Dimyristoyl Lecithin Research Articles

Chlorophyll-Lipid-Interactions in Monomolecular Layers

Mixed monolayers containing chlorophyll a and dim yristoyllecithin (DML) have been investigated by simultaneous thermodynamic, fluorescence and absorption spectroscopic measurements. It has been shown that the solubility limit of chlorophyll a in the fluid, as well as in the solid lipid matrix, is above 20 mol%. This contrasts to previous findings for pheophytin a containing monolayers and indicates the existance of an interaction between the phospholipid head group and the central Mg atom. A fluorescence decrease on solidifying the monolayer was observed and it is suggested that it is due to an enhanced self quenching as a consequence of the reorientation of the porphyrin rings, accompanying the phase transition

Transitions and molecular packing in highly purified 1,2-dipalmitoyl-phosphatidyl choline–water phases. II. The structures of DPPC phases with low water content

The structures of the polar regions are established for a series of bilayer phases with less than four water molecules per lipid molecule and shown to be closely related. For this analysis, the size and symmetry of the unit cells, determined for three phases for which single crystals were grown, were compared with the size and shape of the phosphorylcholine group and the influence of the charges on the packing. The structures of two of these crystalline phases are almost identical with that of a dimyristoyl lecithin for which some details of a structure determination were reported recently. For other phases, reference is made to the chain subcells, which are obtained from x-ray diffraction data and to the polar subcells which can be identified. The polar structures can also be related, directly or indirectly, with those in the crystalline phases from evidence of the changes occurring at the transitions. A square packing of the polar groups is observed for an anhydrous phase and for a transient phase formed at higher temperatures from samples with higher water content. The choline groups are packed in a rectangular array with an hexagonal chain packing for a phase with from one to three water molecules per lipid. In this phase, the chain axes are perpendicular to the bilayer plane and the choline groups and phosphate nonester oxygen atoms are not coplanar. This arrangement is stabilized by the proximity of another bilayer. The crystalline monohydrate phase involves a rectangular packing with coplanar phosphate nonester oxygen atoms and choline groups. This very stable polar structure is found at higher temperatures for the dihydrate and at room temperature for phases with higher water contents.

Partition coefficients of 20 polar molecules between water and trimethyldodecylammonium bromide micelles: Correlation with the water + octanol system

The partition coefficient P between trimethyldodecylammonium bromide micelles and water of 20 nonaromatic polar molecules was determined at 298.15°K by a combination of salting constant and critical micelle concentration determinations; included are amines, alcohols, ethers, esters, ketones, and some high dipole moment molecules. The partition coefficients obtained for the alcohols are about one-half their value in aqueous micellar sodium dodecylsulfate solutions. It is shown that for straight, branched, and cyclic amines, alcohols, esters, and ketones there is an excellent correlation between partition coefficients in the aqueous cationic micelles and in the classical octanol + water system. Some ethers and halogenated alkanes do not follow this correlation; the P values in these cases in the micellar solutions are about one-third of the values predicted from the correlation. The same observation can be made for halogenated alkanes in aqueous sodium dodecylsulfate solutions. The behavior of simple polar solutes in the micellar systems studied resembles that found with dimyristoyl lecithin liposomes. The average solubilization site of the nonaromatic solutes onto the cationic micelles may be looked upon as having the properties of a solvent such as isobutanol saturated with water.

A Study of the Angular Dependence of NMR Relaxation Times in Macroscopically Oriented Lyotropic Liquid Crystal Lamellar Phases

Abstract The angular dependence of 1H and 2H NMR relaxation rates has been investigated for macroscopically oriented multi-bilayers of egg yolk lecithin and dimyristoyl lecithin in the Lα phase. Proton T ip values show a strong dependence on the orientation of the bi-layer normals to the magnetic field, while deuterium T ip and T 1 values and proton T 1's show little or no orientation dependence. The results are interpreted on the basis of a simple modification of the theory of Ukleja, Pirs and Doane for relaxation in oriented thermotropic phases. The results confirm the presence of collective fluctuations in the density of lipid packing in the plane of the bilayer which dominate proton relaxation at low frequencies. There is no evidence of a significant contribution from collective order fluctuations.

Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants.

The three-dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes were analyzed by imaging negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer-aided three-dimensional reconstruction. The information on electron micrographs obtained at 6 degrees angular increments from +60 degrees to -60 degrees was digitized by densitometric scanning, Fourier-transformed, corrected for imaging errors, cross-correlated, and synthesized to the three-dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle, indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16-nm height, 2.0-nm wall thickness, and a 3.0-nm torus at the free end with an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane-binding site, and only small pores of up to 2.0-nm in diameter penetrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of approximately 4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest the MAC may mediate increased membrane permeability by protein channel formation in addition to lipid reorientation.

Hydrophobic membrane interaction of etidocaine, bupivacaine and 2-chloroprocaine. A spin and fluorescent probe study.

It has been suggested that local anesthetics may block sodium conductance through nervous membranes also by hydrophobic interaction, e.g., by expanding the membrane. Decreased anisotropy (fluidization) and depressed phase transition temperatures have been shown by relatively high local anesthetic concentrations. We studied the dose dependence of the effect of three clinically used local anesthetics, with different lipid solubility, on lipid fluidity parameters of four different model membranes. With stearic acid spin labels in dipalmitoyl lecithin vesicles etidocaine (1-5 mM) had the clearest fluidizing effect followed by bupivacaine (5 mM); 2-chloroprocaine was without effect on lipid fluidity. In synaptic plasma membranes a fluidizing effect near the hydrophilic part of the lipid bilayer was similar with etidocaine and bupivacaine (5-10 mM); 2-chloroprocaine had no effect. Bupivacaine at 125 and 250 muM had a small ordering effect, which was not seen at a more hydrophobic site of the membrane. Etidocaine had the strongest fluidizing effect at the latter site of the synaptic plasma membranes. In erythrocyte ghost membranes, probed by stearic acid spin labels near the hydrophilic end, none of local anesthetics affected fluidity at 24 degrees C, while at 37 degrees C etidocaine (1-5 mM) and bupivacaine (5 mM) had a fluidizing effect. Dimyristoyl lecithin vesicles were probed by cis- and trans-parinaric acid. Etidocaine and bupivacaine (5-10 mM) clearly depressed the phase transition temperature evaluated from fluorescence intensity scans. The effect was most marked with bupivacaine (1-10 mM) when cis-parinaric acid was used. While isolated mammalian nerves are blocked by local anesthetic concentrations below 100 muM, this study shows that the clinically used local anesthetics increase fluidity and depress phase transition temperature only at 10-100 times higher concentrations at physiological pH. This kind of hydrophobic membrane interaction may not be important for the nerve blocking effect.

Xenon NMR: chemical shifts of a general anesthetic in common solvents, proteins, and membranes.

The rare gas xenon contains two NMR-sensitive isotopes in high natural abundance. The nuclide 129Xe has a spin of 1/2: 131Xe is quadrupolar with a spin of 3/2. The complementary NMR characteristics of these nuclei provide a unique opportunity for probing their environment. The method is widely applicable because xenon interacts with a useful range of condensed phases including pure liquids, protein solutions, and suspensions of lipid and biological membranes. Although xenon is chemically inert, it does interact with living systems; it is an effective general anesthetic. We have found that the range of chemical shifts of 129Xe dissolved in common solvents is ca. 200 ppm, which is 30 times larger than that found for 13C in methane dissolved in various solvents. Resonances were also observed for 131Xe in some systems; they were broader and exhibited much greater relaxation rates than did 129Xe. The use of 129Xe NMR as a probe of biological systems was investigated. Spectra were obtained from solutions of myoglobin, from suspensions of various lipid bilayers, and from suspensions of the membranes of erythrocytes and of the acetylcholine receptor-rich membranes of Torpedo californica. These systems exhibited a smaller range of chemical shifts. In most cases there was evidence of a fast exchange of xenon between the aqueous and organic environments, but the exchange was slow in suspensions of dimyristoyl lecithin vesicles.

NMR study of synthetic lecithin bilayers in the vicinity of the gel-liquid--crystal transition

1H, 2H, and 31P NMR methods have been employed in the study of dimyristoyl lecithin bilayers hydrated with D2O in the gel (L beta'), intermediate (P beta') and liquid-crystalline (L alpha) phases. For D2O/lipid molar ratios, n, in the range 7 less than or equal to n less than or equal to 11 discontinuities are observed in the deuterium NMR splittings at both main and pretransitions. A partial phase diagram based on NMR and differential scanning calorimetry data is presented. 1H NMR dipolar splittings are observed for macroscopically oriented samples in all three phases. Changes in the 1H splittings are correlated with 2H and 31P data and interpreted to show that the chain tilt in the gel phase undergoes a discontinuous change on transition to the intermediate phase, which brings the chain axes closer to the bilayer normal. An estimate of chain tilt in the gel phase is made on the basis of NMR data and found to be approximately 23 degrees for a sample with n = 11 at 18 degrees C.

Detection of low levels of lipid contamination in lecithin by equilibrium spreading pressures

Lipid contamination of the order of 0.05 mole% in dimyristoyl lecithin is shown to have a profound influence on the equilibrium spreading pressure in the region of the gel-liquid-crystal transition temperature T c . This method is proposed as a means of testing routinely for very low levels of lipid contamination in lecithin.

Molecular motions and phase transitions. NMR relaxation times studies of several lecithins

The spin-lattice relaxation time, T1, and the dipolar energy relaxation time, TD, were measured as a function of temperature. The materials studied were samples of anhydrous L-dipalmitoyl lecithin, DL-dipalmitoyl lecithin, L-dimyristoyl lecithin, DL-dimyristoyl lecithin and their monohydrates, and of anhydrous egg yolk lecithin. It is shown that TD is a much more sensitive parameter than T1 for the determination of the Chapman phase transition. Comparison between T1 and TD provides information about new types of slow molecular motions below and above the phase transition temperature. It is suggested that the relaxation mechanisms for T1 and TD in the gel phase are governed by segmental motion in the phospholipid molecule. A new metastable phase was detected in dimyristoyl lecithin monohydrates. This phase could only be detected from the dipolar energy relaxation times.

Use of the fluorescent probe 1-anilino-8-naphthalene sulfonate to monitor the interactions of chlorophenols with phospholipid membranes (liposomes)

The fluorescence intensity (F 480), at pH 8, of 1-anilino-8-naphthalene sulfonate (ANS) bound to phospholipid membranes (liposomes) was decreased in the presence of mono-, di- and pentachlorophenols (pesticides and chemical intermediates that are toxic to animals). No shifts in emission spectra occurred, and no decreases in ANS fluorescence intensity were observed, in the presence of chlorophenols if liposomes were absent. An exception was 2,6-dichlorophenol which, at pH 8, had no effect on membrane-bound ANS-F 480. Using liposomes prepared from dimyristoyl lecithin, a 50 per cent decrease in membrane-bound ANS-F 480 was produced by 0.046 mM pentachlorophenol and 0.2–0.5 mM dichlorophenols (excluding the 2,6-derivative). These results are consistent with published toxicological data on polychlorinated phenols that show an order of toxicity: pentachlorophenol > 2,4-dichlorophenol > 2,6-dichlorophenol.

Rapid movement of molecules across membranes: Measurement of the permeability coefficient of water using neutron diffraction

We describe a novel approach to the quantitative study of the movement of small molecules across membranes. The method consists of measuring the time-course of coherent neutron diffraction from a stack of membranes, following a sudden change in the isotopic form of the permeant molecule in the surrounding vapour. Our approach offers a number of advantages over conventional techniques; in particular, the minimization of unstirred water layer problems and the expansion of the time-scale of the experimental measurements, so that molecules with high permeabilities can be conveniently studied. In addition, the molecular organization of the membrane, determined from the diffraction measurements at equilibrium, provides a structural basis for analysing the time-course measurements to give single-membrane permeability coefficients. We have measured the time-course of H 2 O/ 2 H 2 O exchange in stacks of lecithin/cholesterol membranes. Based on our knowledge of the structure of the multimembrane stack, we have derived the appropriate diffusion equations. Using this analysis and our experimental measurements, we find the permeability coefficient of water for a single membrane composed of dimyristoyl lecithin and 40 mol % cholesterol at 23°C to be 3·9±0·7 μm/s (mean ± standard error in the mean). Finally, we indicate how our approach might be generalised to measure permeability coefficients using other experimental techniques.

Quantitative determination of natural dipalmitoyl lecithin with dimyristoyl lecithin as internal standard by capillary gas-liquid chromatography

A quantitative determination of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine from dog lungs by means of the corresponding diacyl glycerol trimethylsilyl ether derivative with 1,2-dimyristoyl- sn-glycero-3-phosphocholine as internal standard was carried out by gas-liquid chromatography with glass capillary columns. The individual diacyl glycerol species were identified by using a combination of gas-liquid chromatography and mass spectrometry. In a total of 32 determinations percent deviations from the mean value between ± 2.0 and ± 4.4% were achieved. By using glass capillary columns instead of conventional columns the time required for an analysis was reduced to ca. 30% and separation was considerably improved. Thus the diacyl glycerol species from PC-32 were separated according to both the 1,2 and 1,3 positional isomerism and the degree of saturation of the acyl radicals.

Allogeneic cytolysis of reconstituted membrane vesicles.

The successful use of lipid bilayer model membranes as targets for cytotoxic lymphocytes is described. Lipid vesicles were made from a mixture of dipalmitoyl lecithin, dimyristoyl lecithin, and cholesterol. Membrane proteins of LSTRA or EL4 tumor cells (as source of H-2 antigens), human eye muscle membrane proteins (as supporting proteins), and 51Cr marker were inserted into the lipid vesicles. Incubation of the reconstituted vesicles with lymphocytes sensitized in mixed lymphocyte cultures against allogeneic cells resulted in the specific release of intravesicular 51Cr. Vesicle damage was mediated by thymus-derived lymphocytes. H-2 antigens could be incorporated into vesicles without eye muscle proteins. However, immune damage of the vesicles could not be demonstrated when vesicles inserted with H-2 antigens in the absence of eye muscle proteins were used as targets.

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.

Spontaneous formation of lecithin bilayers at the air–water surface

THE energetics of formation of various surface film states of dimyristoyl lecithin (DML) has been recently obtained from equilibrium spreading pressure πe measurements1. One result from that study, but not reported then, was the formation of a new equilibrium lecithin surface phase. Its existence was deduced solely from formal thermodynamic arguments based on the Gibbs phase rule, and therefore reporting of the new surface phase was deferred until more direct experimental evidence was available for describing its properties. We have now characterised this new surface phase by measuring the surface concentration of DML films and have found the concentration to be double that of a close-packed monomolecular film. Before presenting these results and their implications, we summarise the arguments which led to the prediction of the new lecithin surface phase.

A pulsed NMR study of lipids, bound water and sodium ions in macroscopically-oriented lecithin/water lyotropic liquid crystal model membrane systems

The temperature and orientation dependence of pulsed NMR ‘free induction decay’ signals have been studied in detail for lipid bilayers macroscopically-oriented between glass slides. Results for the lipid molecules ( 1H, 31P), bound water ( 2H 2O) and ions dissolved in the aqueous phase ( 23Na) are presented. Bilayers of egg-lecithin, dimyristoyl lecithin and potassium oleate have been investigated. In the liquid crystal phase all the signals, including those from bound water and ions exhibit a |3 cos 2 ϕ − 1| dependence on orientation of the bilayer normal to the magnetic field. In the case of DML samples, some orientation dependence of both 1H and 2H signals persists in the gel phase, indicating that the lipid molecules retain a degree of reorientational freedom about their long axes in this phase. At the gel-liquid crystal transition the 2H quadrupole spittings undergo a discontinuous change. Results are interpreted in terms of a model in which water molecules are bound to individual lipid head groups and reorient with them, while sodium ions are located in the aqueous channel between bilayers.

Cell-mediated lysis of lipid vesicles containing eye muscle protein: implications regarding pathogenesis of Graves ophthalmopathy.

We prepared artificial vesicles that are lysed upon cell-mediated immunological attack by human lymphocytes. These vesicles are made from a mixture of dimyristoyl lecithin, dipalmitoyl lecithin, and cholesterol, have eye muscle membrane protein (EMP) inserted into the bilayer wall, and contain intravesicular (99m)Tc marker. Injury to the vesicular membrane was assessed by measurement of (99m)Tc release. Thyroglobulin (Tg) and Tg-anti-Tg complex (TgA) bind to EMP-vesicles to an extent equal to or greater than to native eye muscle membranes in vitro; this binding requires the presence of normal human IgG. The role of Tg, TgA, IgG, and peripheral blood lymphocytes in altering membrane permeability was analyzed. Incubation of vesicles for up to 3 hr alone, with added IgG alone, or with further addition of Tg or TgA did not result in (99m)Tc release. Addition of lymphocytes from normal donors to the above four preparations showed release in the presence of TgA. Lymphocytes from each of eight patients with Graves ophthalmopathy caused release not only in the presence of TgA, but also in the presence of Tg. Separation of a patient's lymphocytes into high- and low-affinity rosette-formers (T and K cells, respectively) showed that cell-mediated vesicle lysis in the presence of TgA was greater with K cells than with T cells, while vesicle lysis in the presence of Tg was greater with T cells than with K cells. Vesicles made with inserted Tg but lacking EMP were not lysed by such T cells. Lymphocytes failed to induce permeability changes in vesicles containing other inserted proteins obtained from human nonextraocular muscle, liver, spleen, or adrenal, even if Tg or TgA were present. The results support the concept that muscle cell damage in Graves ophthalmopathy is immunological, cell-mediated, and of two types: (i) K lymphocytes reacting to immune complex, TgA, on the eye muscle cell surface (i.e., antibody-dependent cytotoxicity) and (ii) sensitized T lymphocytes reacting to Tg on the eye muscle cell surface. An antigenic role for EMP is possible, but has not been unequivocally proven.

Interactions of cholesterol esters with phospholipids: cholesteryl myristate and dimyristoyl lecithin.

The ternary phase diagram of cholesteryl myristate--dimyristoyl lecithin--water has been determined by polarizing light microscopy, scanning calorimetry, and x-ray diffraction. Hydrated dimyristoyl lecithin forms a lamellar liquid--crystalline phase (L alpha) at temperatures greater than 23 degrees C into which limited amounts of cholesteryl myristate (less than 5 wt. %) can be incorporated. The amount of cholesterol ester incorporated is dependent upon the degree of hydration of the L alpha phase. Below 23 degrees C dimyristoyl lecithin forms ordered hydrocarbon chain structures (L beta' and P beta') which do not incorporate cholesterol ester. Comparison with other phospholipid--cholesterol ester--water phase diagrams suggests the following general principles: i) the incorporation of cholesterol ester occurs only into liquid crystalling phospholipid bilayers, ii) the extent of incorporation is temperature-dependent, with increasing amounts of cholesterol ester being incorporated at higher temperatures, and iii) unsaturated cholesterol esters induce increased disordering of the phospholipid bilayers.

Rapid conformational changes of cytochrome P -450: Effect of dimyristoyl lecithin

Binding of benzphetamine to purified microsomal cytochrome P-450 from rat liver causes a shift in the heme spin state of the protein to favor the high-spin form. This shift is strongly temperature dependent. A rapid temperature jump of a cytochrome P-450/epsilon benzphetamine mixture, monitored by changes in the Soret absorptions of the heme, reveals two relaxation processes: one in a 50-msec time range (tau(f)) and the other in a 0.3-sec time range (tau(s)). Both relaxations reflect conformational changes of the protein after the substrate binding. No bimolecular reaction of benzphetamine and the enzyme has been resolved. This indicates that there is no absorption change of the heme associated with the initial binding. In the presence of dimyristoyl lecithin, at 25 degrees C tau(f) decreases by nearly one order of magnitude whereas tau(s) decreases to one-third. The enhancement of rates by added phospholipid is both temperature- and concentration-dependent: rates are accelerated only above the gel-liquid crystalline transition temperature, and this effect saturates near the enzyme/lipid ratio of 1:20. In contrast, the lipid does not have significant effect on the equilibrium binding curve of the substrate. These results suggest that the lipid may form an envelope around the enzyme and, depending on its crystalline state, regulates the rate of the substrate-induced conformational changes of cytochrome P-450.