Scophthalmus maximus)

The present study was undertaken to establish whether the formation of 22:6n-3 from 18:3n-3 and/or 20:5n-3 can occur in turbot liver and if this conversion is consistent with the operation of a Delta4 desaturase-independent pathway. At the same, time the effects of feeding a diet devoid of long chain polyunsaturated fatty acids (PUFA) on the patterns of esterification and modification of 18:3n-3, 20:5n-3 and 18:2n-6 by turbot hepatocytes and liver microsomes were examined. For this purpose, two groups of fish (25-30 g) were employed: one was fed a commercial diet containing fish oil (FO) and thus rich in long chain n-3 PUFA and the other was fed an experimental diet based on olive oil (OO). After 5 months of feeding, hepatocytes and liver microsomes isolated from individuals in the two groups of fish were incubated with [1-(14)C]-PUFA [either 18:3n-3, 20:5n-3 or 18:2n-6]. After 3 h of incubation, most radioactivity from all three radiolabelled substrates incorporated into lipids by hepatocytes and microsomes was recovered in the original substrate. The formation of desaturation products of n-3 radiolabelled substrates was higher in hepatocytes isolated from OO-fed than FO-fed fish. Small amounts of radiolabelled 22:6n-3 were formed from [1-(14)C]18:3n-3 and [1-(14)C]20:5n-3, but only by hepatocytes from fish fed OO, which also synthesised a small amount of radiolabelled 24:6n-3 from 14C-20:5n-3. Elongation products predominated over desaturation products in hepatic microsomes from both groups of fish studied, particularly in microsomes from fish fed FO. The results confirm that regardless of the long chain PUFA content of the diet, the production of 22:6n-3 in turbot liver from 18:3n-3 and/or 20:5n-3, and of 20:4n-6 from 18:2n-6, is very limited. The presence of radiolabelled 24:6n-3 in microsomes coupled with the absence of radiolabelled 22:6n-3 suggests that the formation of 22:6n-3 that does occur in turbot liver cells, may involve C24 intermediates and peroxisomal beta-oxidation.     

Brain astrocyte synthesis of docosahexaenoic acid from n-3 fatty acids is limited at the elongation of docosapentaenoic acid

The phospholipids, particularly phosphatidylethanolamine, of brain gray matter are enriched with docosahexaenoic acid (22:6n-3). The importance of uptake of preformed 22:6n-3 from plasma compared with synthesis from the alpha-linolenic acid (18:3n-3) precursor in brain is not known. Deficiency of 18:3n-3 results in a compensatory increase in the n-6 docosapentaenoic acid (22:5n-6) in brain, which could be formed from the precursor linoleic acid (18:2n-6) in liver or brain. We studied n-3 and n-6 fatty acid incorporation in brain astrocytes cultured in chemically defined medium using delipidated serum supplemented with specific fatty acids. High performance liquid chromatography with evaporative light scattering detection and gas liquid chromatography were used to separate and quantify cell and media lipids and fatty acids. Although astrocytes are able to form 22:6n-3, incubation with 18:3n-3 or eicosapentaenoic acid (20:5n-3) resulted in a time and concentration dependent accumulation of 22:5n-3 and decrease in 22:6n-3 g/g cell fatty acids. Astrocytes cultured with 18:2n-6 failed to accumulate 22:5n-6. Astrocytes secreted cholesterol esters (CE) and phosphatidylethanolamine containing saturated and monounsaturated fatty acids, and arachidonic acid (20:4n-6) and 22:6n-3. These studies suggest conversion of 22:5n-3 limits 22:6n-3 synthesis, and show astrocytes release fatty acids in CE.     

Fatty Acid Composition of Human Brain Phospholipids During Normal Development

Abstract: The fatty acid composition of phosphatidylethanolamine (PE), ethanolamine plasmalogens (EPs), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin was studied in 22 human forebrains, ranging in age from 26 prenatal weeks to 8 postnatal years. Phospholipids were separated by two-dimensional TLC, and the fatty acid methyl esters studied by capillary column GLC. Docosahexaenoic acid (22:6n-3) increased with age in PE and PC, whereas arachidonic acid (20:4n-6) remained quite constant. In EP, 22:6n-3 increased less markedly than 20:4n-6, adrenic (22:4n-6) and oleic (18:1n-9) acids being the predominant fatty acids during postnatal age. In PS, 18:1n-9 increased dramatically throughout development, and 20:4n-6 and 22:4n-6 increased only until ∼6 months of age. Although 22:6n-3 kept quite constant during development in PS, its percentage decreased due to the accretion of other polyunsaturated fatty acids (PUFAs). As a characteristic myelin lipid, sphingomyelin was mainly constituted by very long chain saturated and monounsaturated fatty acids. Among them, nervonic acid (24:1n-9) was the major very long chain fatty acid in Sp, followed by 24:0, 26:1n-9, and 26:0, and its accretion after birth was dramatic. As myelination advanced, 18:1n-9 increased markedly in all four glycerophospholipids, predominating in EP, PS, and PC. In contrast, 22:6n-3 was the most important PUFA in PE in the mature forebrain.     

Cholesterol dependent recruitment of di22:6-PC by a G protein-coupled receptor into lateral domains

Bovine rhodopsin was reconstituted into mixtures of didocosahexaenoylphosphatidylcholine (di22:6-PC), dipalmitoylphosphatidylcholine (di16:0-PC), sn-1-palmitoyl-sn-2-docosahexaenoylphosphatidylcholine (16:0, 22:6-PC) and cholesterol. Rhodopsin denaturation was examined by using high-sensitivity differential scanning calorimetry. The unfolding temperature was increased at lower levels of lipid unsaturation, but the highest temperature was detected for native disk membranes: di22:6-PC < 16:0,22:6-PC < di16:0,18:1-PC < native disks. The incorporation of 30 mol% of cholesterol resulted in 2-4 degrees C increase of denaturation temperature in all reconstituted systems examined. From the analysis of van't Hoff's and calorimetric enthalpies, it was concluded that the presence of cholesterol in di22:6-PC-containing bilayers induces a level of cooperativity in rhodopsin unfolding. Fluorescence resonance energy transfer (FRET), using lipids labeled at the headgroup with pyrene (Py) as donors and rhodopsin retinal group as acceptor of fluorescence, was used to study rhodopsin association with lipids. Higher FRET efficiencies detected for di22:6-PE-Py, compared to di16:0-PE-Py, in mixed di22:6-PC-di16:0-PC-cholesterol bilayers, indicate preferential segregation of rhodopsin with polyunsaturated lipids. The effective range of the rhodopsin-lipid interactions facilitating cluster formation exceeds two adjacent lipid layers. In similar mixed bilayers containing no cholesterol, cluster formation was absent at temperatures above lipid phase transition, indicating a crucial role of cholesterol in microdomain formation.     

Phosphatidylserine synthase 2: high efficiency for synthesizing phosphatidylserine containing docosahexaenoic acid

Phosphatidylserine (PS), the major anionic phospholipid in eukaryotic cell membranes, is synthesized by the integral membrane enzymes PS synthase 1 (PSS1) and 2 (PSS2). PSS2 is highly expressed in specific tissues, such as brain and testis, where docosahexaenoic acid (DHA, 22:6n-3) is also highly enriched. The purpose of this work was to characterize the hydrocarbon-chain preference of PSS2 to gain insight on the specialized role of PSS2 in PS accumulation in the DHA-abundant tissues. Flag-tagged PSS2 was expressed in HEK cells and immunopurified in a functionally active form. Purified PSS2 utilized both PE plasmalogen and diacyl PE as substrates. Nevertheless, the latter was six times better utilized, indicating the importance of an ester linkage at the sn-1 position. Although no sn-1 fatty acyl preference was noted, PSS2 exhibited significant preference toward DHA compared with 18:1 or 20:4 at the sn-2 position. Preferential production of DHA-containing PS (DHA-PS) was consistently observed with PSS2 purified from a variety of cell lines as well as with microsomes from mutant cells in which PS synthesis relies primarily on PSS2. These findings suggest that PSS2 may play a key role in PS accumulation in brain and testis through high activity toward DHA-containing substrates that are abundant in these tissues.     

A calorimetric investigation of a series of mixed-chain polyunsaturated phosphatidylcholines: effect of sn-2 chain length and degree of unsaturation.

Although mammalian tissues contain high levels of polyunsaturated fatty acids, our knowledge of the effects of the degree of unsaturation and double-bond location upon bilayer organization is limited. Therefore, a series of mixed-chain unsaturated phosphatidylcholines (PC) comprised of 18:0 at the sn-1 position and various unsaturates at the sn-2 position (18:1n9, 18:2n6, 18:3n6, 18:3n3, 20:2n6, 20:3n6, 20:4n6, 20:5n3, 22:4n6, 22:5n6, or 22:6n3) was studied with differential scanning calorimetry, and their gel to liquid-crystalline phase transitions yielded measurements of Tm, Tonset, delta H, and delta S. Minimal delta H values were obtained for the diene species, 1.7 and 2.9 kcal/mole for 18:2n6 and 20:2n6, respectively. These results are consistent with the dienes having an acyl chain conformation that results in perturbed chain packing. Increasing the degree of unsaturation to three or more double bonds resulted in higher delta H values, 3.7, 4.3, and 4.6 kcal/mole for 18:3n6, 20:3n6, and 20:4n6, respectively, consistent with the occurrence of a gel-state chain conformation(s), which is more tightly packed than the dienes. The 18:0,22:6n3-PC species yielded the highest delta H (6.1 kcal/mole) and delta S(22.7 cal/mol degree) of all the polyunsaturates studied. The distinctive packing properties of phospholipid bilayers containing 22:6n3 may underlie its essential role in the nervous system.     

Thermal behavior of liposomes containing PCs with long and very long chain PUFAs isolated from retinal rod outer segment membranes

About one-fourth the phosphatidylcholines (PC) from retina photoreceptor rod outer segment (ROS) membranes contain docosahexaenoic acid (22:6n-3) at sn-2 and a very long chain polyunsaturated fatty acid (VLCPUFA) (C24 to C36) at the sn-1 position of the glycerol backbone. In order to study the thermotropic behavior of these PCs, subfractions and molecular species of PC (16:0/22:6, 18:0/22:6, 22:6/22:6, 32:5/22:6, 32:6/22:6, 34:5/22:6), were isolated from bovine ROS, and liposomes containing different proportions of these PCs and dimyristoyl-PC (DMPC) or dipalmitoyl PC (DPPC) were compared using the fluorescence probes Laurdan and 1,6-diphenyl-1,3,5-hexatriene (DPH). With both probes, the 22:6n-3 containing PCs from ROS, in all proportions tested, decreased the transition temperature (Tt) of both DMPC and DPPC. Below the transition temperature, coexistence of phases was evidenced in all cases. Liposomes formed with 100% of any of these PCs did not show phase transitions in the temperature range studied (8 degrees C to 50 degrees C). At physiological temperatures, as it is likely to be the case in ROS membranes, all of these PC species were in the liquid-crystalline state. With Laurdan, all dipolyunsaturated PCs seemed to behave similarly: despite the large number of double bonds per molecule, all of them decreased the Tt of DPPC less than did the hexaenoic PCs. With DPH, an ample difference was detected between the dipolyunsaturates, 22:6/22:6-PC and VLCPUFA/22:6-PCs, and between the latter and hexaenoic PCs throughout the temperature range studied. This difference is consistent with the interpretation that the largest "disorder" produced by PCs containing a VLCPUFA like 32:6n-3 at the sn-1 position occurs toward the center of the membrane.     

Studies on polyenoic acid incorporation into human platelet lipid stores: interactions with linoleic and arachidonic acids

Several polyunsaturated fatty acids (C18-C22 acids) have been compared in their uptake by human platelets and their acylation into glycerophospholipid subclasses. This was also studied in the presence of linoleic and/or arachidonic acids, the main fatty acids of plasma free fatty acid pool. Amongst C20 fatty acids, dihomogamma linolenic acid (20:3(n-6)), 5,8,11-icosatrienoic acid (20:3(n-9)) and arachidonic acid (20:4(n-6)) were better incorporated. The uptake of 5,8,11,14,17-icosapentaenoic acid (20:5(n-3)) was significantly lower and comparable to that of C22 fatty acids (7,10,13,16-docosatetraenoic acid (22:4(n-6)) and 4,7,10,13,16,19-docosahexaenoic acid (22:6(n-3)) and linoleic acid (18:2(n-6)). In this respect, linolenic acid (18:3(n-3)) appeared the poorest substrate. The bulk of each acid was acylated into glycerophospholipids although the presence of linoleic and/or arachidonic acids diverted a part towards neutral lipids. This was prominent for 18:3(n-3) and C22 fatty acids. The glycerophospholipid distribution of each acid differed substantially and was not affected by the presence of linoleic and or arachidonic acids, except for 18:3(n-3) and 22:6(n-3) that were strongly diverted towards phosphatidylethanolamine (PE) at the expense of phosphatidylcholine (PC). The main features were an efficient acylation of 20:3(n-9) into phosphatidylinositol (PI) followed by 20:3(n-6) and 20:4(n-6), then by 20:5(n-3) and 22:4(n-6), and finally 22:6(n-3) and C18 fatty acids. This was reciprocal to the acylation into PE and to a lesser extent into PC which remained the main storage species in all cases. We conclude that human platelets may exhibit a certain specificity for taking up polyunsaturated fatty acids both in terms of total uptake and glycerophospholipid subclass distribution. Also the presence of polyunsaturated fatty acids of normal plasma, like linoleic and arachidonic acids, may interact specifically with such an uptake and distribution.     

Metabolism of alpha-linolenic acid in humans

Alpha-linolenic acid (18:3n-3) is essential in the human diet, probably because it is the substrate for the synthesis of longer-chain, more unsaturated n-3 fatty acids eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) which are required for tissue function. This article reviews the recent literature on 18:3n-3 metabolism in humans, including fatty acid beta-oxidation, recycling of carbon by fatty acid synthesis de novo and conversion to longer-chain polyunsaturated fatty acids (PUFA). In men, stable isotope tracer studies and studies in which volunteers increased their consumption of 18:3n-3 show conversion to 20:5n-3 and 22:5n-3, but limited conversion to 22:6n-3. However, conversion to 18:3n-3 to 20:5n-3 and 22:6n-3 is greater in women compared to men, due possibly to a regulatory effect of oestrogen, while partitioning of 18:3n-3 towards beta-oxidation and carbon recycling was lower than in men. These gender differences may be an important consideration in making dietary recommendations for n-3 PUFA intake.     

Selective incorporation of various C-22 polyunsaturated fatty acids in Ehrlich ascites tumor cells

Three 14C-labeled 22-carbon polyunsaturated fatty acids, 7,10,13,16-[14C]docosatetraenoic acid (22:4(n-6)), 7,10,13,16,19-[14C]docosapentaenoic acid (22:5(n-3)), and 4,7,10,13,16,19-[14C]docosahexaenoic acid (22:6(n-3)), were compared with [3H]arachidonic acid (20:4(n-6] and [14C]linoleic acid (18:2(n-6)) to characterize their incorporation into the lipids of Ehrlich ascites cells. The relatively rapid incorporation of the labeled 22-carbon acids into phosphatidic acid indicated that substantial amounts of these acids may be incorporated through the de novo pathway of phospholipid synthesis. In marked contrast to 20:4(n-6), the 22-carbon acids were incorporated much less into choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP). No selective preference was apparent for the (n-3) or (n-6) type of fatty acids. The amounts of the acids incorporated into diacylglycerophosphoethanolamine were in the order of: 22:6(n-3) greater than 20:4(n-6) much greater than 22:5(n-3) greater than or equal to 22:4(n-6) greater than 18:2(n-6), whereas for alkylacylglycerophosphoethanolamine they were in the order of: 22:4(n-6) greater than 22:6(n-3) greater than 22:5(n-3) much greater than 20:4(n-6) greater than 18:2(n-6). Of the mechanisms possibly responsible for the selective entry of 22-carbon acids into ethanolamine glycerophospholipids, the most reasonable explanation was that the cytidine-mediated ethanolamine phosphotransferase may have a unique double selectivity: for hexaenoic species of diacylglycerol and for 22-carbon polyunsaturated fatty acid-containing species of alkylacylglycerol. The relative distribution of fatty acids between newly incorporated and already maintained lipid classes suggested that IGP may function in Ehrlich cells as an intermediate pool for the retention of polyunsaturated fatty acids in glycerolipids.     

Differential postprandial incorporation of 20:5n-3 and 22:6n-3 into individual plasma triacylglycerol and phosphatidylcholine molecular species in humans

The mechanisms by which digested fat is absorbed and transported in the circulation are well documented. However, it is uncertain whether the molecular species composition of dietary fats influences the molecular species composition of meal-derived lipids in blood. This may be important because enzymes that remove meal-derived fatty acids from the circulation exhibit differential activities towards individual lipid molecular species. To determine the effect of consuming oils with different molecular compositions on the incorporation of 20:5n-3 and 22:6n-3 into plasma lipid molecular species. Men and women (18–30 years) consumed standardised meals containing 20:5n-5 and 22:6n-3 (total 450 mg) provided by an oil from transgenic Camelina sativa (CSO) or a blended fish oil (BFO) which differed in the composition of 20:5n-3 and 22:6n-3 – containing molecular species. Blood was collected during the subsequent 8 h. Samples were analysed by liquid chromatography-mass spectrometry. The molecular species composition of the test oils was distinct from the composition of plasma triacylglycerol (TG) or phosphatidylcholine (PC) molecular species at baseline and at 1.5 or 6 h after the meal. The rank order by concentration of both plasma PC and TG molecular species at baseline was maintained during the postprandial period. 20:5n-3 and 22:6n-3 were incorporated preferentially into plasma PC compared to plasma TG. Together these findings suggest that the composition of dietary lipids undergoes extensive rearrangement after absorption, such that plasma TG and PC maintain their molecular species composition, which may facilitate lipase activities in blood and/or influence lipoprotein structural stability and function.