The effect of the phase transition on the hydration and electrical conductivity of phospholipids.

Adsorption isotherms for various saturated phosphatidylcholines have been obtained. Lipids above and below their phase transition temperature differ only in the amount of water adsorbed and not in the nature of their adsorption isotherms. Cholesterol has an effect similar to that of increasing unsaturation in the hydrocarbon chains. Decreasing the length of the hydrocarbon chains for lipids below their phase transition temperature has no effect on the isotherms. If the chain length is short enough so that the lipids are above their transition temperature, however, a large increase in water adsorption occurs. All of the phospholipids exhibit a rapid increase of electrical conductivity for a few water molecules adsorbed per lipid molecule. All of the phospholipids show a saturation in conductivity at greater amounts of adsorbed water; the shape of the saturation region depends on whether the lipids are above or below their phase transition temperature. The activation energy for the electrical conductivity process depends on whether the hydrated lipids are in the "liquid-like" of the crystalline state, being lower for phospholipids in the liquid-like state. If the lipids are hydrated above their phase transition temperatures, their activation energies are lower than if they are hydrated below the transition temperature. Cholesterol lowers the activation energy. The phosphatidylcholines can be characterized by different activation energies, depending both upon their physical state and the presence of unsaturation in their hydrocarbon chains.

The phospholipid head-group orientation: effect on hydration and electrical conductivity.

The water adsorption isotherms have been obtained for egg phosphatidyl-ethanolamine when it is complexed to egg phosphatidylcholine and cholesterol, respectively. In the presence of phosphatidylcholine, the phosphatidylethanol amine water binding is changed to a strong binding as compared to when the phospholipid is in its uncomplexed form. Cholesterol increases the water adorbed by the phospholipid, however, it does not change the nature of the isotherm. Phosphatidylmonomethylethanolamine also exhibits a strong water binding. The electrical conductivity of these phospholipids has been measured concurrently with their hydration. Electrical activation energies have been obtained for the fully hydrated phospholipids and are a function of both the amount of water adsorbed and the orientation of the polar head-group. The results are discussed in terms of a model for water adsorption, previously put forth by the authors.

Chaotropic anion-phosphatidylcholine membrane interactions: an ultra high field NMR study.

NMR studies on the interaction of the linear chaotropic anions, SCN- and SeCN-, with sonicated egg phosphatidylcholine (EPC) vesicles have been carried out at field strengths up to 14.1 Tesla. At 600 MHz, both anions cause splitting or increased splitting of the choline N+(CH3)3, CH2N+ and O3POCH2 1H resonances with SeCN- being somewhat more effective in this action than is SCN-. No changes were observed in the glycerol CH2OP and CH2OCO 1H resonances and the phosphate 31P resonance of the headgroup region. The 13C spectrum was unchanged by the presence of the anions. After 18 h of exposure to the anion, the 1H resonance splittings but not the chemical shift values returned to those prior to anion exposure. Increasing the temperature of the vesicles decreased the anion-induced splitting, but, upon return to the beginning temperature, the chemical shifts did not return to their original values. The results are considered in terms of the 'molecular electrometer' model recently developed by Seelig and co-workers [1].

The water adsorption characteristics of charged phospholipids.

The hydration isotherms of the negatively charged phospholipids, egg phosphatidic acid, bovine heart cardiolipin and two phosphatidylserines as well as one positively charged phospholipid, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, have been obtained gravimetrically. The presence of an electrical charge on these phospholipids does not, in itself, determine whether the water binding to the phospholipids is 'strong' or 'weak'. Interestingly, hysteresis effects were present for certain charged phospholipids suggesting some rearrangement of the lipid molecular organization upon hydration and dehydration, perhaps due to the presence of the ionizable moiety. The hydration isotherms of the charged phospholipids have been analyzed by BET theory, although, of the charged lipids studied, all may not be amenable to the application of BET theory. The hydration isotherms and the resulting parameters obtained from the BET analysis are compared to those found previously for zwitterionic phospholipids, especially egg phosphatidylcholine. The water adsorption characteristics of phospholipids are found to depend mainly on the total head-group structure including the presence of hydrophobic groups as well as electrical charge on the head group.

A temperature study of diacetylenic phosphatidylcholine vesicles.

Dispersions of the diacetylenic phosphatidylcholine, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, DC8,9PC, undergo a change from vesicles to hollow tubes on cooling. We report here a light scattering and multinuclear NMR study of the lipid vesicles over the temperature range 0-50 degrees C. The 'splitting' of the N+(CH3)3 resonance increases with decreasing temperature, consistent with the light scattering measurements which show a decrease in vesicle size with decreasing temperature. The NMR spectrum remains well-resolved over this temperature range, even at temperatures as low as 3 degrees C. Phosphorus NMR also indicates that the 'bilayer structure' is maintained over this temperature range. The various proton resonances and the phosphorous signal from the lipid vesicles broaden as the temperature is lowered. These results will be helpful in developing a model for the tubule-forming ability of DC8,9PC.

The effect of phloretin on the hydration of egg phosphatidylcholine multilayers.

The effect of phloretin on the hydration, structure and interactive properties of supported phospholipid bilayers has been studied by a combination of direct water adsorption measurements and X-ray diffraction. Adsorption isotherms show that over a wide range of relative vapor pressures (from 0 to approximately 1.0) the addition of 20 or 40 mol% phloretin significantly alters the amount of water adsorbed by egg phosphatidylcholine (EPC) multilayers. X-ray diffraction analysis shows that the incorporation of phloretin decreases the width of the EPC bilayer, thereby increasing the area per lipid molecule from approximately 64 A2 for EPC to about 78 A2 for EPC:Ph, 3:2, M:M. Phloretin also decreases the distance between apposing EPC bilayers, most likely because it causes a reduction in repulsive hydration/steric pressure between apposing bilayers. Because phloretin decreases the fluid layer between bilayers by a larger amount than it increases the area per EPC molecule, phloretin has the effect of decreasing the water volume in the multilayers.

Amiodarone-liposome interaction: a multinuclear NMR and X-ray diffraction study.

Amiodarone, a potent antiarrhythmic drug, is widely used in cardiology. Its electrophysiological effects, as well as many of its side effects, seem to involve lipids. We report here a multinuclear NMR and X-ray diffraction study of amiodarone in egg phosphatidylcholine liposomes and lipid multilayers. In proton NMR experiments, amiodarone alters the signal from the lipid trimethyl ammonium group for pH values ranging from 3.2 to 8.4; cholesterol does not cause this alteration. The addition of SCN- changes both the proton and phosphorus NMR spectra of liposomes containing amiodarone. For both proton and carbon NMR, amiodarone modifies the signal from the lipid methylene groups, but to a far lesser extent than does cholesterol. Incorporation of amiodarone in EPC bilayers also modifies the low-angle X-ray diffraction patterns, decreasing the lamellar repeat period at low water contents, but swelling the fluid spaces between bilayers at high water contents. Electron density profiles and modeling studies using the X-ray data indicate that amiodarone decreases the bilayer thickness and adds electron density at the interfacial region of the bilayer. Our analysis of the NMR and X-ray data indicates that the iodine atoms of amiodarone are located near the hydrocarbon/water interface and that the tertiary amine of amiodarone is in the headgroup region of the bilayer.

Liposomes as carriers of iodolipid radiocontrast agents for CT scanning of the liver.

We studied the potential of liposomes to deliver oil soluble radiocontrast agents to the liver and have developed a new preparation for CT liver scanning. The preparation consists of Ethiodol with a large amount of phospholipids. Nuclear magnetic resonance (NMR) spectroscopy was done for this lipid preparation, and the spectra show that the lipids are in the bilayer liposome configuration. Electron microscopy provides direct visualization of the liposomes. X-ray fluorescence measurements suggest that the Ethiodol is incorporated in the liposomes, and since no other particulate configurations are observed, we conclude that the Ethiodol is contained within the hydrophobic region of the liposomes. We used a GE CTT-8800 scanner and a rabbit model to study the liver uptake of the iodine from the Ethiodol. The iodine uptake in the liver was rapid and significant, and an increase in HU number of more than 40 was observed within 20 minutes after i.v. injection of 50 mg I/kg of body weight. Significant image enhancement was obtained. The iodine from the Ethiodol remained in the liver for several hours. Studies in rabbits with hepatic implants of the VX2-carcinoma show that while normal liver concentrates, areas of tumor do not concentrate the Ethiodol liposomes. Tumors not visible on ordinary scans become visible after administration of this combination. The advantages of this liposomal mode of radiocontrast agent administration are small particle size, rapid uptake in the liver, long retention times, a large increase in HU number and low iodine dose.

Relationship between MRI relaxation time and muscle fiber composition.

The relationship between nuclear magnetic resonance relaxation time obtained with magnetic resonance imaging (MRI) and muscle fiber composition of the lateral gastrocnemius was examined in 13 men. There was a positive correlation (r = 0.80, P < or = 0.001) between longitudinal relaxation time and the relative percentage of slow-twitch muscle fibers (type I). There was no relationship between transverse relaxation time and type I percentage (r = 0.17, P = 0.57). These results suggest that MRI longitudinal relaxation time can be used for the noninvasive estimation of muscle fiber composition in humans.

The electrical conductivity of phospholipid films as an iodine-sensing mechanism.

The d.c. electrical conductivity of dry phospholipid films is increased by some 8-11 orders of magnitude by the adsorption of iodine vapor. The conductivity of these films has been found to increase as a function of iodine 'vapor pressure' and the quantitative relationship between electrical conductivity and the adsorbed iodine has been determined. Films composed of phospholipids with unsaturated hydrocarbon chains are some three orders of magnitude more electrically conductive than are films of phospholipids containing saturated hydrocarbon chains. Optical spectroscopic measurements show the development of absorption bands centered near 294 nm and 365 nm, upon iodine adsorption. These bands are much more intense for unsaturated phospholipids than for saturated ones. X-ray diffraction studies show that exposure to iodine decreases the thickness of phospholipid bilayers containing unsaturated hydrocarbon chains but does not change the thickness of bilayers containing only saturated chains. The electrical response of the lipid films, upon exposure to iodine, suggests their possible use as iodine sensors.

The effect of the choline head group on phospholipid hydration.

The hydration behavior of three naturally occurring sphingomyelins (SM) has been studied. Adsorption isotherms for these SM, singly and in combination with other lipids, were obtained and the isotherms analyzed by the application of BET theory. The results are compared with those found previously by us for the phosphatidylcholines (PC). We find that, depending on the SM studied, both "weak" and "strong" water adsorption can be observed and thus, the presence of choline in the phospholipid head group, does not guarantee "strong" water adsorption. When the head group is removed from SM, the resulting ceramides are found to be very weak, water adsorbers. Cholesterol, when present in a mixture with SM, has a rather dramatic effect on the hydration of the SM. A mixed system of PC and SM exhibits water adsorption characteristics very similar to those exhibited by PC itself. We speculate that these hydration results may well play a role in the cell signaling action of SM and, moreover, may be closely associated with the hypothesized formation of lipid "rafts."

The interaction of 1-anilino-8-naphthalenesulfonate with lipids: a nuclear magnetic resonance study.

The proton magnetic resonance (PMR) and phosphorus magnetic resonance (PhMR) spectra of egg phosphatidylcholine in the presence of 1-anilino-8-nahthalenesulfonate (ANS) have been studied. At low ratios of ANS to phospholipid, the spectra indicate that ANS molecules are in the lipid interface region where they interact with the head-group protons. ANS also penetrates into the hydrocarbon region to some extent. As the ANS/phospholipid ratio approaches one, a significant splitting of the head-group signal occurs. This splitting is associated with head-group signals from inner and outer molecules of the phospholipid vesicles. As the ANS/phospholipid ratio is further increased, a gel phase often occurs. The spectra for this gel phase suggest a highly mobile head-group. Further ANS addition results in a PMR spectrum suggestive of ANS-phospholipid micelle formation. The results for a phospholipid-cholesterol complex and for the total lipid extract from a cell membrane show that the ANS effect is more complicated in these cases.

The interaction of 1-anilino-8-naphthalenesulfonate with thyroid lipids and membranes: a nuclear magnetic resonance study.

The proton magnetic resonance (PMR) spectra of thyroid cell membranes and their total lipid extracts, in the presence of 1-anilino-8-naphthalenesulfonate (ANS), have been studied. The addition of ANS causes an shifting of the head group PMR signal, a splitting of the signal into two components and an increase in total spectral intensity. The data suggest that ANS interacts with phospholipid in the membrane as it does in total lipid vesicles. Evidence is also presented for the removal of lipids from the membrane, by ANS, and the subsequent formation of micelles. The membrane results are compared with our earlier work on the interaction of ANS with egg phosphatidylcholine (P.C.) vesicles and the results are used in explaining the inhibition of iodide transport in isolated thyroid slices.

The interaction of MRI contrast agents with phospholipids.

The molecular interactions of three clinically used MRI contrast agents with lipid vesicles, consisting of egg phosphatidylcholine (EPC), have been studied using high-field NMR techniques. At a molar ratio of one contrast agent molecule to five phospholipid molecules, a significant increase in the proton resonance line width occurred for certain lipid head group moieties. A large decrease in the T1 relaxation times for the head group moieties was also observed. These two effects occurred regardless of the ionic status and the chelate structure of the three contrast agents. The structure of the contrast agents did, however, affect the magnitude of the two NMR parameter changes. These NMR effects also differed in magnitude amongst the various head group entities. The NMR effects were greatest for the head group moieties at or near the vesicle-water interface. The results are discussed in terms of the structure of the phospholipid-water interface. Since the use of contrast agents has become routine in clinical MRI, our results are of importance in terms of the interaction of the agents with physiological surfaces, many of which contain phospholipids. The understanding of such interactions should be of value not only for improved diagnostics, but also in the development of new contrast agents.

The interaction of pseudohalides with the phospholipid head-group: a nuclear magnetic resonance study.

Liposomes have been studied by means of high-field magnetic resonance techniques. The choline N+(CH3)3 group showed two proton resonances for phosphatidylcholine whereas the addition of a charged species to the phospholipid resulted in a single N+(CH3)3 resonance. Upon the addition of either of two linear pseudohalide anions, the two resonances for phosphatidylcholine were further split whereas for the mixture of lipids containing a charged species, the single head-group resonance was now split. The presence of a negative charge on the liposome does not prevent the anion-liposome interaction observed for neutral liposomes. Incorporation of cholesterol into the negatively charged liposomes results in a clear initial splitting of the head-group proton signal in a manner very similar to that for neutral liposomes; this head-group signal is then further split upon anion addition. The small splitting involved suggests a weak pseudohalide-liposome interaction whose magnitude depends on the position of the anion in the lyotropic series. The phosphorous NMR signal from the head-group is unaffected by the pseudohalide interaction whereas the carbon signals from the N+(CH3)3 groups are affected, indicating that the initial anion interaction is localized to the region of the choline groups of the liposome. After the initial exposure of the liposome to the anion, however, the splitting decreases with time, indicating that the anions have entered the liposome and interact with both inside and outside head-groups.