Cholesterol and phytosterols effect on sphingomyelin/phosphatidylcholine model membranes--thermodynamic analysis of the interactions in ternary monolayers

In this work thermodynamic analysis of the interactions between lipids in ternary sphingomyelin/DPPC/sterol Langmuir films were performed to compare the effect of cholesterol, beta-sitosterol and stigmasterol on a model membrane. The condensing effect of the respective sterols and the interactions between molecules in ternary mixtures were analyzed on the basis of the excess area per molecule and the excess free energy of mixing values. The stability of the mixed monolayers was verified with the free energy of mixing values. The conclusions on the ordering effect of sterols were drawn from the analysis of the compression modulus values. It was found that the stoichiometry of the mixed films of the highest thermodynamic stability and of the strongest interactions is the same for all the sterols investigated. The results obtained prove that the mammalian sterol induces the strongest contraction of the area and reveals the strongest stabilizing and ordering effect among the investigated sterol. Stigmasterol was found to condense a model membrane in a weaker extent as compared to beta-sitosterol, however, the differences in ordering properties of both phytosterols are less pronounced. The magnitude of the influence of the investigated sterols on a model membrane was thoroughly discussed from the point of view of the structure of their side chain, which determines the geometry of a sterol molecule.     

Ordering effects of cholesterol on sphingomyelin monolayers investigated by high-resolution broadband sum-frequency generation vibrational spectroscopy

After adding cholesterol, the sphingosine backbones (red) of the three nature SMs become more ordered, and the N-linked acyl chain (blue) remains unaltered.     

Edelfosine disturbs the sphingomyelin-cholesterol model membrane system in a cholesterol-dependent way - the Langmuir monolayer study

Synthetic alkyl-lysophospholipids, represented by edelfosine (ED), reveal strong anticancer activity and therefore are promising drugs used in anticancer therapy. Primary target for edelfosine is cellular membrane, which is in contrast to traditional cytostatics affecting DNA. The mechanism of antitumor activity of edelfosine was hypothesized to be related to its accumulation in membrane rafts. Inspired by these findings, we have performed the Langmuir monolayer studies on the influence of edelfosine on systems composed of sphingomyelin (SM) and cholesterol (Chol), being the principal components of membrane rafts. Sphingomyelin-cholesterol proportion in monolayers was varied to reflect the composition of solely membrane rafts (SM/Chol=2:1) and contain excess of cholesterol (SM/Chol=1:1 and 1:2). Into these systems, edelfosine was added in various concentrations. The analysis of surface pressure-area isotherms, complemented with films visualization with Brewster angle microscopy (BAM) allowed us to compare the effect of edelfosine on condensation and ordering of SM/Chol monolayers. The results evidenced that the influence of ED on the interactions in model membranes and its fluidizing effect is highly cholesterol-dependent. The strongest decrease of monolayer ordering was observed for model raft system, while the excess of cholesterol present in the remaining mixtures was found to weaken the fluidizing effect of the drug.     

N-(1-piperidinepropionyl)amphotericin B methyl ester (PAME)--a new derivative of the antifungal antibiotic amphotericin B: searching for the mechanism of its reduced toxicity

N-(1-piperidinepropionyl)amphotericin B methyl ester (in short, PAME), a low-toxicity amphotericin B derivative, has been investigated in Langmuir monolayers at the air/water interface alone and in mixtures with cellular membrane sterols (a mammalian sterol, cholesterol, and a fungal sterol, ergosterol) and a model phospholipid (DPPC). The analysis of the strength of interaction between PAME and both sterols as well as DPPC was based, on surface pressure measurements and analysis of the isothermal compressibility (C(s)(-1)), the mean area per molecule (A(12)), the excess free energy of mixing (DeltaG(Exc)) and the total free energy of mixing (DeltaG(M)). It has been found that the interactions between PAME and sterols are attractive; however, their strength is significantly weaker for mixtures of PAME with cholesterol than with ergosterol. This casts light on the improved selectivity of PAME toward fungal cells. The strongest interactions, found for PAME/DPPC mixtures, proved an important role of DPPC in the mechanism of reduced toxicity of PAME as compared to amphotericin B. Due to stable complex formation between PAME and DPPC the antibiotic is immobilized with DPPC molecules, which reduces the concentration of free antibiotic, which is capable of interacting with membrane sterols.     

Evaluation of the interactions between lipids and γ-globulin protein at the air–liquid interface

The interactions between lipids (cholesterol, distearoylphosphatidylcholine, distearoylphosphatidylethanolamine and sphingomyelin) and the γ-globulin protein have been analyzed using the monolayer technique at the air–liquid interface. The analysis has been carried out using both state equations and an adequate thermodynamic formulation for the surface pressure (π)–molecular area (a) isotherms. Different parameters as the virial coefficients, have been estimated. For the uncharged lipid monolayers, the interactions between the molecules are of an attractive nature, at medium and long distance, and of a steric repulsive nature at short distance. At low surface pressures the lipid molecules form small domains. The net force between γ-Globulin molecules in the monolayers has been found to be attractive. Finally, it can be concluded that when the lipid monolayers are uncharged, there is practically no interaction between the protein and lipid molecules at the mentioned interface.     

Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: influence of the cholesterol content

The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 degrees C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air-water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.     

Properties of β-sitostanol/DPPC monolayers studied with Grazing Incidence X-ray Diffraction (GIXD) and Brewster Angle Microscopy

Although the influence of structurally modified sterols on artificial membranes has been intensively investigated, studies on the properties of stanols, which are saturated analogs of sterols, are very rare. Therefore, we have performed Grazing Incidence X-ray Diffraction (GIXD) experiments aimed at studying in-plane organization of a plant stanol-β-sitostanol monolayer and its mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPC at the air/water interface. The collected GIXD data, resulting in-plane parameters and BAM images provide information on molecular organization and in-plane ordering of the investigated films. It was found that the lateral organization of β-sitostanol/DPPC monolayers depends on their composition. The oblique structure of the in-plane lattice of tilted hydrophobic region of molecules, found for DPPC film, is maintained at 10 mol% of stanol in the system. However, at 30 and 90 mol% of stanol in the mixture, the arrangement of molecules is hexagonal and they are oriented perpendicularly to the interface. With the addition of stanol the extend of the in-plane order of the monolayers decreases. Moreover, in mixtures the ordered domains consist of both monolayer's components. Additionally, β-sitostanol film is of similar in-plane organization as the corresponding sterol monolayer (β-sitosterol) and stanol induces condensing effect on DPPC.     

The interactions between cholesterol and phospholipids located in the inner leaflet of humane erythrocytes membrane (DPPE and DPPS) in binary and ternary films—The effect of sodium and calcium ions

The studies on the influence of cholesterol on phospholipids accumulated in inner leaflet of membrane are performed rather rarely, especially in the presence of electrolytes, which are present in membrane environment. Therefore, in this work the interactions between cholesterol and saturated phosphatidylethanolamine (PE) and phosphatidylserine (PS) were studied in binary (phospholipid/cholesterol) and ternary (PS/PE/cholesterol) monolayers in the presence and absence of sodium and calcium ions. The composition of ternary films was estimated to reflect the proportion of PSs to PEs in inner layer of human erythrocyte membrane. The influence of electrolytes on pure PS and PE films was also analyzed. It was found that both sodium and calcium ions affect the condensation of DPPS films, and influence the interactions in DPPS/cholesterol monolayers. On the other hand, no effect of these ions on DPPE films as well as on DPPE/cholesterol interactions in the mixed systems was observed. The results obtained for ternary mixtures prove that in the presence of Na⁺ the interactions between the lipids are more favorable than in the absence of these ions. This is in contrast to the effect of Ca²⁺. All the results were thoroughly analyzed in the context of the structure of polar heads of the investigated phospholipids.     

The magnitude of condensation induced by cholesterol on the mixtures of sphingomyelin with phosphatidylcholines-Study on ternary and quaternary systems

The studies on the condensing and ordering effect of cholesterol by application of the Langmuir monolayer technique are usually performed on binary lipid/cholesterol systems. The results concerning a quantitative analysis of these effects in multicomponent monolayers are very limited. In this work the condensing and ordering effect of cholesterol in ternary (SM/DSPC/Chol and SM/DOPC/Chol) and quaternary (SM/DSPC/DOPC/Chol) films was investigated. It was evidenced that the systems containing saturated PC (both SM/DSPC and SM/DSPC/Chol) are always more condensed and chain-ordered than the systems containing unsaturated PC (SM/DOPC and SM/DSPC/DOPC and their mixtures with cholesterol). However, the magnitude of condensation provoked by cholesterol at higher surface pressures is stronger on the monolayers containing unsaturated PC. The addition of cholesterol into SM/PC films induces the increase of chain-ordering however, the effectiveness of cholesterol as an ordering agent is determined by the presence/absence of unsaturated phospholipid. The magnitude of the effect of cholesterol on the investigated mixed monolayer was analyzed in the context of the influence of sterol on lipid chains (ordering, straightening and reorientation of chains) as well as the reorientation of polar heads.     

SiO2/Cr monolayers and formation of polyethylene films

1,3,5-tribenzylhexahydro-1,3,5-triazine-CrCl3 (TAC-CrCl3) was supported on monodisperse St?ber silica and on commercial silica particles. The monolayers of these catalyst particles were successfully prepared with Langmuir-Blodgett technique. The effect of TAC-CrCl3 molecules on the ordering and film-forming of the silica particles was investigated. The particles arrange in domains of hexagonal ordering, but the ordering of particles in the monolayer decreases with increasing Cr loading. These hybrid monolayers are active polymerization catalysts. After polymerization, a relatively rough layer of polyethylene molecules covers the catalyst monolayer and this layer is hydrophobic. The monolayers and the polymers coating films were characterized by IR, UV-vis-NIR, and SEM. The method provides a way to fabricate polyethylene films on silica monolayers.     

How does the N-acylation and esterification of amphotericin B molecule affect its interactions with cellular membrane components-the Langmuir monolayer study

This work presents the results of Langmuir monolayers study of two amphotericin B derivatives obtained by N-acylation (N-acetylamphotericin B, Ac-AmB) and esterification (amphotericin B methyl ester, AME) of the parent AmB molecule. The main objective of present investigations was to examine the strength and nature of interactions of Ac-AmB and AME with natural membrane components as compared to AmB, and verify the monolayer results with biological studies in vitro. Our experiments were based on surface pressure-area measurements of mixed monolayers formed by the investigated antibiotics and sterols/DPPC. The interactions were analyzed with the following dependencies: compression modulus-surface pressure, mean molecular area-composition, excess molecular area-composition and excess free energy-composition plots. It has been found that both Ac-AmB and AME form monolayers of a liquid expanded state and their stability is highest as compared to AmB films. The investigated compounds mix in monolayers with natural membrane components within the whole range of the antibiotic mole fraction. The quantitative analysis of the interactions of the investigated antibiotics with sterols and DPPC as well as sterols/DPPC interactions allow us to verify the monolayer results with biological results. A good correlation between both kinds of studies has been found.     

The surface pressure dynamics and appearance of mixed monolayers of cholesterol and different sized polystyrenes at an air-water interface

Synthetic polymers are increasingly being used in situations where they are designed to interact with biological systems. As a result, it is important to investigate the interactions of the polymers with biochemicals. We have used cholesterol, as an example of an important biological surfactant component, to study its interactions with polystyrene. Mixed monolayers of cholesterol and one of two different molecular weight polystyrenes were formed at an air-water interface to investigate their interactions and to determine whether the size of the polystyrene affected the interaction. The pressure-area (pi-A) isocycles of mixed monolayers of cholesterol and polystyrene MW 2700 or polystyrene MW32700 showed that strongest attractive interactions occur at high surface pressures and in polystyrene rich films. The excess area and excess free energy of mixing were most negative at high surface pressures and at high mole fraction of polystyrene. The most stable mixed monolayers were formed with X(PS2700) = 0.9 and X(PS32700) = 0.09. Microscopic observation of the mixed monolayers of cholesterol and polystyrene showed the formation of stable islands in the cholesterol/polystyrene mixtures. These observations, the nature of the inflection points in the isocycles, and the anomalous changes in free energy lead us to conclude that there is a stable rearrangement of polystyrene into compact islands when it is mixed with cholesterol. Any excess cholesterol is excluded from these islands and remains as a separate film surrounding the islands.     

Monolayers (Langmuir films) behavior of multi-component systems composed of a bile acid with different sterols and with their 1:1 mixtures

Different physicochemical properties of Langmuir films (monolayers) composed of 10 mixed systems of a bile acid, deoxycholic acid (DC) with various plant sterols, such as stigmasterol (Stig), beta-sitosterol (Sito) and campesterol (Camp) and a stanol, cholestanol (Chsta) in addition to an animal sterol, cholesterol (Ch) [these sterols and Chsta are abbreviated as St] and DC with 1:1 St mixtures; (Ch+Chsta), (Ch+Stig), (Stig+Chsta), (Ch+Sito) and (Ch+Camp) on the substrate of 5M aqueous NaCl solution (pH 1.2) at 25 degrees C, were investigated in terms of mean surface area per molecule (A(m)), the partial molecular area (PMA), surface excess Gibbs energy (DeltaG((ex))), interaction parameter (I(p)) as well as activity coefficients (f(1) and f(2)) in 2-D phase of each binary (or ternary) component system and elasticity (Cs(-1)) of formed films; these were analyzed on the basis of the respective surface pressure (pi) versus A(m) isotherms as a function of mole fraction of Sts (X(st)) in the DC/St(s) mixtures at discrete surface pressures. Notable findings are: (i) all the binary component systems did form patched film type monolayers consisting of (a) DC-dominant film solubilizing a trace amount of St molecules and (b) St dominant film dissolving a small amount of DC molecules, (ii) DC in 2-D phase exhibited a transition from LE film to LC film at a constant pressure (pi(C)(1)) accompanied by compression and (iii) DeltaG((ex)) as well as I(p) was found to be greatly dependent on (a) the combinations of DC with different St species and (b) to be markedly varied by a difference in mixing ratio of DC to Sts. Compressibility (or elasticity) analyses and fluorescence microscopy images could support the above findings as well as interpretation.     

Miltefosine-cholesterol interactions: a monolayer study

Mixed Langmuir monolayers of miltefosine (hexadecylphosphocholine) and cholesterol have been investigated by recording surface pressure-area (pi-A) isotherms at different subphase pHs (2, 6, and 10) and temperatures (10, 20, 25, and 30 degrees C). The change of both pH and temperature within the investigated range does not modify significantly the behavior of mixed films. The most pronounced effect involves condensation of the miltefosine monolayer by cholesterol, which diminishes in the following order: pH 6 > pH 2 > pH 10. The analyses of pi-A and compressibility modulus dependencies indicate the existence of interactions in the investigated system; at pH 2 and 6, the strongest were found to occur for the mixed film of miltefosine molar fraction (XM) between 0.6 and 0.7 (mean value, 0.66). Such a composition corresponds to the stable complex formation wherein 2 miltefosine molecules and 1 molecule of cholesterol are strongly bound together, mainly by attractive hydrophobic forces between their apolar tails. However, at pH 10 the highest stability occurs for mixtures containing a smaller proportion of miltefosine (XM = 0.5), which means that on alkaline subphases the ability to condense the miltefosine monolayer by cholesterol is less efficient as it requires a higher proportion of cholesterol (1:1 as compared to 1:2 at pH 2 and 6) to attain the maximum stability of the mixed film. The attractive forces between miltefosine and cholesterol are also weaker at pH 10 due to a greater solvatation of the miltefosine polar group. A similar trend is observed on increasing subphase temperature, when monolayers are more expanded.     

Enantiomeric interactions between liquid crystals and organized monolayers of tyrosine-containing dipeptides

We have examined the orientational ordering of nematic liquid crystals (LCs) supported on organized monolayers of dipeptides with the goal of understanding how peptide-based interfaces encode intermolecular interactions that are amplified into supramolecular ordering. By characterizing the orientations of nematic LCs (4-cyano-4'-pentylbiphenyl and TL205 (a mixture of mesogens containing cyclohexane-fluorinated biphenyls and fluorinated terphenyls)) on monolayers of l-cysteine-l-tyrosine, l-cysteine-l-phenylalanine, or l-cysteine-l-phosphotyrosine formed on crystallographically textured films of gold, we conclude that patterns of hydrogen bonds generated by the organized monolayers of dipeptides are transduced via macroscopic orientational ordering of the LCs. This conclusion is supported by the observation that the ordering exhibited by the achiral LCs is specific to the enantiomers used to form the dipeptide-based monolayers. The dominant role of the -OH group of tyrosine in dictating the patterns of hydrogen bonds that orient the LCs was also evidenced by the effects of phosphorylation of the tyrosine on the ordering of the LCs. Overall, these results reveal that crystallographic texturing of gold films can direct the formation of monolayers of dipeptides with long-range order, thus unmasking the influence of hydrogen bonding, chirality, and phosphorylation on the macroscopic orientational ordering of LCs supported on these surfaces. These results suggest new approaches based on supramolecular assembly for reporting the chemical functionality and stereochemistry of synthetic and biological peptide-based molecules displayed at surfaces.     

Thermodynamic characteristics and Langmuir-Blodgett deposition behavior of mixed DPPA/DPPC monolayers at air/liquid interfaces

The role of dipalmitoylphosphatic acid (DPPA) as a transfer promoter to enhance the Langmuir-Blodgett (LB) deposition of a dipalmitoylphosphatidylcholine (DPPC) monolayer at air/liquid interfaces was investigated, and the effects of Ca2+ ions in the subphase were discussed. The miscibility of the two components at air/liquid interfaces was evaluated by surface pressure-area per molecule isotherms, thermodynamic analysis, and by the direct observation of Brewster angle microscopy (BAM). Multilayer LB deposition behavior of the mixed DPPA/DPPC monolayers was then studied by transferring the monolayers onto hydrophilic glass plates at a surface pressure of 30 mN/m. The results showed that the two components, DPPA and DPPC, were miscible in a monolayer on both subphases of pure water and 0.2 mM CaCl2 solution. However, an exception occurs between X(DPPA)=0.2 and 0.5 at air/CaCl2-solution interface, where a partially miscible monolayer with phase separation may occur. Negative deviations in the excess area analysis were found for the mixed monolayer system, indicating the existence of attractive interactions between DPPA and DPPC molecules in the monolayers. The monolayers were stable at the surface pressure of 30 mN/m for the following LB deposition as evaluated from the area relaxation behavior. It was found that the presence of Ca2+ ions had a stabilization effect for DPPA-rich monolayers, probably due to the association of negatively charged DPPA molecules with Ca2+ ions. Moreover, the Ca2+ ions may enhance the adhesion of DPPA polar groups to a glass surface and the interactions between DPPA polar groups in the multilayer LB film structure. As a result, Y-type multilayer LB films containing DPPC could be fabricated from the mixed DPPA/DPPC monolayers with the presence of Ca2+ ions.     

Cholesterol Alters the Phase Separation in Model Membranes Containing hBest1

Human retinal pigment epithelial (RPE) cells express the transmembrane Ca²⁺-dependent Cl⁻ channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (C_s⁻¹) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca²⁺ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔG_mix^π values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔG_mix^π are retained and equalized with the values of ΔG_mix^π in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.     

Comparison of the interaction of dihydrocholesterol and cholesterol with sphingolipid or phospholipid Langmuir monolayers

We report here a study of the interaction of dihydrocholesterol (DChol) with palmitoyl-oleoyl-phosphatidylcholine (POPC) or sphingomyelin (SM) in Langmuir monolayers. DChol and cholesterol (Chol) have very close chemical structures, and DChol is often used in place of Chol because of its better stability. Surface pressure measurements and experiments of desorption induced by beta-cyclodextrin show that POPC-DChol monolayers behave similarly to POPC-Chol ones: condensing effects of DChol and Chol on POPC and desorption percentages are in the same range. Moreover Brewster angle microscopy (BAM) experiments performed on these monolayers show that on the whole they are both homogenous. The analysis of mean molecular areas versus DChol percentage shows that this sterol is also able to induce SM condensation at low surface pressure. The condensation of SM molecules is particularly strong at 30 mol% of DChol. At higher surface pressure, the condensation efficiency of DChol decreases and monolayers behave more ideally, even if an inflection point is always observed at 30 mol% of DChol. However, desorption percentages, clearly lower than those obtained with POPC-DChol monolayers, show that DChol is kept at the interface. At last BAM images show also differences in the behaviour of SM-DChol and SM-Chol monolayers. These differences could be due to the different compressibility and conformation of the A/B rings in the two sterols and the rigidity of the sphingosine chain. They suggest that the use of DChol in place of Chol has to be done carefully in the presence of SM.     

Energetics of cholesterol transfer between lipid bilayers

It is believed that natural biological membranes contain domains of lipid ordered phase enriched in cholesterol and sphingomyelin. Although the existence of these domains, called lipid rafts, is still not firmly established for natural membranes, direct microscopic observations and phase diagrams obtained from the study of three-component mixtures containing saturated phospholipids, unsaturated phospholipids, and cholesterol demonstrate the existence of lipid rafts in synthetic membranes. The presence of the domains or rafts in these membranes is often ascribed to the preferential interactions between cholesterol and saturated phospholipids, for example, between cholesterol and sphingomyelin. In this work, we calculate, using molecular dynamics computer simulation technique, the free energy of cholesterol transfer from the bilayer containing unsaturated phosphatidylcholine lipid molecules to the bilayer containing sphingomyelin molecules and find that the affinity of cholesterol to sphingomyelin is higher. Our calculations of the free-energy components, energy and entropy, show that cholesterol transfer is exothermic and promoted by the favorable change in the lipid-lipid interactions near cholesterol and not by the favorable energy of cholesterol-sphingomyelin interaction, as assumed previously.     

Mixed monolayers composed of PC/PS/Chol. Interaction with opioid molecules

The miscibility of phosphatidylserine, phosphatidylcholine, and cholesterol in monolayers were studied. The influence of sodium and calcium ions in this system was determined. The compression isotherms of mixed monolayers of the above cited three components spread on subphases containing opiate molecules are elucidated. Moreover, the penetration kinetics of opiate molecules in these mixed monolayers was also recorded. The results show that the presence of cholesterol always lowers the penetration of opioid molecules; this effect is weaken for meperidine, the most hydrophobic of the molecules assayed.Abreviations PS phosphatidylserine - PC phosphatidylcholine - PI phosphatidylinositol - Chol cholesterol