Liquid crystalline/gel state phase separation in docosahexaenoic acid-containing bilayers and monolayers.

The phase behavior of lipid mixtures containing 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0, 22:6 PC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with bilayers using differential scanning calorimetry (DSC), and with monolayers monitoring pressure/area isotherms and surface elasticity, and lipid domain formation followed by epifluorescence microscopy. From DSC studies it is concluded that DPPC/18:0, 22:6 PC phase separates into DPPC-rich and 18:0, 22:6 PC-rich phases. In monolayers, phase separation is indicated by changes in pressure-area isotherms implying phase separation where 18:0, 22:6 PC is 'squeezed out' of the remaining DPPC monolayer. Phase separation into lipid domains in the mixed PC monolayer is quantified by epifluorescence microscopy using the fluorescently labeled phospholipid membrane probe, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl). These results further describe the ability of docosahexaenoic acid to participate in lipid phase separations in membranes.

Effect of retinol and retinoic acid on permeability, electrical resistance and phase transition of lipid bilayers.

Retinol and retinoic acid have been incorporated into the artificial membrane systems, planar bimolecular lipid membranes and liposomes, and their effects on several membrane parameters have been measured. 1. Retinol and retinoic acid increased the permeability of egg lecithin liposomes to K+, I- and glucose when incorporated into the membranes at levels as low as 0.5 membrane mol%. Retinoic acid influenced permeability more than did retinol for each of the solutes tested. 2. Retinol and retinoic acid both decreased the electrical resistance of egg lecithin-planar bimolecular lipid membranes from 0.5 to 8 membrane mol%. Retinoic acid effected a larger change than did retinol. 3. Retinol and retinoic acid increased the permeability of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine liposomes to water at 1.0 and 3.0 membrane mol%. A larger effect on water permeability was measured for retinoic acid than for retinol. 4. Retinol and retinoic acid at 1.0 and 3.0 membrane mol% were shown to lower the phase-transition temperature of liposomes composed of dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. Phase-transition temperatures were monitored by abrupt changes in water permeability and liposome size associated with the transition. Retinoic acid lowered the phase-transition temperature of dimyristoylphosphatidylcholine liposomes more than did retinol, while both retinoids had almost the same effect on dipalmitoylphosphatidylcholine liposomes.

Docosahexaenoic acid-containing phosphatidylcholine affects the binding of monoclonal antibodies to purified Kb reconstituted into liposomes.

Class I major histocompatibility complex (MHC I) molecules are transmembrane proteins that bind and present peptides to T-cell antigen receptors. The role of membrane lipids in controlling MHC I structure and function is not understood, although membrane lipid composition influences cell surface expression of MHC I. We reconstituted liposomes with purified MHC I (Kb) and probed the effect of lipid composition on MHC I structure (monoclonal anti-MHC I antibody binding). Four phospholipids were compared; each had a phosphocholine head group, stearic acid in the sn-1 position, and either oleic, alpha-linolenic, arachidonic, or docosahexaenoic acid (DHA) in the sn-2 position. The greatest binding of monoclonal antibody AF6-88.5, which detects a conformationally sensitive epitope in the extracellular region of the MHC I alpha-chain, was achieved with DHA-containing proteoliposomes. Other epitopes (CTKb, 5041.16.1) showed some sensitivity to lipid composition. The addition of beta2-microglobulin, which associates non-covalently with the alpha-chain and prevents alpha-chain aggregation, did not equalize antibody binding to proteoliposomes of different lipid composition, suggesting that free alpha-chain aggregation was not responsible for disparate antibody binding. Thus, DHA-containing membrane lipids may facilitate conformational change in the extracellular domains of the alpha-chain, thereby modulating MHC I function through effects on that protein's structure.

Electron spin resonance study of the interactions of retinoids with a phospholipid model membrane.

The effects of up to 20 mol% incorporation of all-trans-retinol (vitamin A), retinal (vitamin A aldehyde) and retinoic acid (vitamin A acid) on acyl chain order and dynamics in liquid crystalline dipalmitoylphosphatidylcholine membranes at pH 7.5 were studied by electron spin resonance (ESR) of 5-, 7-, 10-, 12- and 16-doxyl spin-labelled stearic acids intercalated into the membrane. Order parameters S and correlation times tau c determined from the ESR spectra demonstrate that the influence of retinoic acid differs from retinol or retinal. Whereas the latter two retinoids have negligible effect (less than 1%) on acyl chain order towards the membrane surface (5 position), retinoic acid reduces the order parameter by as much as 8% at 20 mol% incorporation. All three retinoids restrict acyl chain motion to a similar extent approaching the center of membrane (10, 12 and 16 positions), where up to 22% increases in order parameter and correlation time were observed. Complementary osmotic swelling and carboxyfluorescein release measurements show that the enhancement in permeability of egg phosphatidylcholine membranes to erythritol and carboxyfluorescein is greater with all-trans-retinoic acid than all-trans-retinol or retinal.

Docosahexaenoic acid-induced alteration of Thy-1 and CD8 expression on murine splenocytes.

Here we test whether the incorporation of docosahexaenoic acid (DHA, 22:6), an (n-3) fatty acid, into lymphocyte membranes affects the expression of the surface proteins Thy-1.2 and CD8. DHA was incorporated into splenocytes by three methods: feeding mice diets containing menhaden (fish) oil, fusing splenocytes with DHA-containing phosphatidylcholine vesicles, and culturing splenocytes with DHA. Thy-1.2 and CD8 expression were measured by flow cytometry and complement-mediated lysis using a panel of monoclonal antibodies. As (n-3) fatty acid incorporation into the lymphocytes increased, the expression of one Thy-1.2 epitope and one CD8 epitope decreased; the expression of two CD8 epitopes increased. Although diet-induced changes in surface protein expression may result from selective migration of cell populations or the diet's effect on protein biosynthesis, fusion with lipid vesicles demonstrated that DHA-containing phospholipids can mediate a direct and immediate effect. The decrease in Thy-1.2 expression was sustained for more than a week after removal of (n-3) fatty acids from the diet, most likely due to retention of membrane-bound (n-3) fatty acids. Because Thy-1.2 and CD8 participate in T cell activation, modulation of their expression by DHA suggests that DHA, when serving as a membrane structural element, may alter immune function.

Docosahexaenoic acid reverses cyclosporin A-induced changes in membrane structure and function.

The use of a fish oil vehicle for cyclosporin A (CsA) can decrease the toxic effects of CsA but the mechanism is unclear. Here we examine the mechanism by which docosahexaenoic acid (DHA), a fish oil-derived polyunsaturated fatty acid, can alter the toxic effects of CsA on mouse organ function, endothelial macromolecular permeability, and membrane bilayer function. Mice given CsA and fish oil showed increased liver toxicity, kidney toxicity, incorporation of DHA, and evidence of oxidized fatty acids compared to control animals. We hypothesized that the toxic effects of CsA were primarily a result of membrane perturbation, which could be decreased if DHA were not oxidized. The presence of CsA (10 mol%) alone increased dipalmitoylphosphatidylcholine membrane permeability by seven fold over control (no CsA, no DHA). However, if non-oxidized DHA (15 mol%) and CsA were added to the membrane, the permeability returned to control levels. Interestingly, if the DHA was oxidized, the antagonistic effect of DHA on CsA was completely lost. While CsA alone increased endothelial permeability to albumin, the combination of non-oxidized DHA and CsA had no effect on endothelial macromolecular permeability. However the combination of oxidized DHA and CsA was no different than the effects of CsA only. CsA increased the fluorescence anisotropy of DPH in the liquid crystalline state of DPPC, while DHA decreased fluorescence anisotropy. However the combination of CsA and DHA was no different than DHA alone. We conclude that non-oxidized DHA can reverse the membrane perturbing effects of CsA, and the increases in endothelial macromolecular permeability, which may explain how fish oil is capable of decreasing the toxicity of CsA.

Docosahexaenoic acid in phosphatidylcholine mediates cytotoxicity more effectively than other omega-3 and omega-6 fatty acids.

We reported previously that docosahexaenoic acid (22:6)-containing phosphatidylcholine (PC), but not oleic acid-containing PC nor 22:6-containing phosphatidylethanolamine, is toxic to tumor cells in vitro. To test whether other polyunsaturated fatty acids share 22:6's cytotoxic activity, we treated cultured T27A murine leukemia cells with PC liposomes composed of stearic acid in the sn-1 position and alpha-linolenic acid (alpha-18:3), arachidonic acid (20:4), or eicosapentaenoic acid (20:5) in the sn-2 position. PC containing 22:6 in both positions was also tested. Following treatment, the cells were monitored for fatty acid composition, liposome uptake and viability. Here we demonstrate that cytotoxicity is unique to 22:6-containing PCs and is not shared by PCs with other polyunsaturated omega-3 and omega-6 fatty acids. Because PCs with fatty acids other than 22:6 were taken up by cells but did not kill the cells, we propose that 22:6-containing PCs incorporated into cellular membranes produce unique changes in the membrane structure incompatible with cell survival. PC liposomes containing 22:6 are potential drug delivery vehicles that may, by virtue of their cytotoxicity, serve concomitantly as adjunct cancer therapy.

A possible biochemical mode of action for benzimidazole anthelmintics.

Albendazole (ABZ), cambendazole (CBZ), oxibendazole (OBZ), and thiabendazole (TBZ) are potent, orally active, broad spectrum anthelmintics widely used in human and veterinary medicine. As members of the benzimidazole series, they are closely related chemically, and it is likely that they exert their anthelmintic effects in an identical fashion. We have examined the effects of these anthelmintics on the electrical resistance of planar bimolecular lipid membranes and compared the results with those obtained with a known uncoupler, 2,4-dinitrophenol (2,4-DNP). All drugs tested markedly reduced membrane resistance at concentrations lower than 0.1 microM and were better proton conductors than 2,4-DNP by at least an order of magnitude. The sequence of proton conducting efficiency was ABZ greater than OBZ greater than TBZ greater than CBZ greater than 2,4-DNP. From 1 to 40 microM, ABZ and CBZ substantially decreased P/O (phosphorous/oxygen) ratios in coupled rat liver mitochondria in a concentration-dependent fashion using beta-hydroxybutyrate as the substrate. 2,4-DNP was also shown to decrease P/O ratios, but less effectively than the benzimidazole anthelmintics. These experiments indicate that the benzimidazole anthelmintics are lipid-soluble proton conductors that are effective in artificial (phospholipid bilayer) and natural (rat liver mitochondria) membrane systems. Dissipation of the transmembrane proton gradient should result in diminished levels of cellular ATP. In vivo treatment with a therapeutically effective dose of ABZ caused a severe disturbance in the energy balance of Hymenolepis diminuta; this was evident from a distinct drop in ATP levels, and from a decline in the ATP/ADP ratios, adenylate energy charge (AEC) and available adenylate energy (AAE) values.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron spin resonance study of the interaction of alpha-tocopherol with phospholipid model membranes.

The effect of up to 20 mol% incorporation of alpha-tocopherol on acyl chain order and dynamics in liquid crystalline phosphatidylcholine (PC) membranes was studied as a function of acyl chain unsaturation by electron spin resonance (ESR) of 5-, 7-, 12- and 16-doxyl spin labelled stearic acids intercalated into the membrane. Order parameters S in the upper portion of the chain (positions 5 and 7) and correlation times tau C in the lower portion (positions 12 and 16) determined from the ESR spectra indicate that in general alpha-tocopherol restricts acyl chain motion within the membrane. The magnitude of the increases in order appears to be dependent upon phospholipid molecular area, being the greatest (up to 15%) in saturated dimyristoylphosphatidylcholine (14:0-14:0 PC) which possesses a relatively small area per molecule as opposed to much smaller increases (less than 3%) in unsaturated PC membranes of larger molecular area. This behavior is interpreted as incompatible with the hypothesis of Lucy and coworkers (A.T. Diplock and J.A. Lucy (1973) FEBS Lett. 29, 205-210), who proposed that membranes are structurally stabilized by interactions between the phytyl side chain of alpha-tocopherol and the polyunsaturated chains of phospholipids.

Docosahexaenoic acid increases permeability of lipid vesicles and tumor cells.

Docosahexaenoic acid (DHA), a long-chain polyunsaturated omega 3 fatty acid, is tested to determine its mode of action as an anti-cancer agent. We demonstrate that DHA can increase the permeability of phospholipid vesicles, as monitored by vesicle swelling in isosmolar erythritol and leakage of sequestered carboxyfluorescein, and T27A tumor cells, as monitored by swelling in isosmolar erythritol and release of sequestered 51Cr. DHA was incorporated into lipid vesicles as either the free fatty acid or as 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine. DHA was incorporated into the tumor cells by fusion with vesicles made from the mixed-chain phosphatidylcholines. DHA is demonstrated here to be much more effective in increasing permeability than is oleic acid, the major unsaturated fatty acid normally found in tumor plasma membranes. It is proposed that incorporation of DHA makes tumor plasma membranes substantially more permeable, which may explain, in part, its anti-tumor properties.

Effect of docosahexaenoic acid on mouse mitochondrial membrane properties.

Long-chain polyunsaturated (n-3) fatty acids have been proposed to be involved in a wide variety of biological activities. In this study, mitochondrial docosahexaenoic acid (DHA) levels were increased by either dietary manipulation or by fusing the mitochondria with phospholipid vesicles made from 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0/22:6 PC). The fused mitochondria exhibited a DHA-induced decrease in respiratory control index (RCI) and membrane potential and an increase in proton movement. The modified mitochondria also demonstrated an increase in fluidity (as detected by 1,6-diphenyl-1,3,5-hexatriene anisotropy) and changes in membrane structure detected by the fluorescence probes MC540 and pyrene decanoate. Proton movement in lipid vesicles made from mitochondrial lipid extracts was shown to be enhanced by incorporated 18:0/22:6 PC. Mitochondria were isolated from young (5-mon) and old (24-mon) mice which were maintained on either a diet rich in saturated fats (hydrogenated coconut oil) or rich in n-3 polyunsaturated fats (menhaden oil). Mitochondrial bioenergetic function was followed by RCI, state 3 respiration, ATP level, and phosphate uptake. In addition, lipid composition, phospholipid area/molecule and extent of lipid peroxidation were also determined. Decreases in RCI for the menhaden oil diet-modified mitochondria paralleled those in which DHA levels were enhanced by fusion with phospholipid vesicles. RCI reductions are attributed to DHA-induced increases in H+ movement, producing diminished mitochondrial membrane potentials. One purpose of this project was to determine if the deleterious effects of aging on mitochondrial bioenergetic function could be reversed by addition of n-3 fatty acids. The experiments reported here indicate that incorporation of long-chain polyunsaturated n-3 fatty acids into mitochondrial membranes does not appear likely to reverse the effects of age on mitochondrial function.

Geraniol interferes with membrane functions in strains of Candida and Saccharomyces.

Geraniol, an olefinic terpene, was found to inhibit growth of Candida albicans and Saccharomyces cerevisiae strains. Geraniol was shown to enhance the rate of potassium leakage out of whole cells and also was shown by fluorescence polarization to increase C. albicans membrane fluidity. Biophysical studies using differential scanning calorimetry, fluorescence polarization and osmotic swelling of phospholipid vesicles demonstrated that geraniol decreased the phase-transition temperature of dipalmitoylphosphatidylcholine vesicles, affected fluidity throughout the bilayer, particularly the central portion of the bilayers, and caused an increase in bilayer permeability to erythritol. Geraniol may have potential use as an antifungal agent.

Facilitated diffusion as a method for selective accumulation of materials from the primordial oceans by a lipid-vesicle protocell.

A model is proposed for the selective accumulation of amino acids, sugars, nucleotides, cations and protons from the primordial oceans into a lipid vesicle type of protocell. The model is built on facilitated diffusion using simple, primordial, lipid-soluble carriers. The advantages a lipid vesicle protocell would have had over the other potential types of protocells are discussed.

Acquired os odontoideum. A case report.

A 7-year-old boy presented with os odontoideum after a 5 story fall at 19 months of age, at which time roentgenograms documented a normal odontoid. Current flexion-extension films revealed a marked altlantoaxial instability related to the acquired "os." The treatment consisted of an atlantoaxial arthrodesis. Acquired os odontoideum is a recognized, though uncommon entity in which it is hypothesized that trauma produces vascular insufficiency, with subsequent resorption of the central portion of the odontoid, resulting in the classical features of os odontoideum. It is often associated with instability, with potential danger to the spinal cord. Instability in cases of os odontoideum is a positive indication for atlantoaxial arthrodesis.

Abscisic acid enhances aggregation and fusion of phospholipid vesicles.

The plant hormone abscisic acid (ABA) is shown to enhance the aggregation and fusion of small unilamellar lipid vesicles composed of 80 mol% dimyristoylphosphatidylcholine (DMPC) and 20 mol% dimyristoylphosphatidylcholine (DMPE). Aggregation and fusion did not occur with single component (100 mol%) DMPC vesicles. Fusion was followed by two fundamentally different techniques, fluorescence resonance energy transfer which monitors intermixing of bilayers and ANTS-DPX which monitors intermixing of the sequestered aqueous interiors. It is suggested that a previously unreported role of ABA may be as a membrane fusagen.

Anomalous changes in forward scatter of lymphocytes with loosely packed membranes.

BACKGROUND: Forward scatter (FSC) is generally associated with cell size and has been suggested as a way to differentiate apoptotic from viable cells. Among spleen cells cultured for 48 h, a population of cells (population B) was found to have decreased forward and increased side scatter relative to freshly purified cells (population A). Interestingly, population B was not present early in analysis; this report explores the change in FSC of population B. METHODS: Using a Coulter (Hialeah, FL) Epics Elite ESP flow cytometer, changes in forward scatter and lipid packing of spleen cells were measured. RESULTS: Over time, the FSC of unfixed cells in population B increased from that of the debris field, to reach a stable value by 30 sec (population A's FSC remained constant). When fixed, populations A and B exhibited constant FSC. Population B cells displayed altered lipid packing as reported by MC540, and the FSC changes were mimicked by Nonidet P-40 treatment of freshly purified spleen cells. CONCLUSIONS: Data emphasize the importance of delaying measurements on unfixed cells until FSC readings have stabilized, and suggest that flow cytometry may be a useful tool in studying lipid packing.

Omega-3 fatty acid-containing liposomes in cancer therapy.

omega-3 fatty acids are associated with reduced growth and incidence of certain cancers, and in this report we demonstrate that a fish oil diet (rich in omega-3 fatty acids) enhances the longevity of mice bearing the myeloid leukemia T27A. We have proposed that the omega-3 fatty acid docosahexaenoic acid (DHA, 22:6 delta 4,7,10,13,16,19) may induce structural changes in tumor cell plasma membranes resulting in reduced tumor growth in vitro. Here, we test whether liposomes containing DHA (18:0, 22:6 PC) have antitumor effects in vivo, leading to enhanced longevity of the tumor-bearing host. Male BALB/c mice (6-8 weeks old) were inoculated intraperitoneally with a T27A tumor dose known to cause 100% mortality of syngeneic (BALB/c) mice in less than 2 weeks. Small unilamellar vesicles (liposomes) were prepared, composed of phosphatidylcholine (PC) with 18:0 in the sn-l position and one of the following fatty acids in the sn-2 position: 18:0, 18:1 omega 9 (oleic), 18:3 omega 3 (alpha-linolenic), 20:4 omega 6 (arachidonic), 22:6 omega 3 (docosahexaenoic). The liposomes were injected intraperitoneally into tumor-bearing mice at various times: concurrently with the tumor inoculum, at select times during tumor growth, and when the mice were moribund. Mouse survival was then charted. DHA-containing lipid vesicles (18:0, 22:6 PC) caused a statistically significant increase in survival of the tumor-bearing mice when compared with 18:0, 18:1 PC. Lipid vesicles of 18:0, 18:0 PC showed no benefit, and 18:0, 20:4 PC was not significantly different than 18:0, 18:1 PC. Lipid vesicles containing a different omega-3 fatty acid, 18:0, 18:3 PC, also effectively enhanced tumor-bearing mouse survival. The greatest benefit was achieved if either the liposome treatments were spaced throughout the tumor growth period, or if the tumor inoculum was suspended in the liposome preparation (without further liposome treatments). Neither lipid peroxidation nor prolonged inflammatory responses appeared to be pertinent, leaving membrane structural changes as a feasible mode of liposome action. With antitumor properties of their own, omega-3 fatty acid-containing lipid vesicles may offer an important new avenue in combination cancer therapies.