Lipid Dispersions Research Articles

Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824.

Butanol, at sub-growth-inhibitory levels, caused a ca. 20 to 30% increase in fluidity of lipid dispersions from Clostridium acetobutylicum. When grown in the presence of butanol or into stationary phase, C. acetobutylicum synthesized increased levels of saturated acyl chains at the expense of unsaturated chains.

Antitumor activity of lipophilic prodrugs of mitomycin C entrapped in liposome or O/W emulsion.

Nine lipophilic la-N-substituted prodrugs of mitomycin C were formulated in lipid dispersion dosage forms and their fundamental antitumor activities were evaluated. The prodrugs were efficiently incorporated into liposome or O/W emulsion according to their increased lipophilicities , while mitomycin C was hardly entrapped into them. Almost complete incorporation was observed in nonyloxycarbonyl and cholesteryloxycarbonyl mitomycin C which showed partition coefficients over 8000 in chloroform/water system. The release rate from these dosage forms determined by a dynamic dialysis method decreased with an increase in the partition coefficients of the derivatives. All prodrugs entrapped in liposome or O/W emulsion showed significant antitumor activities against L1210 leukemia in i.p.-i.p. system except for cholesteryloxycarbonyl mitomycin C. In spite of considerable antitumor activities showen in the forms of liposome and emulsion, saline suspension of nonyloxycarbonyl mitomycin C failed to exhibit any activity because of its poor aqueous solubility. These results suggested the utility of the combining delivery system of lipophilic prodrug with physical device such as liposome and O/W emulsion.

The effects of ionizing radiation on the peroxide content of a pure polyunsaturated lipid dispersion and of lipids and membranes derived from Acholeplasma laidlawii.

Dispersions of a pure unsaturated phospholipid, dilinoleoylphosphatidyl choline, formed conjugated diene hydroperoxides when irradiated in air with 7 MeV electrons (150 Gy and 300 Gy). Peroxide formation was optimized when the dispersions were irradiated in air at 37 degrees C at a dose rate of 5 Gy/min. No significant loss of linoleic acid from the irradiated phospholipid dispersions was observed after doses of 150 or 300 Gy. Small amounts of thiobarbituric acid-reactive material were formed in irradiated unsaturated phospholipid dispersions. However, lipids or membranes isolated from 48 hour cultures of Acholeplasma laidlawii grown in media supplemented with either linoleic or linolenic acid did not appear to be peroxidized by irradiation under the same conditions.

Ultrasonic and jet aerosolization of phospholipids and the effects on surface activity.

Surface active aerosols were produced from aqueous dispersions of mixed lipids (CLL), extracted from bovine lung lavage. Particle size distributions were measured as a function of humidity for two types of aerosol generators: ultrasonic and jet. Lipid dispersions before aerosolization were prepared by sonication in an ice bath and by mechanical vortexing. Over a range of high humidity greater than 60-70%, ultrasonic nebulization gave CLL aerosols with mass median aerodynamic diameters (MMAD) of 1.4 +/- 0.1 micron, compatible with predicted alveolar deposition fractions of 0.2-0.3 according to current deposition models. For humidities of 30-95%, jet nebulization gave MMAD values of 0.4-0.5 micron, which have lower predicted alveolar deposition. The surface pressure-time (pi - t) adsorption characteristics at 35 +/- 2 degrees C of CLL dispersions prepared initially by vortexing or sonication were not significantly affected by ultrasonic nebulization over a 1-2 h time period. In addition, the dynamic surface tension lowering of both kinds of CLL dispersion was not affected by ultrasonic nebulization (minimum surface tension less than 1 dyne/cm at 37 degrees C and 100% humidity). Current interest in the treatment of the respiratory distress syndrome (RDS) with exogenous surfactant replacement has focused largely on the delivery of surfactant replacement has focused largely on h delivery of surfactants to infants by tracheal instillation at birth. However, the ability to form multi-component surfactant aerosols with appreciable alveolar deposition fractions and high surface activity may help to expand the utility of replacement therapy to patients with aerated lungs.

The effect of dietary lipids on the thermotropic behaviour of rat liver and heart mitochondrial membrane lipids

Diets supplemented with relatively high levels of either saturated fatty acids derived from sheep kidney fat (sheep kidney fat diet) or unsaturated fatty acids derived from sunflower seed oil (sunflower seed oil diet) were fed to rats for a period of 16 weeks and changes in the thermotropic behaviour of liver and heart mitochondrial lipids were determined by differential scanning calorimetry (DSC). The diets induced similar changes in the fatty acid composition in both liver and heart mitochondrial lipids, the major change being the ω6 to ω3 unsaturated fatty acid ratio, which was elevated in mitochondria from animals on the sunflower seed oil diet and lowered with the mitochondria from the sheep kidney fat dietary animals. When examined by DSC, aqueous buffer dispersions of liver and heart mitochondrial lipids exhibited two independent, reversible phase transitions and in some instances a third highly unstable transition. The dietary lipid treatments had their major effect of the temperature at which the lower phase transition occurred, there being an inverse relationship between the transition temperature and the ω6 to ω3 unsaturated fatty acid ratio. No significant effect was observed for the temperature of the higher phase transition. These results indicate that certain domains of mitochondrial lipids, probably containing some relatively higher melting-point lipids, independently undergo formation of the solidus or gel phase and this phenomenon is not greatly influenced by the lipid composition of the mitochondrial membranes. Conversely, other domains, representing the bulk of the membrane lipids and which probably contain the relatively lower melting point lipids, undergo solidus phase formation at temperatures which reflect changes in the membrane lipid composition which are in turn, a reflection of the nature of the dietary lipid intake. These lipid phase transitions do not appear to correlate directly with those events considered responsible for the altered Arrhenius kinetics of various mitochondria membrane-associated enzymes.

Mechanism of action of Bacillus thuringiensis var israelensis insecticidal δ-endotoxin

Bacillus thuringiensis var israelensisδ-endotoxin protein active against mosquitoes was inactivated by prior incubation with lipids extracted from Aedes albopictus cells. Experiments with lipid dispersions and multilamellar liposomes showed that the toxin binds to phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine provided these lipids contain unsaturated fatty acids. Phosphatidyl serine binds toxin less efficiently and phosphatidyl inositol, cardiolipin, cerebroside and cholesterol show no affinity for the toxin. The results suggest an insecticidal mechanism in which interaction of toxin with specific plasma membrane lipids causes a detergent-like rearrangement of the lipids, leading to disruption of membrane integrity and eventual cytolysis.

Effect of liposomal size on calorimetric behavior of mixed-chain phosphatidylcholine bilayer dispersions

The effect of liposomal size on the endothermic transition profiles of the saturated mixed-chain phosphatidylcholines 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine and 1-stearoyl-2-caproyl-sn-glycero-3-phosphocholine has been investigated. Liposomal bilayer dispersions of progressively smaller average diameter were prepared by extrusion of coarse multilamellar preparations of these lipids through polycarbonate membrane filters of decreasing pore size from 3-to 0.2-micrometers diameter. These samples were then investigated by high-sensitivity differential scanning calorimetry and negative-strain electron microscopy. It was found that coarse dispersions of the above lipids are composed of liposomes whose average diameter is considerably smaller than that typically associated with multilamellar liposomes of synthetic phosphatidylcholines. This fact, coupled with an analysis of the size dependence of the transition cooperativity, leads to the conclusion that the small size of the liposomes limits the transition cooperativity in coarse dispersions of these mixed-chain phosphatidylcholines. Thus, the broad and highly asymmetric transition profiles that have been observed in previous studies of these phosphatidylcholines are postulated to arise largely from this size dependence, rather than from characteristic packing properties of the phospholipid acyl chains as has been previously suggested.

Structural studies of plant membrane lipid dispersions subjected to oxidation in the presence of decomposing peroxychromate

Total membrane polar lipid extracts of broad bean ( Vicia faba) leaves have been dispersed in phosphate buffer and oxidised in the presence of decomposing potassium peroxychromate. Changes in the organisation of the membrane structures formed by these lipids resulting from the oxidation of unsaturated fatty acvl residues were assessed by different biophysical methods. Fluorescence polarisation values of 1,6-diphenylhexatriene intercalated into oxidised dispersions indicated considerable restriction in probe motion. Temperature-dependence studies suggested that this restriction was not due to the removal of unsaturated residues by oxidation leaving predominantly gel-phase lipid. This view was confirmed by wide-angle. X-ray diffraction studies, which showed diffuse reflections in control and oxidised preparations centred around 0.46 nm as compared to a sharp reflection at about 0.41 nm in a hydrogenated sample, and electron microscopic studies. Freeze-fracture and negatively stained dispersions of unoxidised lipids show typical lamellar structures with inverted micelles of lipid sandwiched within the bilayer structure. Oxidation of the lipid destroys the bilayer arrangement and leads to a generally amorphous structure. These results are discussed in terms of the role of oxidation in senescing plant tissue.

Studies on the uptake of exogenous phosphoglycerides by Escherichia coli B cells.

The uptake of exogenous lipids by Escherichia coli B was studied under various conditions. Lysophosphoglycerides were absorbed more readily than diacyl analogues when sonicated dispersions of a single lipid were used. When these same lipids were admixed with coliform lipids and dispersed as vesicles, the uptake of lysophosphoglyceride diminished and was approximately equal to that of diacyl analogues. Neutral lipids such as diglyceride, fatty acid, and cholesterol were also absorbed when admixed and dispersed as vesicles with coliform lipids. The uptake of lysophosphoglyceride was stimulated slightly by all divalent cations tested except Mg2+; monovalent cations were ineffective. Uptake was accompanied by conversion of lysophosphoglyceride to diacyl analogues. In the presence of Ca2+, lysophosphatidylethanolamine also formed a more polar lipid product yet unidentified. The uptake of lipid did not cause release of 3H label into the medium from cells that had been grown in [3H]acetate-containing medium. Also, the [3H]phosphoglyceride content of such labelled cells remained constant. Thus the uptake process did not involve exchange of membrane lipid with the medium or an enhanced hydrolysis of endogenous lipids, but it represented a net gain of lipid by the cell. The uptake did not seem to involve a stable adsorption of lipid at the surface of the cell as could be judged from electron microscopic examination of the cells after incubation; the cell surfaces were devoid of adsorbed vesicle or liposomal types of structures and did not display evidence of expansion. [3H]Cholesterol-[32P]phospholipid mixtures were taken up without a change in isotopic ratio. This result together with the other evidence presented indicate a net uptake of exogenous lipid by a process likely involving fusion.

Phospholipids as adjuncts for calcium ion stimulated release of chromaffin granule contents: implications for mechanisms of exocytosis.

Structure-function relationships for the lipid component of chromaffin granules isolated from the bovine adrenal medulla have been investigated by employing 31P nuclear magnetic resonance (NMR), freeze-fracture, and spectrophotometric techniques. Two aspects have been studied in detail, namely, the structural preferences of lipids in the isolated granule membrane and derived liposomal model membrane systems as well as the influence of exogenous lipid (in the form of sonicated vesicle systems) on the Ca2+-stimulated release of granule contents. It is shown that at least 90% of endogenous granule membrane phospholipids assume a liquid-crystalline bilayer configuration at physiological temperatures. Liposomal dispersions of total granule lipid also exhibit bilayer structure, consistent with a structural role of phospholipids in vivo. Incubation of intact isolated granules in the presence of up to 10 mM Ca2+ does not induce significant release of contents above background levels. However, it is shown that incubation of granules in the presence of sonicated phospholipid systems which undergo structural transitions in the presence of Ca2+ can cause immediate and total release of granule contents at Ca2+ levels of 2 mM or more. This behavior is attributed to disruption of granule membrane integrity due to fusion of the vesicle systems with the chromaffin granules. Direct evidence for such fusion is obtained by freeze-fracture electron microscopy. On the basis of this information and with the assumption that the inner leaflet of the adrenal cell plasma membrane is composed predominantly of phosphatidylethanolamine and phosphatidylserine, a mechanism of Ca2+-stimulated exocytotic release of catecholamines in vivo is proposed.

Fluorescence quenching study of mercury compounds and liposome interactions: Effect of charged lipid and pH

In relation to ecotoxicological research on the bioaccumulation and transfer processes of mercury compounds, a study of the interactions between these contaminants and the membrane barriers was investigated. The fluorescence quenching measurement of pyrene (collisional process)—a hydrophobic probe embedded into lipidic dispersions (PC, PS, PC-PS)—was performed with two mercury compounds (HgCl 2 and CH 3HgCl) versus pH (5.0 and 9.5), lipidic composition of artificial membranes, and chemical speciation. The results show that the accessibility of the mercurials to the hydrophobic core is highly influenced by the parameters studied; these results can be correlated to those observed in mercury bioaccumulation processes in natural environments.

Specific heats of lipid dispersions in single phase regions

Differential scanning calorimetry has been used for the first time to measure the specific heat, C p, as a function of temperature in the single phase regions above and below the main phase transition temperature, T m, for dispersions of saturated phosphatidylcholines and phosphatidylethanolamines. Within error limits C p, when expressed per gram, does not vary in any systematic way with chain length or headgroup. Its temperature dependence in both single phase regions qualitatively resembles that of n-alkanes. Contributions to C p from intrachain vibrations and interchain van der Waals' interactions have been calculated and account for nearly all the measured C p at temperatures above T m. However, these contributions do not yield the observed temperature dependence below T m. It is conjectured that such a temperature dependence arises from the unhindering of chain vibrations as the lipids undergo thermal expansion, and the result of a preliminary calculation which supports this conjecture is presented.

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.

The structure of unsaturated lipid dispersions in aqueous systems influences susceptibility to oxidation

The susceptibility of unsaturated lipids dispersed in aqueous systems to oxidation in the presence of decomposing peroxychromate has been examined. Almost complete oxidation of unsaturated monoenoic fatty acyl residues present in detergents such as Tween 80, which form micellar structures, was observed, whereas multibilayer liposomes of egg-yolk phosphatidylcholine could be oxidized only partially despite the presence of more susceptible polyenoic fatty acids. More vigorous methods of phospholipid dispersion or disturbing the structure of partially oxidized dispersions increased susceptibility of peroxidation, suggesting that substrate molecules located in the centre of large multibilayer dispersions are not accessible to free radicals generated in the bulk water.

Artificial black membranes from bipolar lipids of thermophilic Archaebacteria

The membrane of thermophilic archaebacteria is characterized by the presence of unusual isoprenoid bipolar lipids. The molecular organization of these lipids is still a matter of study. Important information could come from forming artificial black membranes. Black films can be formed from n-alkane or squalene dispersions of bipolar lipids extracted from the membrane of Caldariella acidophila. Membrane formation occurred only above a critical temperature (approximately 70 degrees C) corresponding to the physiological one. At lower temperatures, special solvent systems (n-alkanes or squalene, butanol and n-alkanes or squalene, butanol chloroform) were required. To characterize the physical parameters of these membranes, conductance and capacitance measurements were performed. Conductance was in the range of 10(-8) - 10(-7) omega -1 cm -2 , where specific capacitance at T = 72 degrees C was Cs = 0.685 +/- 0.004 microF/cm2 and Cs = 0.658 +/- 0.08 microF/cm2, corresponding to a dielectric thickness of 27 and 29 A for squalene and dodecane dispersions, respectively. Capacitance was shown to vary as the square of membrane potential, as usual in lipid bilayers. Values of the proportionality constant alpha have been compared to those of solvent-containing and solvent-free bilayers. The behavior of capacitance as a function of temperature is also shown by lowering temperature; the occurrence of complex structural changes was indicated. All the experimental data suggest that the presence of solvent is very low. Two possible molecular configurations of the films are discussed.

Conversion of a Lipoprotein from Egg Yolk into a Membranous Form in the Presence of Yolk Lipid

Treatment of the major lipoprotein (density O� 95 gjml) from hen's egg yolk with aqueous dispersions of yolk lipid resulted in a mixture that contained new large globular particles with membranous outer layers. These particles, referred to as 'membranous globules', had diameters in the range 50-200 nm when viewed in the electron microscope and in appearance, properties, and composition resembled the 'insoluble yolk globules' that are a natural part of whole yolk

Effects of amphotericin B on membrane permeability--kinetics of spin probe reduction.

The effect of the polyene antibiotic amphotericin B on the permeability of both unilamellar and multilamellar model membranes is investigated. The method measures the loss of the electron paramagnetic resonance signal of a spin probe, trapped in the aqueous compartment of a lipid dispersion, upon addition of ascorbate ions to the bulk aqueous phase. Amphotericin B causes large increases in the permeability of cholesterol-containing egg phosphatidylcholine membranes, whereas the effects are small in the absence of sterol and do not depend on surface charge. The effect of amphotericin depends upon the antibiotic:sterol mole ratio. The antibiotic appears to be unable to cross the membrane, acting only on the outermost bilayer of a multibilayer dispersion. When a phospholipid in the gel phase is used, amphotericin B causes large increases in permeability, independently of the presence or absence of sterol. It is suggested that the mechanism of action of amphotericin B is different for lipids in the liquid crystalline or gel states.

Modification of a vibrating-tube density meter for precise temperature scanning

We have devised a method of temperature scanning with a vibrating-U-tube density meter in which temperature fluctuations are much reduced compared to those using a constant or programmable thermostat. The standard error of a density measurement is 5 × 10 −7 g/ml. Volume changes associated with conformational changes of macromolecular systems can be precisely measured. Using this instrument the volume expansion-melting curves of lipid dispersions have been obtained. The curves are similar in shape and resolution to the excess heat-capacity curves derived from differential scanning calorimetry performed on the same sample. Temperature scanning allows measurements of expansivity as well as apparent volume throughout a temperature range of interest.

19F-NMR and fluorescence polarization of yeast plasma membrane and isolated lipids

Summary 16-Fluoropalmitic acid was incorporated biosynthetically into the plasma membrane of yeast and the F-NMR spectra of the intact membrane and of aqueous dispersions of membrane lipids were compared. The greater degree of resonance broadening in the intact membrane reveals a lesser degree of mobility of the acyl chains. Fluorescence polarization studies with a probe that monitors the polar environment and one that detects viscosity changes in the inner core of the bilayer indicate hindered motions of the polar head groups and acyl chains in the intact membrane as compared to liposomes prepared from membrane lipids. The observations indicate that the bulk of lipids in the yeast membrane experiences immobilization by proteins and that membrane proteins influence lipid-lipid interactions and inhibit lipid phase transition.

SOME IN VITRO OBSERVATIONS ON FACTORS AFFECTING RENNET (CHYMOSIN) CLOTTING OF CALF MILK REPLACERS

The formation of a firm rennet (chymosin) curd in the abomasum appears to have a useful physiological function in the newborn calf. The results of in vitro experiments with calf milk replacers conducted to study the effect of various factors on rennet clot formation demonstrated that low-pressure (L) dispersion of lipid into skim milk powder replacers resulted in markedly higher (P < 0.05) curd firmness values with rennet than homogenization (H) at all total solid (10, 15 and 20%) and lipid (10, 20, 30 and 40% TS) levels tested. At higher total solids levels, curd firmness, clot weight and percentage of replacer lipid in clot were significantly (P < 0.05) increased. The L dispersion method also promoted rennet coagulation and firmer curds than H when skim was partially replaced by mixtures offish protein-whey or Promine D-whey. The results indicate that reconstitution of milk replacers at high total solids levels (e.g., 20%), in conjunction with L dispersion of lipid, would be beneficial for promoting rennet coagulation, curd firmness and high concentrations of protein and lipid in the clot.