Solubility Limit Of Cholesterol Research Articles

Lipid loss and compositional change during preparation of simple two-component liposomes

Liposomes are used as model membranes in many scientific fields. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures can produce liposomes at specific concentrations and lipid compositions, and researchers often assume that the concentration and composition of their liposomes are similar or identical to what would be expected if no lipid loss occurred. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward HPLC-ELSD method to quantify the lipid concentration and composition of liposomes and apply that method to study the preparation of simple cholesterol/POPC liposomes. We examine common liposome preparation steps, including vortexing during resuspension, lipid film hydration, extrusion, freeze-thaw, and sonication. We found that the resuspension step can play an outsized role in determining the lipid loss (up to ∼50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses (∼10–20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 min and increasing cholesterol concentrations up to 50 mol % had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition, as long as the lipid mixture was below the cholesterol solubility limit. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation for cholesterol/POPC liposomes.

In vitro determination of the solubility limit of cholesterol in phospholipid bilayers

Cholesterol has limited solubility in phospholipid bilayers. The solubility limit is strongly dependent on the nature of the lipid with which the cholesterol is mixed while properties of the crystals formed can be modified by phospholipid-cholesterol interactions. In this review we summarize the various methods that have been developed to prepare hydrated mixtures of cholesterol and phospholipid. We point out some of the factors that determine the form adopted when cholesterol crystallizes in such mixtures, i.e. two- or three-dimensional, monohydrate or anhydrous. These differences can greatly affect the ability to experimentally detect the presence of these crystals in a membrane. Several methods for detecting cholesterol crystals are discussed and compared including DSC, X-ray and GIXRD diffraction methods, NMR and EPR spectroscopy. The importance of the history of the sample in determining the amount and nature of the cholesterol crystals formed is emphasized.

In vitro determination of the solubility limit of cholesterol in phospholipid bilayers

In vitro determination of the solubility limit of cholesterol in phospholipid bilayers

Comment on "Cholesterol solubility limit in lipid membranes probed by small angle neutron scattering and MD simulations" by S. Garg et al., Soft Matter, 2014, 10, 9313.

In a recent article, Garg et al. used neutron scattering techniques to determine the limiting amount of cholesterol which vesicles of either POPS or POPC can accommodate. This amount was called "the cholesterol solubility limit". In light of extensive literature on cholesterol phase separation in phospholipid bilayers, the way in which "solubility limit" is defined in this article and the conclusions derived are misleading and require some clarification.

Reply to the ‘Comment on “Cholesterol Solubility Limit in Lipid Membranes probed by Small Angle Neutron Scattering and MD simulations”’ by R. Epand, Soft Matter, 2015, 11, DOI: 10.1039/C4SM02819H

In the comment by Epand et al. on our recent article, it is stated that the term “cholesterol solubility limit” is misused.

Response to “Reply to the ‘Comment on “Cholesterol Solubility Limit in Lipid Membranes probed by Small Angle Neutron Scattering and MD Simulations by Ursula Perez-Salas, Soft Matter, 2014, 10, 9313–9317”’”

As authors of the “Comment on ‘Cholesterol solubility limit in lipid membranes probed by small angle neutron scattering and MD simulations’”, we wish to comment on both the form and content of the Reply cited above.

Observation of Stripe Superstructure in the β-Two-Phase Coexistence Region of Cholesterol-Phospholipid Mixtures in Supported Membranes.

Visualization of phase coexistence in the β region of cholesterol-phospholipid mixtures consisting of high cholesterol concentrations has proved elusive in lipid bilayers. Here, using the solvent-assisted lipid bilayer approach to prepare supported membranes with high cholesterol fractions close to the cholesterol solubility limit, we report the observation of coexisting liquid phases using fluorescence microscopy. At ∼63 mol % cholesterol, supported membranes consisting of mixtures of DOPC and cholesterol exhibit large-area striping reminiscent of the stripe superstructures that characterize the proximity of the second critical point in the miscibility phase diagram. The properties of the two phases are consistent with condensed complex-rich and cholesterol-rich liquids. Both phases exhibit long-range lateral mobility, and diffusion through a given phase is favored over hopping across the phase boundary, producing an "archipelago effect" and a complex percolation path.

Cholesterol solubility limit in lipid membranes probed by small angle neutron scattering and MD simulations.

The solubility limits of cholesterol in small unilamellar vesicles made of POPS and POPC were probed using Small Angle Neutron Scattering (SANS) and coarse grained (CG) molecular dynamics (MD) simulations. SANS, being non-invasive, allowed the direct and quantitative measurement of cholesterol in intact vesicles. Our experimental measurements reveal a 61% mole fraction solubility limit of cholesterol in POPC, consistent with previous studies. However, in POPS the solubility limit of cholesterol is found to be 73% mole fraction. Previous work reports solubility limits of cholesterol in POPS varying significantly, ranging from 36% up to 66%. The CG MD simulations are in remarkable quantitative agreement with our experimental results showing similar solubility limits. Further, neither experiments nor simulations show evidence of stable nanodomains of cholesterol in POPS membranes as suggested in some previous reports.

Ergosterol and Stigmasterol Interact with Phosphatidylcholine Lipid Bilayers Less Favorably than Cholesterol

Sterols are a class of membrane lipids that is important to maintain plasma membrane structure and functions in eukaryotic cells. The maximum solubility of sterol in a lipid bilayer is the highest mole fraction of sterol that can be incorporated into a lipid bilayer before sterol precipitates from the bilayer to form crystals. A higher maximum solubility indicates more favorable interactions between the sterol and lipid bilayer. In this study, the maximum solubilities of ergosterol and stigmasterol in DOPC and DSPC lipid bilayers were measured using light scattering and further confirmed using optical microscopy. We found that correlation function of scattering intensities from two independent detectors can be used to sensitively determine the solubility limits of sterols. The validity of our new technique was confirmed by measuring the solubility limit of cholesterol in DOPC and DPPC lipid bilayers. We found that the maximum solubilities of ergosterol and stigmasterol are higher in PC lipid bilayers with saturated chains (DSPC) than that in PC bilayers with unsaturated chains (DOPC). Compared with cholesterol, ergosterol and stigmasterol both have much lower solubility limits in PC lipid bilayers. Our results suggest that minor differences in sterol structure could result in large differences in sterol-PC interactions.

Large Domain Formation in POPC/POPE Lipid Bilayers at the Maximum Solubility Limit of Cholesterol

Lipid bilayers composed of POPC and POPE usually stay as 1-phase mixtures at room temperature, with or without incorporation of modest amount of cholesterol. However, at the maximum solubility limit of cholesterol, possibility of domain formation has been suggested based on the detail molecular packing patterns of phospholipids with cholesterol. The maximum solubility of cholesterol in a lipid bilayer is the highest mole fraction of cholesterol that can be incorporated into a lipid bilayer before cholesterol crystals precipitate. Previously, the maximum solubility of cholesterol in mixtures of POPC/POPE lipid bilayers has been measured using a cholesterol oxidase (COD) reaction rate assay. The maximum solubility of cholesterol was determined to be 67 mol % in POPC bilayers and 50 mol % in POPE bilayers. In mixtures of POPE/POPC, the maximum solubility of cholesterol increases linearly as a function of the ratio POPC/(POPE+POPC). In this study, planar lipid bilayers of POPC and POPE at their cholesterol solubility limits were incubated on mica surfaces inside hydrated chambers. Although the bilayers appeared having uniform fluorescence intensity initially, we found that large lipid domains became visible by fluorescence microscopy after several days of incubation. Previously, it has been suggested that cholesterol may form a “hexagonal” regular distribution pattern at the maximum solubility limit in POPE bilayers and a “maze” pattern at the maximum solubility in POPC bilayers. Thus, it is possible that the lipid mixtures separated into POPC-rich and POPE-rich lipid domains, each having their signature lipid packing pattern and mole fraction of cholesterol.

Ceramide Drives Cholesterol Out of the Ordered Lipid Bilayer Phase into the Crystal Phase in 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/Cholesterol/Ceramide Ternary Mixtures

The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.

Lateral Distribution of Cholesterol in Dioleoylphosphatidylcholine Lipid Bilayers: Cholesterol-Phospholipid Interactions at High Cholesterol Limit

Lateral organization of cholesterol in dioleoyl-phosphatidylcholine (DOPC) lipid bilayers at high cholesterol concentration (>45 mol%) was investigated using steady-state fluorescence anisotropy and fluorescent resonance energy transfer techniques. The recently devised Low Temperature Trap method was used to prepare compositionally uniform cholesterol/DOPC liposomes to avoid the problem of lipid demixing. The fluorescence anisotropy of diphenylhexatrience chain-labeled phosphatidylcholine (DPH-PC) in these liposomes exhibited local maxima at cholesterol mol fractions of 0.50 and 0.57, and a sharp drop at 0.67. For the liposomes labeled with both dehydroergosterol and DPH-PC, the fluorescent resonance energy transfer efficiency from dehydroergosterol to DPH-PC displayed a steep jump at cholesterol mol fraction of 0.5, and dips at 0.57 and 0.68. These results indicate the presence of highly ordered cholesterol regular distribution domains at those observed critical compositions. The observed critical mol fraction at 0.67 agreed favorably with the solubility limit of cholesterol in DOPC bilayers as independently measured by light scattering and optical microscopy. The regular distribution at 0.57 was previously predicted from a Monte Carlo simulation based on the Umbrella model. The results strongly support the hypothesis that the primary requirement for cholesterol-phospholipid mixing is that the polar phospholipid headgroups need to cover the nonpolar body of cholesterol to avoid the exposure of cholesterol to water.

Bilayer structure of ganglioside/cholesterol mixed system in the presence of Ca2+

We employed small-angle X-ray scattering measurement using synchrotron radiation to study structural properties of monosialoganglioside (GM1)/cholesterol (chol) mixtures in aqueous dispersions, in the presence of 0–50 mM CaCl2. Various molar ratios of GM1/chol were examined from 1:0 to 1:5. Without CaCl2 the radius of gyration (Rg ) of the aggregate increased linearly with increasing cholesterol content up to GM1/chol = 1/1. At cholesterol content above GM1/chol = 1/1, Rg of the aggregate was almost constant, and a sharp diffraction peak at q = 0.18 Å−1, characteristic of cholesterol monohydrate crystals, was observed, suggesting the solubility limit of cholesterol in GM1 aggregates is about GM1/chol = 1/1. For the samples containing cholesterol of GM1/chol = 1/0.6 and above, the diffraction patterns showed a set of peaks that correspond to ~85 Å periodic lamellar structures in the presence of CaCl2 at concentrations higher than ~10 mM. The observed lamellar peak positions and the intensity ratio of the lamellar peaks (1st–4th) was not changed by the addition of CaCl2 up to 100 mM, demonstrating once formed multilayered structure of GM1/chol was not altered under the present conditions. Electron density profiles for this GM1/chol bilayer were obtained. The Ca2+-induced vesicle-to-lamellar phase separation of this system was found to be highly reversible by the Ca2+ chelation with ethylenediamine-tetraacetic acid.

The arrangement of cholesterol in membranes and binding of NAP-22

Cholesterol forms crystals when the mol fraction of sterol in a membrane bilayer exceeds a certain value. The solubility limit of cholesterol is very dependent on the nature of the phospholipid with which it is mixed. NMR methods have proven useful in quantifying the amount of cholesterol monohydrate crystals present in mixtures with phospholipids. A protein, NAP-22, present in high abundance in the synaptic cell membrane and synaptic vesicle, promotes the formation of cholesterol crystallites in lipid mixtures in which cholesterol would be completely dissolved in the membrane in the absence of protein. This finding, along with effects of the protein on the phase transitions of mixtures of phosphatidylcholine (PC) and cholesterol indicate that NAP-22 facilitates the formation of cholesterol-rich domains. This protein will bind only to membranes of PC that contain either cholesterol or phosphatidylethanolamine (PE). The process requires the presence of a myristoyl group on the N-terminus of NAP-22. The phenomenon also does not occur with a 19 amino acid myristoylated peptide corresponding to the amino terminal segment of NAP-22. The basis of the selectivity of NAP-22 for interacting with membranes of specific composition is suggested to be due to the accessibility of the myristoyl group.

Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.

Distinct effects of three bile salts on cholesterol solubilization by oleate-monoolein-bile salt micelles

Micellar cholesterol solubilities in bile salt-monoolein-oleic acid systems have been determined. Whatever the bile salt/oleyl compounds ratio, taurochenodeoxycholate solubilizes more cholesterol than taurocholate and much more than tauroursodeoxycholate. At pH 6.7, the cholesterol solubility limit is about the same with either oleate or monoolein. Cholesterol solubility falls in oleate-bile acid mixtures as the pH is raised. The capacity for supersaturation with cholesterol is greater for bile salt-monoolein than for bile salt-oleate micelles. For the latter it decreases as pH increases.