Molar Ratio Of Phosphatidylcholine Research Articles

Correction to: Antiatherosclerotic Effects of CSL112 Mediated by Enhanced Cholesterol Efflux Capacity.

HomeJournal of the American Heart AssociationVol. 12, No. 5Correction to: Antiatherosclerotic Effects of CSL112 Mediated by Enhanced Cholesterol Efflux Capacity Open AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toOpen AccessCorrectionPDF/EPUBCorrection to: Antiatherosclerotic Effects of CSL112 Mediated by Enhanced Cholesterol Efflux Capacity Originally published21 Feb 2023https://doi.org/10.1161/JAHA.121.027554Journal of the American Heart Association. 2023;12:e027554This article corrects the followingAntiatherosclerotic Effects of CSL112 Mediated by Enhanced Cholesterol Efflux CapacityOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: February 21, 2023: Ahead of Print In the article by Bronwyn A. Kingwell et al, “Antiatherosclerotic Effects of CSL112 Mediated by Enhanced Cholesterol Efflux Capacity,” which published online April 12, 2022 (J Am Heart Assoc. 2022;11:e024754. DOI: 10.1161/JAHA.121.024754) and was included in the April 19, 2022 issue of the journal, a correction was needed.On page 4 of the article, in the third line of the section “Plaque Stabilization,” the sentence reading “CSL111 is a predecessor compound formulated with higher apoA‐I/phosphatidylcholine molar ratio than CSL112.” was incorrect. It has now been corrected to read “CSL111 is a predecessor compound formulated with higher phosphatidylcholine/apoA‐I molar ratio than CSL112.”The authors regret the error.The online version of the article has been updated and is available here:https://www.ahajournals.org/doi/10.1161/JAHA.121.024754 Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesAntiatherosclerotic Effects of CSL112 Mediated by Enhanced Cholesterol Efflux CapacityBronwyn A. Kingwell, et al. Journal of the American Heart Association. 2022;11 March 7, 2023Vol 12, Issue 5Article InformationMetrics Copyright © 2023 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley BlackwellThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.https://doi.org/10.1161/JAHA.121.027554PMID: 36802969 Originally publishedFebruary 21, 2023 PDF download

Resolution V fractional factorial design for screening of factors affecting weakly basic drugs liposomal systems

This study aims to investigate factors affecting weakly basic drugs liposomal systems. Resolution V fractional factorial design (2V5−1) is used as an example of screening designs that would better be used as a wise step before proceeding with detailed factors effects or optimization studies. Five factors probable to affect liposomal systems of weakly basic drugs were investigated using Amisulpride as a model drug. Factors studied were; A: Preparation technique B: Phosphatidyl choline (PhC) amount (mg) C: Cholesterol: PhC molar ratio, D: Hydration volume (ml) and E: Sonication type. Levels investigated were; Ammonium sulphate-pH gradient technique or Transmembrane zinc chelation-pH gradient technique, 200 or 400 mg, 0 or 0.5, 10 or 20 ml and bath or probe sonication for A, B, C, D and E respectively. Responses measured were Particle size (PS) (nm), Zeta potential (ZP) (mV) and Entrapment efficiency percent (EE%). Ion selective electrode was used as a novel method for measuring unentrapped drug concentration and calculating entrapment efficiency without the need for liposomal separation. Factors mainly affecting the studied responses were Cholesterol: PhC ratio and hydration volume for PS, preparation technique for ZP and preparation technique and hydration volume for EE%. The applied 2V5−1 design enabled the use of only 16 trial combinations for screening the influence of five factors on weakly basic drugs liposomal systems. This clarifies the value of the use of screening experiments before extensive investigation of certain factors in detailed optimization studies.

Phosphatidylcholine-rutin complex as a potential nanocarrier for food applications

The aim of the present research was to encapsulate rutin with phosphatidylcholine (PC) to form PC-rutin complex known as phytosome to overcome the fortification difficulties such as low water solubility and bioavailability. Rutin loaded nanophytosome (RNP) was prepared in different molar ratios of rutin to PC. Colloidal properties of RNPs such as particle size, polydispersity index, encapsulation efficiency and stability, turbidity, and effects of thermal processes and pH on the physical stability of RNPs were assessed. Phytosome structure and chemical stability of rutin were investigated by FT-IR spectroscopy and HPLC/UV–Vis spectrophotometry, respectively. RNPs with the ruitn: PC molar ratios of 1:3 showed the most physical and chemical stability (during 30days of storage) with fine particle sizes (<100nm) and encapsulation efficiency of 99%. The turbidity and pH of food models/samples were remained unchanged at the fortification level. RNPs showed the ability of masking undesirable features of rutin resulting the potential applications in fortification of food products with water insoluble nutraceuticals.

Preparation and Characterization of Rutin-loaded Nanophytosomes

Background: Plant-derived materials are increasingly gaining attention as dietary supplements and due to their role in medicinal application. Rutin, a phenolic antioxidant, is a member of bioflavonoids which has been demonstrated to scavenge superoxide radicals and is believed to be a vital nourishing supplement due to its ability to strengthen and modulation of the permeability of the blood vessels walls. However, Rutin shows poor absorption when administered orally and its bioavailability is an important restrictive factor. Purpose: The present study was aimed to prepare and characterize a stable Rutin-loaded nanophytosomal formulation to improve its antioxidant property and bioavailability. Methods: Rutin-loaded nanophytosomes were prepared by phosphatidylcholine (PC) and cholesterol by thin layer hydration method. The physicochemical properties of prepared nanophytosomes were evaluated using particle size analyses, Furrier transformation infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). Stability of the prepared nanophytosomes was also investigated during three weeks of storage period. Results: Results showed that formulation with the Rutin: PC molar ratio of 1:2 possess lowest particle size and the incorporation of cholesterol improved the physical stability of nanophytosome for over three weeks. FTIR and DSC analysis showed the formation of Rutin-phospholipid complex during the formulation development process. Conclusion: Results of the present study showed that nanophytosome can be introduced as a useful carrier for fortifying herbal medicinal compounds for application the formulation of various food and pharmaceutical products.

Development of ibuprofen nanoliposome for transdermal delivery: Physical characterization, in vitro/in vivo studies, and anti-inflammatory activity

Objective Ibuprofen is an established non-steroidal anti-inflammatory drug commonly used for general inflammation. However, it causes gastrointestinal troubles when administered orally, thereby decreasing patient compliance. Transdermal application of vesicular formulation of Ibuprofen will provide better patient compliance and efficacy. Methods Six different compositions of lipid constituents have been formulated into nanovesicles using thin-film hydration method and dispersed into gel using Carbopol 934. The formulations were characterized based on physicochemical parameters using photon correlation spectroscopy, transmission electron microscopy, in vitro drug release, ex vivo skin permeation using human skin, and in vivo studies. Results The formulation, ibuprofen liposomal gel-5 (ILG-5), had nanoliposome of smallest size (159 nm) and polydispersity index (0.331). This formulation showed moderate zeta potential and the highest encapsulation. All the formulations including IG showed a considerable amount of drug release through in vitro synthetic membrane. ILG-5 showed maximum permeation during skin permeation studies. IG showed no permeation in ex vivo settings. ILG-5 has shown the highest C max and AUC during in vivo permeation study. Conclusions The present work clearly shows the superiority of nanoliposome formulation over non-vesicular formulations and that lipid composition containing 7/3/1 molar ratio of phosphatidylcholine, cholesterol, and dicetyl phosphate is optimum for nanoliposome preparations, in the cases where controlled delivery of drug is needed for a sufficient period of time.

Detailed Structure of Hairy Mixed Micelles Formed by Phosphatidylcholine and PEGylated Phospholipids in Aqueous Media

Aqueous dispersions of mixed egg yolk phosphatidylcholine (PC) and poly(ethylene glycol) (PEG) modified distearoyl phosphatidylethanolamine (DSPE) were investigated with the purpose of determining shape, size, and conformation of the formed mixed micelles. The samples were prepared at a range of DSPEPEG to PC molar ratios ([DSPEPEG/PC] from 100:0 to 30:70) and with, respectively, DSPEPEG2000 and DSPEPEG5000, where 2000 and 5000 refer to the molar masses of the PEG chains. Particle shape and internal structure were studied using small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). The contrast of the micelles is different for X-rays and neutrons, and by combining SANS and SAXS, complementary information about the micelle structure was obtained. The detailed structure of the micelles was determined in a self-consistent way by fitting a model for the micelles to SANS and SAXS data simultaneously. In general, a model for the micelles with a hydrophobic core, surrounded by a dense hydrophilic layer that is again surrounded by a corona of PEG chains in the form of Gaussian random coils attached to the outer surface, is in good agreement with the scattering data. At high DSPEPEG contents, nearly spherical micelles are formed. As the PC content increases the micelles elongate, and at a DSPEPEG/PC ratio of 30:70, rodlike micelles longer than 1000 angstroms are formed. We demonstrate that by mixing DSPEPEG and PC a considerable latitude in controlling the particle shape is obtained. Our results indicate that the PEG chains in the corona are in a relatively unperturbed Gaussian random coil conformation even though the chains are far above the coil-coil overlap concentration and, therefore, interpenetrating. This observation in combination with the observed growth behavior questions that the "mushroom-brush"transition is the single dominating factor for determining the particle shape as assumed in previous theoretical work (Hristova, K.; Needham, D. Macromolecules 1995, 28, 991-1002).

β 2 -Glycoprotein I (Apolipoprotein H) Modulates Uptake and Endocytosis Associated Chemiluminescence in Rat Kupffer Cells

g 2-Glycoprotein I (g 2GPI) is known to influence macrophage uptake of particles with phosphatidylserine containing surfaces, as apoptotic thymocytes and unilamellar vesicles in vitro. Nevertheless, effects upon macrophage activation induced by this interaction are still unknown. g 2GPI influence upon the reactive species production by Kupffer cells was evaluted in order to investigate whether g 2GPI modulates the macrophage response to negatively charged surfaces. Chemiluminescence of isolated non-parenchymal rat liver cells was measured after phagocytosis of opsonized zymosan or phorbolmyristate acetate (PMA) stimulation, in the presence and absence of large unilamellar vesicles (LUVs) containing 25mol% phosphatidylserine (PS) or 50mol% cardiolipin (CL) and complementary molar ratio of phosphatidylcholine (PC). g 2GPI decreased by 50% the chemiluminescence response induced by opsonized zymosan, with a 66% reduction of the initial light emission rate. PMA stimulated Kupffer cell chemiluminescence was insensitive to human or rat g 2GPI. Albumin (500 w g/ml) showed no effect upon chemiluminescence. g 2GPI increased PS/PC LUV uptake and degradation by Kupffer cells in a concentration-dependent manner, without leakage of the internal contents of the LUVs, as shown by fluorescence intensity enhancement. LUVs opsonized with antiphospholipid antibodies (aPL) from syphilitic patients increased light emission by Kupffer cells. Addition of g 2GPI to the assay reduced chemiluminescence due to opsonization with purified IgG antibodies from systemic lupus erythematosus (SLE or syphilis (Sy) patient sera. A marked net increase in chemiluminescence is observed in the presence of Sy aPL antibodies, whereas a decrease was found when SLE aPL were added to the assay, in the presence or absence of g 2GPI. At a concentration of 125 w g/ml, g 2 GPI significantly reduced Kupffer cell Candida albicans phagocytosis index and killing score by 50 and 10%, respectively. The present data strongly suggest that particle uptake in the presence of g 2GPI is coupled to an inhibition of reactive species production by liver macrophages during the respiratory burst, supporting the role of g 2GPI as a mediator of senescent cell removal.

Probing for preferential interactions among sphingolipids in bilayer vesicles using the glycolipid transfer protein.

We have investigated the intervesicular transfer of galactosylceramide between unilamellar bilayer vesicles composed of differing sphingomyelin and phosphatidylcholine molar ratios. To monitor glycolipid transfer from donor to acceptor vesicles, we used a fluorescence resonance energy transfer assay involving anthrylvinyl-labeled galactosylceramide (AV-GalCer) and perylenoyl-labeled triglyceride. The transfer was mediated by glycolipid transfer protein (GLTP), purified from bovine brain and specific for glycolipids. The initial transfer rate and the total accessible pool of glycolipid in the donor vesicles were both measured. An increase in the sphingomyelin content of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) vesicles decreased the transfer rate in a nonlinear fashion. Decreased transfer rates were clearly evident at sphingomyelin mole fractions of 0.22 or higher. The pool of AV-GalCer available for GLTP-mediated transfer also was smaller in vesicles containing high sphingomyelin content. In contrast, AV-GalCer was more readily transferred from vesicles composed of POPC and different disaturated phosphatidylcholines. Our results show that GLTP acts as a sensitive probe for detecting interactions of glycosphingolipids with neighboring lipids and that the lateral mixing of glycolipids is probably affected by the matrix lipid composition. The compositionally driven changes in lipid interactions, sensed by GLTP, occur in membranes that are either macroscopically fluid-phase or gel/fluid-phase mixtures. Gaining insights into how changes in membrane sphingolipid composition alter accessibility to soluble proteins with affinity for membrane glycolipids is likely to help increase our understanding of how sphingolipid-enriched microdomains (i.e., "rafts" and caveolae) are formed and maintained in cells.

Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression

The ability of different phosphatidylcholine (PC) species to inhibit cytokine-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) was investigated. PC species containing palmitoyl- in the sn-1 position and palmitoyl- (DPPC), arachidonyl- (PAPC), linoleoyl- (PLPC) or oleoyl- (POPC) in the sn-2 position were compared. These PC species were studied as components of reconstituted high density lipoproteins (rHDL) (containing apolipoprotein A-I [apoA-I] as the sole protein) or as small unilamellar vesicles (SUVs). The rHDL containing PLPC and PAPC inhibited VCAM-1 expression in activated HUVECs by 95 and 70%, respectively, at an apoA-I concentration of 16 micrometer. At this concentration of apoA-I, POPC rHDL inhibited by only 16% and DPPC rHDL did not inhibit at all. These differences could not be explained by differential binding of the rHDL to HUVECs. The same hierarchy of inhibitory activity was observed when these PC species were presented to the cells as SUVs but only when the SUVs also contained an antioxidant. It was concluded that rHDL PC is responsible for their inhibitory activity and that this varies widely with different PC species.

Vesicle to micelle structural transitions involved in the interaction of dodecylbetaine with liposomes: Transmission electron microscopy and light scattering studies

The interaction of the amphoteric surfactant dodecylbetaine (C 12-Bet) with phosphatidylcholine (PC) liposomes was studied by means of transmission electron microscopy (TEM) and changes in the mean particle size (quasi-elastic light scattering, QELS) and in static light scattering (SLS) of the system were observed during liposome solubilization. A good correlation was found between the TEM diameter of the particles and the mean hydrodynamic diameter (HD) determined by OELS. The aggregates resulting in this interaction were dependent on the C 12-Bet concentration in the system. Thus, an initial vesicle growth occurred when the surfactant concentration was 16.66 mol%. Additional surfactant amounts led first to the formation of largest vesicles (HD 306 nm for 37.50 mol% C 12-Bet concentration) and second to a fall in the vesicle diameter an in the SLS of the system. Thus, for 47.36 mol% (Re SAT, surfactant to PC molar ratio for liposome saturation), the TEM picture still showed the presence of vesicles albeit with signs of disintegration and vesicle fusion. When additional amounts of surfactant were added to the system the size curve started to show a bimodal distribution. Thus, for 52.38 mol% a sharp curve appeared at 9 nm corresponding to the formation of small particles and the TEM picture showed vesicle disintegration with formation of tubular structures as a crucial intermediate. Up to a surfactant mole percent of 60% progressive vesicle disintegration took place with increasing presence of small aggregates (tubular structures and bilayer fragments) and without formation of complex multilayered structures. The gradual increase in the proportion of these small aggregates (mixed micelles) led to the complete solubilization of liposomes.

Remodelling of reconstituted high density lipoproteins by lecithin: cholesterol acyltransferase

Discoidal reconstituted high density lipoproteins (rHDL) with a diameter of 7.9 nm, a molar ratio of egg phosphatidylcholine (PC): unesterified cholesterol (UC): cholesteryl esters (CE): apolipoprotein (apo) A-I of 33:7:0:1 and containing two molecules of apoA-1 per particle were incubated with lecithin:cholesterol acyltransferase (LCAT) in the presence of low density lipoproteins as a source of additional UC and PC for the LCAT reaction. After 24 h of incubation, the rHDL had a diameter of 8.8 nm, a molar ratio of PC:UC:CE:apoA-I of 16:3:23:1 and contained three rather than two molecules of apoA-I per particle. The fact that there was no change in the concentration of rHDL-associated apoA-I indicated that the increase from two to three molecules of apoA-I per particle was achieved at the expense of a one-third reduction in the number of rHDL particles in a process that must have involved particle fusion. When the incubations were repeated in the presence of exogenous, lipid-free apoA-I, the resulting rHDL were identical in size and composition to those generated in its absence. Under these conditions, however, the increase from two to three molecules of apoA-I per rHDL particle coincided with a 50% increase in the concentration of rHDL-associated apoA-I. Thus, when lipid-free apoA-I is available, the LCAT-mediated increase in number of apoA-I molecules per rHDL particle is achieved by a direct incorporation of lipid-free apolipoprotein without any need for particle fusion and therefore without a reduction in the number of rHDL particles.

Participation of cholesterol in hypochlorite-induced oxidation of cholesterol-phosphatidylcholine liposomes

The effect of cholesterol on hypochlorite-induced lipid peroxidation is studied in cholesterol-phosphatidylcholine liposomes. Cholesterol is shown to promote the accumulation of lipid peroxidation products in the presence of 0.1–3 mM hypochlorite anion in the incubation medium. The content of 2-thiobarbituric acid-reactive products is maximal at a cholesterol: phosphatidylcholine molar ratio of 1∶1. On the other hand, in the presence of low hypochlorite concentrations (10–100 μM) cholesterol is found to inhibit lipid peroxidation.

Transmembrane Ca2+ gradient-mediated phosphatidylcholine modulating sarcoplasmic reticulum C(a2+)-ATPase.

The sarcoplasmic reticulum (SR) C(a2+)-ATPase was purified and reconstituted into the sealed phospholipids vesicles with or without transmembrane Ca2+ gradient. The role of phospholipids, especially phosphatidylcholine (PC), in the modulation of C(a2+)-ATPase by transmembrane Ca2+ gradient was investigated. The results are as follows. (i) Incubated with phospholipids, the enzyme activity of the delipidated C(a2+)-ATPase is inhibited by Ca2+ and the highest inhibition is observed in the presence of PC. (ii) When there exists a transmembrane Ca2+ gradient (higher Ca2+ concentration inside vesicles, 1,000 mumol/L:50 mumol/L, similar to the physiological condition), the inhibition of C(a2+)-ATPase by transmembrane Ca2+ gradient can be only observed in the vesicles containing PC:PE, but not in those containing PS:PE or PG:PE. The highest inhibition is obtained at a 50:50 molar ratio of PC:PE (iii) By comparing the effects of PC differing in acyl chains, higher inhibition of C(a2+)-ATPase is observed in vesicles containing DPPC:PE and DOPC:PE, while no inhibition in DMPC:PE vesicles (iv) If the transmembrane Ca2+ gradient is in the inverse direction, the enzyme activity of C(a2+)-ATPase is inhibited whenever reconstituted with acidic or neutral phospholipids.

Effect of protein energy malnutrition on the lipid composition and leucine uptake of small intestinal brush border vesicles of growing rhesus monkeys.

Mild to moderate protein energy malnutrition (PEM) was induced in young developing rhesus monkeys by giving them half of the casein-based synthetic diet which was given to control animals. After a body weight reduction of 30-40%, the PEM animals were sacrificed. The small intestine was removed, flushed with ice-cold saline, everted and divided into equal proximal, middle and distal segments. Brush border membrane vesicles (BBMV) were prepared from all three segments and assayed for marker enzymes, e.g. sucrase and alkaline phosphatase, to assess their purity. Sucrase was found to be purified 23-fold and alkaline phosphatase 12-fold compared to the respective homogenates in all three parts. In PEM animals, uptake of [U-14C]L-leucine into the BBMV was diminished in all three segments and cholesterol and phospholipid levels also decreased significantly. As a result there was an elevation in the molar ratio of cholesterol to phospholipid, and the sphingomyelin: phosphatidylcholine molar ratio also increased. This signified a decrease in lipid fluidity and amino acid uptake in PEM in the small intestine. Histologically, a mild to moderate grade of partial villus atrophy was observed in the intestine. The diminished uptake and lipid fluidity of the membrane and the histological changes returned to their control values after nutritional rehabilitation.

The Effects of Coexisting Lipids on the Action of Bacillus thuringiensis Phosphatidylinositol-Specific Phospholipase C toward Liposomal Substrate.

The hydrolytic activity of phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) from Bacillus thuringiensis was studied in detail toward mixed liposomes consisting of PI and one of other phospholipids and cholesterol. Among PI-liposomes, small unilamellar vesicles (SUV) were the most sensitive to PI-PLC; the enzymatic hydrolysis of PI in SUV was not less than 10-fold that in large unilamellar vesicles (LUV) or in multilamellar vesicles (MLV). Thus, in a survey of the effects of coexisting lipids on PI-PLC activity, PI-SUV was used. Phosphatidylcholine (PC) was stimulative for the enzyme activity toward PI-SUV at any molar ratio of PC to PI. Also, the effects of the addition of sphingomyelin (SM), phosphatidylethanolamine (PE) and cholesterol on the enzymatic hydrolysis of PI were studied in detail on the basis of concentration of total lipids or PI.

Effect of calcium on kinetic and structural aspects of dilution-induced micellar to lamellar phase transformation in phosphatidylcholine-cholate mixtures.

Previously, we have shown [Almog, S., Kushnir, T., Nir, S., & Lichtenberg, D. (1986) Biochemistry 25, 2597-2605] that the distribution of cholate between phosphatidylcholine (PC) vesicles and aqueous media apparently obeys a single distribution coefficient, K. In PC-cholate mixed micellar systems, the monomer concentration does not rise much above the cholate's critical micelle concentration (cmc). Consequently, for vesicular systems, the cholate:PC molar ratio in the mixed aggregates (Re) is given by Re = [cholate]/([PC] + 1/K) whereas for mixed micellar systems Re = ([cholate] - cmc)/[PC]. Dilution of mixed micellar systems results in a decrease of Re, due to an increase in the fraction of monomeric PC. If the decrease in Re is to values lower than 0.3, micellar to lamellar transformation occurs. This process involves a sequence of three steps, namely, micellar equilibration followed by vesiculation and subsequent vesicle size growth via a lipid transfer mechanism. The ultimate size of the resultant vesicles is an increasing function of Re. This work is devoted to the effect of calcium on the dilution-induced vesicle formation. Its major findings and conclusions are as follows: (i) Calcium reduces the cmc of the detergent and raises its distribution coefficient between PC vesicles and the aqueous medium. Thus, for any given cholate and PC concentrations, calcium causes an increase of Re. (ii) The rate of all the steps which ultimately lead to an apparent equilibrium vesicle size distribution increases dramatically with increasing calcium concentration. Thus, equilibration is attained in seconds to minutes rather than many hours required in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of saccharides with lipid bilayer vesicles: stabilization during freeze-thawing and freeze-drying

The fusion of small unilamellar vesicles of phosphatidylcholines during freeze-thawing and freeze-drying / rehydration, and the suppression of fusion under these conditions by various saccharides, was investigated by gel filtration on Sepharose 4B, quasielastic light scattering, high-resolution 1H-NMR, ESR spin labeling, and differential scanning calorimetry. Freeze-thawing and freeze-drying of aqueous small unilamellar vesicle suspensions in the presence of sufficient sucrose had no significant effect on the average size and size distribution of small unilamellar vesicles. In the presence of sucrose the structural integrity and the permeability properties of the phosphatidylcholine bilayers were retained during freeze-thawing and freeze-drying. A comparison of the stabilizing effect of sucrose with that of trehalose and glucose showed that the stabilization is not sugar-specific but is a general property of saccharides. The fraction of small unilamellar vesicles recovered after freeze-thawing depended on the saccharide / phosphatidylcholine molar ratio. The mechanism of the cryoprotective effect involves binding of the sugar to the phospholipid polar group, probably through hydrogen bonding.

Kinetic and structural aspects of reconstitution of phosphatidylcholine vesicles by dilution of phosphatidylcholine-sodium cholate mixed micelles.

Dilution of mixed micellar dispersions of egg phosphatidylcholine (PC) and sodium cholate beyond a critical value results in formation of cholate-containing PC vesicles. The structure of the resultant vesicles and some mechanistic aspects of this process have been investigated by the use of light scattering and nuclear magnetic resonance techniques. The main findings and conclusions are the following: Both the state of aggregation (micellar or vesicular) and the apparent equilibrium size distribution of micelles or vesicles obtained by dilution of the PC-cholate mixed micellar dispersions are a function of the cholate to PC molar ratio in the mixed aggregates (micelles or vesicles). When this effective ratio (Re) is higher than 0.4, the dispersion is micellar, and the size of the mixed micelles increases with decreasing Re; when Re less than 0.3, the dispersion is essentially vesicular, and the mean hydrodynamic radius of the vesicles is an increasing function of Re; in dispersions with 0.3 less than Re less than 0.4, mixed micelles and vesicles coexist. Addition of cholate to vesicular dispersions, to Re values below 0.3, results in vesicle size growth through a concentration-independent lipid-exchange mechanism. Addition of cholate to higher Re values results in micellization (solubilization) of the vesicles. On the other hand, dilution of vesicular dispersions does not affect the size of the vesicles. Apparent equilibration of a mixed micellar dispersion following dilution to Re values below 0.3 is slow (many hours). The overall process involves a series of three subsequent categories of steps: (i) a rapid (approximately 1-2 min) prevesiculation equilibration of micellar sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of cholesterol in the activity of reconstituted Ca-ATPase vesicles containing unsaturated phosphatidylethanolamine.

The effect of cholesterol on the activity of sarcoplasmic reticulum Ca-ATPase, reconstituted in proteoliposomes containing soybean phosphatidylethanolamine (PE), egg phosphatidylcholine (PC), and cholesterol, was examined. The protein incorporation efficiency increased with PE content but appeared to be independent of cholesterol content. At low cholesterol, PE stimulated calcium uptake. The coupling efficiency of the proteoliposomes increased with an increase in cholesterol content at each PC/(PC + PE) ratio and was more pronounced for those proteoliposomes containing high PE. Dynamic fluorescence measurements of the incorporated lipophilic probe, diphenyl-1,3,5-hexatriene, revealed a decrease in the motion and an increase in the order of the phospholipid fatty acyl chains in proteoliposomes with high cholesterol content. A complementary observation was made using electron spin resonance of the spin label, 2,2-dimethyl-5-dodecyl-5-methyloxazolidine N-oxide. Freeze-fracture electron microscopy studies on proteoliposomes containing 0.20 molar ratio of PC/(PC + PE) and cholesterol revealed predominantly vesicular structures with occasional bilayer defects at high cholesterol content. It is postulated that the cholesterol-induced enhancement of the Ca-transport function of the Ca-ATPase is related to the hydration-related bilayer-destabilizing characteristic of the cholesterol molecule as revealed by 31P NMR.

Effect of dietary cholesterol on microsomal membrane composition, dynamics and kinetic properties of UDPglucuronyl transferase

The effect of cholesterol administration in vivo on the lipid composition, dynamic properties of the microsomal membrane of guinea pig livers and the kinetic properties of UDPglucuronyl transferase were studied. Cholesterol administration in the diet evoked an increase of microsomal cholesterol, but no significant changes in the fatty-acid composition of total lipids or of each phospholipid class. Instead, the phosphatidylethanolamine, phosphatidylcholine molar ratio of the membrane was markedly decreased from 0.57 to 0.38. This decline was not enough to counterbalance the overall ‘ordering’ effect of cholesterol and consequently, the fluorescence anisotropy of the membranes labeled with 1,6-diphenylhexatriene was increased. The lateral diffusion evaluated by measuring the pyrene excimer formation was decreased by the cholesterol incorporation. These physical changes were associated with changes in the kinetic properties of UDPglucuronyl transferase: V max increased, while the K m of the different steps of the reaction decreased in the modified microsomes. Furthermore, a shift of the non-michaelian kinetics to michaelian, equivalent to a decrease of a negative homotropic effect and apparent cooperativity of UDPglucuronic acid was observed since the Hill coefficient changed, approaching 1. A non-michaelian kinetics of this enzyme is an indication of boundary lipids in the gel phase and a shift to michaelian, a change of the surrounding lipids to a liquid-crystalline structure. In consequence, our results suggest that cholesterol incorporation in the microsomal membrane while producing a condensing effect of bulk lipids would produce an opposite effect on the UDPglucuronyl transferase boundary lipids.