Yolk Lecithin Research Articles

ESR study of the liposome membrane physical parameters in the heating-cooling cycles.

Changes of dynamic and structural parameters of egg yolk lecithin (EYL) liposome membranes in the heating-cooling cycles have been studied using the ESR spin probe method. The investigations were conducted in the range of temperatures from -18 degrees C to +60 degrees C. It has been found that in the range of temperatures -15 degrees C to +45 degrees C in both the heating and the cooling run the spectroscopic parameters changed practically along the same curve (reversible changes). However, after exceeding this range of temperatures one of the parameters (partition coefficient of the spin probe 2,2,6,6--tetramethylpiperidine -1-oxyl; TEMPO) changed along a closed curve, showing the phenomenon of thermal hysteresis. In the heating process the TEMPO content in liposome membranes was smaller than this in the cooling process. We assume that during the heating, the lipid molecules of the outer liposome layers dissolve in the aqueous medium. In the cooling process they can aggregate and form new liposomes, what in turn increases the surface of liposome membranes, accessible for the TEMPO probe (active surface).

Molecular dynamics and phase transitions in phospholipid monolayers at liquid-liquid interfaces

Stablen-hexadecane/water andn-tetradecane/water macroemulsions containing monolayers of natural (egg yolk lecithin, EY) and synthetic (dimyristoylphosphatidylcholine, DMPC) phospholipids at liquid-liquid interfaces were prepared. The existence of the monolayers was proved by studying the reduction kinetics of a surface-active spin probe with ascorbate anions. Spin labeled derivatives of stearic acid in which the nitroxide group is locared at different distances from the polar head (5-, 12-, and 16-doxylstearic acids) were used to study the temperature dependences of the molecular ordering, rotational mobility, and local polarity in the monolayers in emulsions and also in bilayers in liposomes obtained from the same lipids. In the EY monolayers, the degree of spin probe solubilization is higher, while the order parameters (S) and rotational correlation times (τ) are lower than those in EY bilayers. The differences between these parameters for mono- and bilayers increase with an increase in the distance of the reporter group from the aqueous phase. In the DMPC monolayers, a first-order phase transition was detected by measuring the temperature dependences ofS and τ. The temperature region of the phase transition in monolayers is shifted to lower temperatures with respect to that for bilayers and depends on the nature of the oil phase. It was concluded that the phospholipid monolayers in emulsions incorporate hydrocarbon molecules, whose concentration in the DMPC monolayers increases on going from the low-temperature (gel) to the high-temperature (liquid crystal) phase.

Increase in Deformability of Human Erythrocytes through the Action of β-Lysophospholipid Rich in n-3 Polyunsaturated Fatty Acid Content

Lysophosphatidylcholine (LPC) that binds to docosahexaenoic acid (DHA) at the sn-2 position (ƒA-DHA-LPC) was prepared by the Lipozyme mediated selective partial hydrolysis of DHA-en- riched egg yolk lecithin. The ƒA-DHA-LPC was found to be rapidly incorporated into erythrocyte cells and increase their deformability at 5ƒEM/erythrocyte.

Stability of liposomal formulations: action of amphiphilic molecules

The destabilization of vesicles obtained by cyclic freezing-thawing and extrusion (FATVET) bilayer membranes consisting of L- α-dipalmitoyl phosphatidylcholine (DPPC) by the bile salt sodium deoxycholate (DOC) was studied from turbidimetric, entrapped solute release and steady-state fluorescence anisotropy measurements. Based on turbidity measurements, bilayer membranes in a rigid gel state are more resistant to lysis by deoxycholate than are bilayers in a fluid liquid-crystal state. Thus, the [detergent] total/[phospholipid] mole ratio, R t sat, needed to saturate the bilayers at 25°C ( T< T m) is three times greater than that at 50°C ( T> T m) also, R t sol, which corresponds to complete solubilization of the bilayers, is twice higher at 25°C than it is at 50°C. However, this does not mean that bilayer saturation or complete solubilization calls for increased DOC concentrations, as shown by the fact that the effective mole ratios for both events, R e sat and R e sol, are identical at 25°C and 50°C. The ability of DOC molecules to intercalate themselves between DPPC molecules therefore appears to be decreased by tightly packed phospholipid molecules. Thus, the partition coefficient for the sub-solubilization range, R e, is almost 3 times lower at 25°C than it is at 50°C. On the other hand, the R e sat, R e sol and K values for FATVETs{egg yolk lecithin (EYL)} at 25°C are virtually identical with those for FATVETs{DPPC} at 50°C. Hence, for a series of phospholipid analogues, the partition coefficient K for a surfactant/liposome system is bound to depend primarily on the physical state of the bilayer. The retention properties of FATVETs{DPPC} are impaired at sub-lytic DOC levels thus, 5-fluorouracil (5-FU) leakage under these conditions amounts to R e 50%=0.09, which is somewhat smaller than R e sat (1.2–1.4). This may be the result of the packing and structural changes undergone by lamellae prior to solubilization of the bilayers. The 1,6-diphenyl-1,3,5-hexatriene (DPH) steady-state fluorescence anisotropy results also suggest that DOC alters acyl chain packing, diminishes transition cooperativeness and decreases the orderliness of acyl chains.

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile

Background/Aims: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstones pathogenesis are unknown. We have studied the effects of calcium and a major biliary protein, anionic polypeptide fraction, on the process of cholesterol crystallization in bile. Methods: Anionic polypeptide fraction was purified from human bile. Model bile composed of cholesterol, egg yolk lecithin and sodium taurocholate was prepared in a lipid concentration (18 mM, 37 mM, and 120 mM, respectively) simulating lithogenic human gallbladder bile. The crystallization process was observed by phase contrast light microscopy, and sequential separation of precipitable cholesterol structures by sucrose density gradient ultracentrifugation. Results: Addition of calcium, or anionic polypeptide fraction alone, or both together did not influence the crystal observation time of bile (the time which elapsed from initiation of supersaturation to the first appearance of crystals). However, the rate and quantity of cholesterol precipitation and crystal formation were affected by both. Calcium increased in a dose-dependent manner the cholesterol monohydrate crystal mass before apparent equilibrium was reached. This effect was inhibited by anionic polypeptide fraction, which increased the amount of cholesterol within precipitable phospholipid vesicles, and decreased the rate of crystal formation. Fluorescence-labeled anionic polypeptide fraction revealed that anionic polypeptide fraction (with and without calcium) was primarily associated with vesicle aggregates. Conclusions: Our data demonstrate that calcium and anionic polypeptide fraction have opposing effects on the process of cholesterol crystallization and the resultant crystal mass without influencing the crystal observation time of bile. These findings suggest that biliary proteins, in addition to being crystallization effectors by themselves, may further influence cholesterol crystallization and gallstone formation by interacting with calcium and possibly other elements that coexist in bile.

Effects on the early development of the pearl sac by cytochrome c, yolk lecithin and polyvinyplyrro

Mantle pieces ofHyriopisis cumingii was treated by three kinds of solution, These mantle pieces were inserted together with paraffin nucleuses into the connective tissue of three groups ofHyriopisis cumingii. These operated animals were cultured in a pool for a month. Several pearl sacs were put out and immersed by Bouin's solution after every five days. Sections of each animals were made by histological method. Those without treatment were in a control group. Observations of these sections showed that cytochrome c and yolk lecithin have an accelerated roles on pearl sac development and pearl layer secretion. No effects on pearl sac development by polyvinyplyrrolidone (PVP).

Quantitative Examination of the Simple Micelle–Mixed Micelle Coexistence for the Taurocholate–Lecithin System by GP-HPLC

Gel-permeation, high-performance liquid chromatography (GP-HPLC) studies were conducted to quantify simple micelles and mixed micelles present in model biles. A TSK-gel G2000SW column was preequilibrated with a buffered mobile phase: phosphate buffer, pH 7.4, 0.1MNaCl, and 30 mM[14C]taurocholate (TC). Simulated biles with different TC and/or egg-yolk lecithin (L) levels were then introduced onto the GP column. The injected biles without L (TC alone) yielded positive peaks or negative valleys (relative to the baseline) at ∼9.5 min, depending on the injected TC concentration. A theoretical mass balance equation quantitatively accounted for the observed simple micelle peaks (or valleys). When TC-L coexisting simple micelle–mixed micelle model biles were injected onto the column, in addition to the simple micelle peaks at ∼9.5 min, mixed micelle peaks were observed at retention times in the region ∼7.5 to 8.0 min. Here, also, there was good mass balance between the expected and the observed TC peaks. The average molecular weights were estimated as ∼28,000–45,000 Da for the mixed micelles and ∼5500 Da for the simple micelles, respectively. The TC-to-L molar ratio (K) for the mixed micelle peak showed a curvilinear decrease as a function of L concentration and leveled off at around 1.5. These results are consistent with those obtained from previous equilibrium dialysis studies, although theKvalues from present studies are slightly lower (∼20%); a possible explanation for this discrepancy is presented.

Plastoquinol and α-tocopherol quinol are more active than ubiquinol and α-tocopherol in inhibition of lipid peroxidation

Comparative studies of antioxidant activities of such natural prenyllipids as plastoquinol-9 (PQH 2-9), α-tocopherol quinol ( α-TQH 2), ubiquinol-10 (UQH 2-10) and α-tocopherol ( α-T) in egg yolk lecithin liposomes have been performed. The investigated compounds showed oxidation under molecular oxygen in the order UQH 2-10> α-TQH 2>PQH 2-9>> α-T. The corresponding second order rate constants have been determined in Tris buffer (pH=6.5) and were 0.413, 0.268, 0.154 and 0.022 M −1/s, respectively. The inhibition order of Fe 2+-H 2O 2 -induced lipid peroxidation, corrected for the amount of prenyllipids oxidized during the initiation period, was α-TQH 2>PQH 2-9> α-T>UQH 2-10 for 5 mol% of the antioxidants content in liposomes. The radicals formed in the initiation phase of the reaction caused oxidation of 27.5–33% α-T, 40–64% UQH 2-10, 42–85% PQH 2-9 and 43–80% α-TQH 2, depending on the antioxidant concentration in liposomes (5–1 mol%, respectively) which reflects approximately their reactivity against radicals derived from the Fenton reaction. The antioxidant activity of the investigated prenylquinols, in relation to the activity of α-T, in natural membranes is discussed.

Interaction of sodium alkyl sulphates with egg yolk lecithin vesicles / Wechselwirkungen von Na-Alkylsulfaten mit Vesikeln aus Eigelblecithin

The interaction of three anionic surfactants - sodium alkyl (decyl-, dodecyl-, tetradecyl-) sulphates - with unilamellar liposomes from egg yolk lecithin was studied fluorometrically by monitoring the leakage of the marker 6-carboxyfluorescein. An increase in leakage rate was observed with increasing surfactant concentration ; very small quantities of surfactant depending on its alkyl chain could disrupt the vesicles thus release the whole entrapped marker immediately after mixing with vesicles. Light scattering results suggest an increase in vesicle size followed by the formation of mixed micelles of surfactant and phospholipid molecules. The presence of cholesterol in the vesicle decreased the leakage of the marker due to its rigidifying effect.

Concentration of Docosahexaenoic Acid-containing Phospholipid through Lipozyme IM-mediated Hydrolysis

A selective partial hydrolyses of egg-yolk lecithin from fish-oil fed hens and squid-skin lecithin were carried out with immobilized Rhizomucor miehei lipase (Lipozyme IM) in n-hexane media in order to concentrate docosahexaenoic acid (DHA) -containing phospholipids. DHA were abundant in phospholipid fractions that had recovered from the partial hydrolysates than the corresponding substrates i.e. egg-yolk lecithin and squid lecithin. Thus, it was clear that DHA ester bonds were less susceptible against hydrolysis than other fatty acid ester bonds. Optimum water activity (aw) for Lipozyme IM to concentrate DHA-containing phospholipids was aw=0.44. Under this aw, DHA showed to three fold increase compared to that of the original egg-yolk lecithin from fish-oil fed hens, after 8 h reaction at 40°C. Water activity-controlled partial hydrolysis with Lipozyme IM was borned out to be a useful method for concentrating functional phospholipids.

Destabilisation of liposomes by bovine serum albumin; Sepharose 2B-Cl experiment

The stability of liposomes (2∶1 egg yolk lecithin:cholesterol, mole ratio; diameter about 100 nm) at increasing bovine serum albumin (BSA) concentration was study. The influence of introducing positive or negative charge to the liposomal bilayers was tested. The results indicated appreciable destructive effects of serum albumin on the liposomal membranes of neutral and negatively charged liposomes. Near-physiological concentration (30 mg mL−1) of albumin dissolved more then 50% of liposomes.

Transformation of sterols byMycobacterium vaccae: effect of lecithin on the permeability of cell envelopes to sterols

An enhancement of Β-sitosterol transformation to androstendione byMycobacterium vaccae observed in medium containing egg-yolk lecithin, was associated with the incorporation of a considerable amount of lecithin into the cell envelope lipids. By GC/MS measurements, fatty acids ranging from 14 to 22 carbon atoms were identified in the lipids removed from the cells by organic solvents. Octadecenoic (18:1), 2-methyl-octadecenoic (2-Me 18:1), and hexadecanoic (16:0) acids were the major components of the lipid preparation obtained from both the control cells, and the cells grown in lecithin-containing medium. However, in the fatty acid pattern of the latter a distinct increase in the C18:1 component, concomitant with the decrease in the 2-Me 18:1 fatty acid was demonstrated. The C16 fatty acid fraction also showed a higher content of methyl-branched components in the control cell preparation. The enrichment in unsaturated fatty acids increases fluidity of lipids, whereas the decrease in methyl-branched fatty acids may affect the conformation of the surface lipidic components, which may result in enhanced sterol penetration through the cell wall barrier in the presence of lecithin.

Antioxidant Activity of Reduced Menadione in Solvent Solution and in Model Membranes

The antioxidant activity of reduced menadione was investigated and compared with that of alpha-tocopherol both in solvent solution and in large unilamellar vesicles by using azocompounds as free radical generators. The results show that: i) reduced menadione behaves as a chain-breaking antioxidant; ii) its inhibition rate constant is similar to that of alpha-tocopherol in homogeneous solution, whereas it is 4 times larger in egg yolk lecithin vesicles; iii) the stoichiometric factor is found lower than 1 in both systems, since a substantial portion of menadiol is consumed by autoxidation and does not contribute to radical trapping; iv) when both alpha-tocopherol and menadiol are present in vesicles, reduced menadione can spare alpha-tocopherol. Data presented here suggest that the reduced form of vitamin K may protect, when present, cellular membranes from free radical damage.

Effect of xanthophyll pigments on fluorescence of chlorophyll a in LHC II embedded to liposomes

A major light-harvesting pigment-protein complex of Photosystem II (LHC II) isolated from rye leaves was incorporated to egg yolk lecithin liposomes containing various amounts (0-0.6 mol % with respect to lipid) of xanthophyll pigments: violaxanthin, lutein or zeaxanthin. It appeared that the addition of xanthophylls increased the level of the chlorophyll a fluorescence emission despite the screening effect of additional pigments in the system. Another phenomenon documented by the 77 K fluorescence emission spectra was the xanthophyll-related desaggregation of LHC II within the lipid phase of liposomes. The analysis of the parameters of chlorophyll a fluorescence decay in comparison to steady state fluorescence emission let us conclude that the fluorescence reabsorption in aggregated LHC II was a major cause of the lower level of fluorescence emission in that state of antenna proteins. Chlorophyll singlet excitations quenching brought about by violaxanthin, lutein and zeaxanthin is also documented in liposomes containing incorporated LHC II and exogenous xanthophyll pigments, and in liposomes containing xanthophyll pigments and chlorophyll b.

Preparation of Membrane Extrusion Vesicles of Egg Yolk Lecithin and Their Characterization

Membrane extrusion vesicles of egg yolk lecithin were prepared and their diameters and lamellarity were determined from dynamic light scattering measurement and the fluorescent probe method, respectively. The fluorescent probe lipid used was dimyristoylphosphatidylethanolamine 4-nitrobenz-2-oxa-1, 3-diazole (NBD-DMPE). The mean radius of the vesicles sharply decreased with the number of extrusions and became ca. 60 nm at more than ten cycles of extrusion through a 0.1 mm pore-size membrane filter. For a unilamellar vesicle with 60 nm radius and bilayer thickness of 3 nm, the external lipid fraction should be 0.53. The experimentally obtained value was 0.52±0.03 at a molar ratio of NBD-DMPE in total lipid less than 0.3%. At higher NBD-DMPE (0.3%1.0%), the external marker lipid fraction was 0.550.61 immediately following preparation and subsequently decreased slowly to ca. 0.5. It thus follows that 1) vesicles after ten cycles of extrusion are unilamellar, 2) NBD-DMPE becomes distributed in the outer layer of the lecithin bilayer during extrusion, and 3) flip-flop motion of NBD-DMPE occurs after extrusion, leading to even distribution of the marker lipid in the vesicles.

Bimodal effect of amphiphilic biocide concentrations on fluidity of lipid membranes.

Using the spin label method (ESR) it has been shown that biologically active, amphiphilic compounds (quaternary ammonium salts (AS) containing polar heads with single and double positive charge caused, at low concentrations, decrease fluidity of liposome membranes formed with egg yolk lecithin (EYL). At higher concentrations an increase in fluidity was observed. With compounds having a single positive charge minimum fluidity of membrane structure occurs in the range of 1 to 3%, with compounds containing double positive charge-in the range of 4-6%. That effect does not depend on polar head size and length of alkyl chains of the AS used. Analysis of the electrostatic interaction between positive charges and dipole system suggest that at low ion concentrations the binding energy of the system increases, while it decreases at high concentrations. For the model presented, maximum of binding energy of the system occurs at 3% of positive monovalent ions and at 6% of positive divalent ions admixed.

Binding and relaxation behaviour of prodan and patman in phospholipid vesicles: a fluorescence and 1H NMR study

The relative location, binding behaviour and the solvent relaxation behaviour of the polarity sensitive membrane probes 6-propionyl-2-(dimethylamino)naphthalene and 6-palmitoyl-2-[[trimethylammoniumethyl]methylamino]naphthalene chloride in vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine or egg yolk lecithin have been compared using steady-state and time-resolved fluorescence as well as high resolution NMR measurements. The reconstructed time-resolved emission spectra show unambiguously that the observed spectral shifts in vesicle systems have to be assigned to time-dependent solvent relaxation processes rather than to a probe relocation mechanism. All fluorescence as well as the NMR relaxation data suggest a deeper localization of Patman in the membrane, sensing a less polar and/or more restricted probe environment.

Visual Acuity and Fatty Acid Status of Term Infants Fed Human Milk and Formulas with and without Docosahexaenoate and Arachidonate from Egg Yolk Lecithin1

Preterm infants fed formulas with docosahexaenoic acid (DHA, 22:6n-3) during the interval equivalent to the last intrauterine trimester and beyond have higher circulating DHA and transiently higher visual acuity compared with infants fed formulas containing linolenic acid. In term infants several nonrandomized studies of infants receiving DHA from human milk suggest a relationship between DHA status and acuity, but the evidence for a cause-and-effect relationship is mixed. In the present study, term infants were randomly assigned to a standard term formula (n = 20) or the same formula with egg yolk lecithin to provide DHA (0.1%) and arachidonic acid (AA, 20:4n-6, 0.43%) (n = 19) at levels reported in milk of American women. A third group of infants was breast fed for > or = 3 mo (n = 19). Grating visual acuity (Teller Acuity Card procedure) and plasma and red blood cell (RBC) phosphatidylcholine (PC) and phosphatidylethanolamine (PE) DHA and AA were determined at corrected ages of 2, 4, 6, 9 (acuity only), and 12 mo past term = 40 wk postmenstrual age (PMA). At 2 mo breast-fed infants and infants fed the supplemented formula had higher grating acuity than term infants fed standard formula. As in preterm infants, the increase was transient. Plasma PC DHA and AA and RBC PE AA increased by 2 mo in supplemented infants, but RBC PE DHA in supplemented infants was not higher than in controls until 4 mo and beyond. Despite normal intrauterine accumulation of DHA and AA, infants fed formula with 2% linolenic acid and 0.1% DHA had better 2-mo visual acuity than infants fed formula with 2% linolenic acid.

Influence of the redox state of ubiquinones and plastoquinones on the order of lipid bilayers studied by fluorescence anisotropy of diphenylhexatriene and trimethylammonium diphenylhexatriene

The measurements of diphenylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) fluorescence anisotropy in egg yolk lecithin (EYL) and of DPH anisotropy in dipalmitoylphosphatidylcholine (DPPC) liposomes containing different concentrations of oxidized and reduced ubiquinone (UQ) and plastoquinone (PQ) homologues have been performed. All the oxidized UQ homologues strongly induced ordering of EYL membrane structure, whereas in DPPC liposomes, above the phase transition temperature, the most pronounced effect showed UQ-4. PQ-2 and PQ-9 were less effective than the corresponding ubiquinones in this respect. The reduced forms of UQ and PQ homologues increased the order of membrane lipids to a smaller extent than the corresponding quinones both in the interior of the membrane and closer to its surface. Nevertheless, the investigated prenylquinols showed stronger increase in the membrane order than α-tocopherol or α-tocopherol acetate, which could be connected with binding of prenylquinol head groups to phospholipid molecules by hydrogen bonds. The strong ordering influence of ubiquinones on the membrane structure was attributed to methoxyl groups of the UQ quinone rings.

Lipid oxidation in bilayer liposomes induced by radicals from the surrounding water phases

Some features of the radiation chemistry of organized assemblies were studied in aqueous dispersions of small unilamellar vesicles of egg yolk lecithin. The kinetics for the reaction of OH radicals with the bilayer was determined by pulse radiolysis. The conversion of OH radicals into N 3 radicals results in a remarkable reduction of the radiolysis of the hydrophylic part of the phospholipid and in an enhanced degradation of the most radiosensitive group of polyunsaturated fatty acid residues. The transverse proton relaxation of the choline head group is very sensitive to the radical attack on the bilayer.