William D. Ehringer

Docosahexaenoic Acid Increases Permeability of Lipid Vesicles and Tumor Cells

Docosahexaenoic acid (DHA), a long-chain polyunsaturated ω3 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 isomolar erythritol and leakage of sequestered carboxylfluorescein, and T27A tumor cells, as monitored by swelling in isomolar erythritol and release of sequestered51Cr. 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.

Use of merocyanine (MC540) in quantifying lipid domains and packing in phospholipid vesicles and tumor cells

The fluorescent probe merocyanine (MC540) reports qualitatively on several membrane events. Here we demonstrate that MC540 fluorescence can quantify the degree of coexisting liquid-crystalline and gel states in mixed monotectic phosphatidylcholine (PC) bilayers. The probe exhibits disparate fluorescence wavelength maximas and and intensities when incorporated into liquid-crystalline and gel state membranes. The fluorescence measurements partitioning of the EPR spin probe TEMPO between the aqueous environment and the membrane fluid phase. While both techniques can accurately assess the phase transition of synthetic PCs, only MC540 can distinguish between liquid-crystalline phases of different composition. MC540 fluorescence for single-component PC bilayers correlates quantitatively with estimates of the area/molecule determined from surface area/pressure isotherms of lipid monolayers, whereas partitioning of TEMPO fails to assess the relative degree of lipid packing in various fluid state membranes. Additionally, MC540 fluorescence characterizes the interaction of cholesterol with membranes made from condensable (18:0, 18:1-PC) and non-condensable (18:0, 22:6-PC) lipids. Finally MC540 distinguishes tumor cell membranes differing only in the amount of docosahexaenoic acid (DHA). Thus we conclude that MC540 can be used quantitatively to study phospholipid packing and membrane phases with lipid vesicles and to sense subtle differences in the arrangement of phospholipids in biological membranes.

A comparison of the effects of linolenic (18:3Ω3) and docosahexaenoic (22:6Ω3) acids on phospholipid bilayers

Abstract The class of long chain polyunsaturated fatty acids known as omega-3 are believed to be involved in prevention of a number of human afflictions. The mode of action for two of the most common omega-3 fatty acids, linolenic 18 : 3 Δ9,12,15 and docosahexaenoic 22 : 6 Δ4,7,10,13,16,19 (DHA), is not known. One suggestion is that they may be incorporated into membranes and there provide some specific function. Here we compare the effects of DHA and its metabolic precursor linolenic acid on the membrane properties of fluidity, fusion and permeability. The fatty acids were investigated as both free fatty acids and mixed chain 18 : 0, 18 : 3 and 18 : 0, 22 : 6 phosphatidylcholines (PCs). Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a series of anthracene stearic acid probes indicates 20 mol% incorporation of either fatty acid into dipalmitoylphosphatidylcholine bilayers broadens and depresses the temperature of the phase transition, but has almost no effect on fluidity in the liquid crystalline state. Similar fluidity was also observed in the liquid crystalline bilayers of the mixed chain PCs using the same set of fluorescent fatty acid probes. In contrast, DHA as a free fatty acid or as part of a mixed chain PC, causes a much greater enhancement than linolenic acid of the rates of fusion and permeability as monitored by fluorescence resonance energy transfer and aqueous compartment mixing (fusion) and by lipid vesicle swelling in isotonic erythritol, (permeability). These experiments establish a clear distinction between the effects of linolenic acid and DHA in membranes.

Omega‐3 fatty acid modification of membrane structure and function. II. Alteration by docosahexaenoic acid of tumor cell sensitivity to immune cytolysis

Docosahexaenoic acid (DHA, 22:6) is a long-chain omega-3 fatty acid abundant in cold water fish; it is the most unsaturated fatty acid found in biologic systems and is reported to alter membrane structure. To explore DHAs effect on membrane function, we have fused tumor cells with synthetic phosphatidylcholine (PC) containing stearic acid in the sn-1 position and DHA in the sn-2 position (18:0, 22:6 PC) and have found the lipid-modified tumor cells to be more sensitive to cytolysis by alloreactive cytotoxic T lymphocytes. Cold target competition experiments suggested that fusion of tumor plasma membranes with 18:0, 22:6 PC produced a qualitative change in expression of surface antigens recognized by cytotoxic T lymphocytes. We monitored the expression of various epitopes on tumor cells by complement-mediated lysis and radioimmunoassay with monoclonal antibodies against H-2 class I antigens. Our results suggest that membrane-bound DHA increases the expression of some epitopes while decreasing the expression of others and that different tumor lines vary in the magnitude of DHAs effect. Our findings are consistent with a model in which DHA-containing phospholipids segregate into membrane domains, in turn altering the expression of membrane proteins.

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 diets 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.

Interaction of α-tocopherol with fatty acids in membranes and ethanol

Abstract The techniques of fluorescence polarization, ultraviolet light absorbance and fluorescence quenching by acrylamide are used to probe the structural role of α-tocopherol in phospholipid bilayers. Using 1,6-diphenyl-1,3,5-hexatriene (DPH) and a series of (anthroyloxy)stearic acid (AS) fluorescence probes, α-tocopherol is shown to increase fluidity and decrease order of gel state bilayers, and to decrease fluidity and increase order of bilayers in the liquid crystalline state. More complex behavior is noted for bilayers made from mixed acyl chain phosphatidylcholines (PCs) where the sn -1 position is saturated and the sn -2 position unsaturated compared to bilayers composed of PCs where both acyl chains are either saturated or unsaturated. Complexation between α-tocopherol and either free fatty acids or fatty acids esterified to the sn -2 position of PCs is indicated by ultraviolet light absorbance in both organic solution and in lipid bilayers. The strength of the complexes, expressed as interaction constants, are dependent upon the number of acyl chain unsaturations from 0 (stearic acid), to 6 (docosahexaenoic acid). Relation of the strength of these complexes to the degree of acyl chain unsaturation is confirmed by monitoring the fatty acid protection from acrylamide bleaching of α-tocopherol. These experiments suggest that the extent of acrylamide bleaching is related to the extent of association with the fatty acids.

Omega‐3 Fatty Acid Modification of Membrane Structure and Function. I. Dietary Manipulation of Tumor Cell Susceptibility to Cell‐ and Complement‐Mediated Lysis

Omega-3 fatty acids, abundant in fish oil, are reported to alter membrane properties when incorporated into membrane phospholipids. We report that dietary omega-3 fatty acids, incorporated into tumor cell membranes, alter tumor recognition and cytolysis by the immune system. Mice were fed diets rich in corn oil, hydrogenated coconut oil, or menhaden (fish) oil. T27A leukemia cells were grown as an ascites tumor in these mice and harvested for biochemical and immunologic assays. The incorporation of the long-chain omega-3 fatty acid docosahexaenoic acid (22:6) into tumor plasma membranes correlated with an increased susceptibility to tumor cytolysis by alloreactive cytotoxic T lymphocytes and decreased expression of a class I major histocompatibility complex epitope, monitored by complement-mediated lysis and radioimmunoassay. Thus the immunologic phenotype of this ascites tumor reflected the source of oil present in the diet during tumor growth.

ω3 fatty acids increase spontaneous release of cytosolic components from tumor cells

Mice fed menhaden (fish) oil or coconut oil-rich diets were inoculated intraperitoneally with a rapidly growing leukemia, T27A. After one week, the tumor cells were harvested, and51Cr was used to label intracellular molecules. Spontaneous release of51Cr was used as a measure of plasma membrane permeability. Compared to cells from mice fed coconut oil (rich in saturated fatty acids), tumor cells from mice fed menhaden oil (rich in long chain polyunsaturated ω3 fatty acids) showed an increased level of spontaneous51Cr release, which was exacerbated by increased temperature and reduced by extracellular protein. At physiological salt concentrations, the releated51Cr was detected in particles of ∼2700 daltons. Enhanced permeability correlated with the incorporation of dietary (fish oil) ω3 polyunsaturated fatty acids docosahexaenoic and eicosapentaenoic acid into the tumor cells. The results demonstrate that ω3 fatty acids are incorporated into cellular constituents of tumor cells and change properties associated with the plasma membrane. This result suggests that dietary manipulation may be used to enhance tumor cell permeability and contribute to tumor eradication.

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.