Comparative molecular dynamics study of lipid membranes containing cholesterol and ergosterol

Sterol molecules are essential for maintaining the proper structure and function of eukaryotic cell membranes. The influence of cholesterol (the principal sterol of higher animals) on the lipid bilayer properties was extensively studied by both experimental and simulation methods. In contrast, the effect of ergosterol (the principal fungal sterol) on the membrane structure and dynamics is much less recognized. This work presents the results of comparative molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine bilayer containing approximately 25 mol % of cholesterol or ergosterol. A detailed analysis of the molecular properties (e.g., bilayer thickness, lipid order, diffusion, intermolecular interactions, etc.) of both sterol-induced liquid-ordered membrane phases is presented. Presence of sterols in the membrane significantly changes its property, especially fluidity and molecular packing. Moreover, in accordance with the experiments, our calculations show that, compared to cholesterol, ergosterol has higher ordering effect on the phospholipid acyl chains. This different influence on the properties of the lipid bilayer stems from differences in conformational freedom of sterol side chains. Additionally, obtained models of lipid membranes containing human and fungal sterols, constituting the result of our work, can be also utilized in other chemotherapeutic studies on interaction of selected ligands (e.g., antifungal compounds) with membranes.     

The replacement of cholesterol by phytosterols and the increase of total sterol content in model erythrocyte membranes

The activity of phytosterols on human organism includes the ability of these compounds to incorporate into membranes. In the consequence the plant sterols are able to increase total sterol concentration in membrane or/and to replace cholesterol molecules. The aim of this work was to compare the influence of both these effects on the properties of model erythrocyte membranes. Moreover, the interactions between the plant sterols (β-sitosterol and stigmasterol) and saturated–monounsaturated phosphatidylcholine were investigated and the condensing and ordering potency of these phytocompounds on membrane phospholipids were thoroughly analyzed. It was found that the addition of the plant sterols into model membrane modifies the condensation, ordering and interactions in the system. Moreover, the replacement of mammalian sterol by phytosterol more strongly influences the model system than even a 10% increase of total sterol concentration induced by the incorporation of the plant sterol, at constant content of cholesterol. The investigated plant sterols at their lower concentration in the mixed system are of similar effect on its properties. At higher content stigmasterol was found to modify the properties of model membrane more strongly than β-sitosterol.     

Ordering effects of cholesterol and its analogues

Without any exaggeration, cholesterol is one of the most important lipid species in eukaryotic cells. Its effects on cellular membranes and functions range from purely mechanistic to complex metabolic ones, besides which it is also a precursor of the sex hormones (steroids) and several vitamins. In this review, we discuss the biophysical effects of cholesterol on the lipid bilayer, in particular the ordering and condensing effects, concentrating on the molecular level or inter-atomic interactions perspective, starting from two-component systems and proceeding to many-component ones e.g., modeling lipid rafts. Particular attention is paid to the roles of the methyl groups in the cholesterol ring system, and their possible biological function. Although our main research methodology is computer modeling, in this review we make extensive comparisons between experiments and different modeling approaches.     

Ordering effects of cholesterol and its analogues

Without any exaggeration, cholesterol is one of the most important lipid species in eukaryotic cells. Its effects on cellular membranes and functions range from purely mechanistic to complex metabolic ones, besides which it is also a precursor of the sex hormones (steroids) and several vitamins. In this review, we discuss the biophysical effects of cholesterol on the lipid bilayer, in particular the ordering and condensing effects, concentrating on the molecular level or inter-atomic interactions perspective, starting from two-component systems and proceeding to many-component ones e.g., modeling lipid rafts. Particular attention is paid to the roles of the methyl groups in the cholesterol ring system, and their possible biological function. Although our main research methodology is computer modeling, in this review we make extensive comparisons between experiments and different modeling approaches.     

Significance of sterol structural specificity. Desmosterol cannot replace cholesterol in lipid rafts

Desmosterol is an immediate precursor of cholesterol in the Bloch pathway of sterol synthesis and an abundant membrane lipid in specific cell types. The significance of the difference between the two sterols, an additional double bond at position C24 in the tail of desmosterol, is not known. Here, we provide evidence that the biophysical and functional characteristics of the two sterols differ and that this is because the double bond at C24 significantly weakens the sterol ordering potential. In model membranes, desmosterol was significantly weaker than cholesterol in promoting the formation or stability of ordered domains, and in mammalian cell membranes, desmosterol associated less avidly than cholesterol with detergent-resistant membranes. Atomic scale molecular dynamics simulations showed that the double bond gives rise to additional stress in the tail, creating a rigid structure between C24 and C27 and favoring tilting of desmosterol distinct from cholesterol. Functional effects of desmosterol in cell membranes were assessed upon acutely exchanging approximately 70% of cholesterol to desmosterol. This led to impaired raft-dependent signaling via the insulin receptor, whereas non-raft-dependent protein secretion was not affected. We suggest that the choice of cholesterol synthesis route may provide a physiological mechanism to modulate raft-dependent functions in cells.     

Phloretin-Induced Reduction in Dipole Potential of Sterol-Containing Bilayers

The phloretin-induced reduction in the dipole potential of planar lipid bilayers containing cholesterol, ergosterol, stigmasterol, 7-dehydrocholesterol and 5α-androstan-3β-ol was investigated. It is shown that effects depend on the type and concentration of membrane sterol. It is supposed that the effectiveness of phloretin in reducing the dipole potential of the bilayers that contain cholesterol, ergosterol and 7-dehydrocholesterol correlates with the ordering and condensing effects. The role of the concentration-dependent ability of different sterols to promote lateral heterogeneity in membranes is also discussed.     

Predicting the effect of steroids on membrane biophysical properties based on the molecular structure

The relationship between sterol structure and the resulting effects on membrane physical properties is still unclear, owing to the conflicting results found in the current literature. This study presents a multivariate analysis describing the physical properties of 83 steroid membranes. This first structure-activity analysis supports the generally accepted physical effects of sterols in lipid bilayers. The sterol chemical substituents and the sterol/phospholipid membrane physical properties were encoded by defining binary variables for the presence/absence of those chemical substituents in the polycyclic ring system and physical parameters obtained from phospholipid mixtures containing those sterols. Utilizing Principal Coordinates Analysis, the steroid population was grouped into five well-defined clusters according to their chemical structures. An examination of the membrane activity of each sterol structural cluster revealed that a hydroxyl group at C3 and an 8-10 carbon isoalkyl side-chain at C17 are mainly present in membrane active sterols having rigidifying, molecular ordering/condensing effects and/or a raft promoting ability. In contrast, sterol chemical structures containing a keto group at C3, a C4-C5-double bond, and polar groups or a short alkyl side-chain at C17 (3 to 7 atoms) are mostly found in sterols having opposite effects. Using combined multivariate approaches, it was concluded that the most important structural determinants influencing the physical properties of sterol-containing mixtures were the presence of an 8-10 carbon C17 isoalkyl side-chain, followed by a hydroxyl group at C3 and a C5-C6 double bond. Finally, a simple Logistic Regression model predicting the dependence of membrane activity on sterol chemical structure is proposed.     

Effect of sphingomyelin headgroup size on molecular properties and interactions with cholesterol

Sphingomyelins (SMs) and sterols are important constituents of the plasma membrane and have also been identified as major lipid components in membrane rafts. Using SM analogs with decreasing headgroup methylation, we systemically analyzed the effect of headgroup size on membrane properties and interactions with cholesterol. An increase in headgroup size resulted in a decrease in the main phase transition. Atom-scale molecular-dynamics simulations were in agreement with the fluorescence anisotropy experiments, showing that molecular areas increased and acyl chain order decreased with increasing headgroup size. Furthermore, the transition temperatures were constantly higher for SM headgroup analogs compared to corresponding phosphatidylcholine headgroup analogs. The sterol affinity for phospholipid bilayers was assessed using a sterol-partitioning assay and an increased headgroup size increased sterol affinity for the bilayer, with a higher sterol affinity for SM analogs as compared to phosphatidylcholine analogs. Moreover, the size of the headgroup affected the formation and composition of cholesterol-containing ordered domains. Palmitoyl-SM (the largest headgroup) seemed to attract more cholesterol into ordered domains than the other SM analogs with smaller headgroups. The ordering and condensing effect of cholesterol on membrane lipids was also largest for palmitoyl-SM as compared to the smaller SM analogs. The results show that the size of the SM headgroup is crucially important for SM-SM and SM-sterol interactions. Our results further emphasize that interfacial electrostatic interactions are important for stabilizing cholesterol interactions with SMs.     

Cholesterol Induces Specific Spatial and Orientational Order in Cholesterol/Phospholipid Membranes

Background

In lipid bilayers, cholesterol facilitates the formation of the liquid-ordered phase and enables the formation of laterally ordered structures such as lipid rafts. While these domains have an important role in a variety of cellular processes, the precise atomic-level mechanisms responsible for cholesterol''s specific ordering and packing capability have remained unresolved.

Methodology/Principal Findings

Our atomic-scale molecular dynamics simulations reveal that this ordering and the associated packing effects in membranes largely result from cholesterol''s molecular structure, which differentiates cholesterol from other sterols. We find that cholesterol molecules prefer to be located in the second coordination shell, avoiding direct cholesterol-cholesterol contacts, and form a three-fold symmetric arrangement with proximal cholesterol molecules. At larger distances, the lateral three-fold organization is broken by thermal fluctuations. For other sterols having less structural asymmetry, the three-fold arrangement is considerably lost.

Conclusions/Significance

We conclude that cholesterol molecules act collectively in lipid membranes. This is the main reason why the liquid-ordered phase only emerges for Chol concentrations well above 10 mol% where the collective self-organization of Chol molecules emerges spontaneously. The collective ordering process requires specific molecular-scale features that explain why different sterols have very different membrane ordering properties: the three-fold symmetry in the Chol-Chol organization arises from the cholesterol off-plane methyl groups allowing the identification of raft-promoting sterols from those that do not promote rafts.     

Membrane cholesterol dynamics: cholesterol domains and kinetic pools

Nonreceptor mediated cholesterol uptake and reverse cholesterol transport in cells occur through cellular membranes. Thus, elucidation of cholesterol dynamics in membranes is essential to understanding cellular cholesterol accumulation and loss. To this end, it has become increasingly evident that cholesterol is not randomly distributed in either model or biologic membranes. Instead, membrane cholesterol appears to be organized into structural and kinetic domains or pools. Cholesterol-rich and poor domains can even be observed histochemically and physically isolated from epithelial cell surface membranes. The physiologic importance of these domains is 2-fold: (i) Select membrane proteins (receptors, transporters, etc.) are localized in either cholesterol-rich or cholesterol-poor domains. Consequently, the structure and properties of the domains rather than of the bulk lipid may selectively affect the function of proteins residing therein. (ii) Kinetic evidence suggests that cholesterol transport through and between membranes may occur through specific domains or pools. Regulation of the size and properties of such domains may be controlling factors of cholesterol transport or accumulation in cells. Recent technologic advances in the use of fluorescent sterols have allowed examination of cholesterol domain structure in model and biologic membranes. These techniques have been applied to examine the role of high-density lipoprotein, cholesterol lowering drugs, and intracellular lipid transfer proteins in membrane sterol domain structure and sterol movement between membranes.     

From hopanoids to cholesterol: Molecular clocks of pentameric ligand-gated ion channels

Pentameric ligand-gated ion channels (pLGICs) and their lipid microenvironments appear to have acquired mutually adaptive traits along evolution: 1) the three-ring architecture of their transmembrane (TM) region; 2) the ability of the outermost TM ring to convey lipid signals to the middle ring, which passes them on to the central pore ring, and 3) consensus motifs for sterol recognition in all pLGICs. Hopanoids are triterpenoid fossil lipids that constitute invaluable biomarkers for tracing evolution at the molecular scale. The cyanobacterium Gloeobacter violaceus is the oldest known living organism in which the X-ray structure of its pLGIC, GLIC, reveals the presence of the above attributes and, as discussed in this review, the ability to bind hopanoids. ELIC, the pLGIC from the bacillus Erwinia chrysanthemi is the only other known case to date. Both prokaryotes lack cholesterol but their pLGICs exhibit the same sterol motifs as mammalian pLGIC. This remarkable conservation suggests that the association of sterols and hopanoid surrogate molecules arose from the early need in prokaryotes to stabilize pLGIC TM regions by means of relatively rigid lipid molecules. The conservation of these phenotypic traits along such a long phylogenetic span leads us to suggest the possible co-evolution of these sterols with pLGICs.     

Lateral distribution of cholesterol in membranes probed by means of a pyrene-labelled cholesterol: effects of acyl chain unsaturation

The lateral distribution of cholesterol in membranes in the fluid state was investigated by studying the variation of the molar absorption coefficient of pyrene-labelled cholesterol (Py-chol) vs. its concentration in vesicles made of phosphatidylcholine, with variable acyl chain unsaturations. Absorption measurements indicated non-ideal mixing of Py-chol in unsaturated lipids, a process mainly controlled by the cholesterol moiety of the probe. Similar abilities of cholesterol and Py-chol in perturbing the phase properties of pure saturated phosphatidylcholine were observed by DSC experiments. Immiscibility of sterols was corroborated by fluorescence polarization measurements, which indicated a weaker ordering effect of cholesterol in unsaturated membranes. The sizes and the quantities of sterol oligomers formed were calculated. A model for the lateral distribution of cholesterol in membranes is proposed and is applied to known cholesterol/phosphatidylcholine phase diagrams. Finally, the results are discussed with regard to recent models of biological membrane organization, (i.e. rafts).     

Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide.

Ordered lipid domains enriched in sphingolipids and cholesterol (lipid rafts) have been implicated in numerous functions in biological membranes. We recently found that lipid domain/raft formation is dependent on the sterol component having a structure that allows tight packing with lipids having saturated acyl chains (Xu, X., and London, E. (2000) Biochemistry 39, 844-849). In this study, the domain-promoting activities of various natural sterols were compared with that of cholesterol using both fluorescence quenching and detergent insolubility methods. Using model membranes, it was shown that, like cholesterol, both plant and fungal sterols promote the formation of tightly packed, ordered lipid domains by lipids with saturated acyl chains. Surprisingly ergosterol, a fungal sterol, and 7-dehydrocholesterol, a sterol present in elevated levels in Smith-Lemli-Opitz syndrome, were both significantly more strongly domain-promoting than cholesterol. Domain formation was also affected by the structure of the sphingolipid (or that of an equivalent "saturated" phospholipid) component. Sterols had pronounced effects on domain formation by sphingomyelin and dipalmitoylphosphatidylcholine but only a weak influence on the ability of cerebrosides to form domains. Strikingly it was found that a small amount of ceramide (3 mol %) significantly stabilized domain/raft formation. The molecular basis for, and the implications of, the effects of different sterols and sphingolipids (especially ceramide) on the behavior and biological function of rafts are discussed.     

Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function

Ceramide is a membrane lipid involved in a number of crucial biological processes. Recent evidence suggests that ceramide is likely to reside and function within lipid rafts; ordered sphingolipid and cholesterol-rich lipid domains believed to exist within many eukaryotic cell membranes. Using lipid vesicles containing co-existing raft domains and disordered fluid domains, we find that natural and saturated synthetic ceramides displace sterols from rafts. Other raft lipids remain raft-associated in the presence of ceramide, showing displacement is relatively specific for sterols. Like cholesterol-containing rafts, ceramide-rich "rafts" remain in a highly ordered state. Comparison of the sterol-displacing abilities of natural ceramides with those of saturated diglycerides and an unsaturated ceramide demonstrates that tight lipid packing is critical for sterol displacement by ceramide. Based on these results, and the fact that cholesterol and ceramides both have small polar headgroups, we propose that ceramides and cholesterol compete for association with rafts because of a limited capacity of raft lipids with large headgroups to accommodate small headgroup lipids in a manner that prevents unfavorable contact between the hydrocarbon groups of the small headgroup lipids and the surrounding aqueous environment. Minimizing the exposure of cholesterol and ceramide to water may be a strong driving force for the association of other molecules with rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft structure and function, altering liquid ordered properties as well as molecular composition. In this regard, certain previously observed physiological processes may be a result of displacement. In particular, a direct connection to the previously observed sphingomyelinase-induced displacement of cholesterol from plasma membranes in cells is proposed.     

2H NMR evidence for antibiotic-induced cholesterol immobilization in biological model membranes

The interaction of the polyene antibiotic filipin with membrane sterols has been studied by deuterium nuclear magnetic resonance of the molecular probes [2,2,3,4,4,6-2H6]cholesterol and 1-myristoyl-2-[4',4',14',14',14'-2H5]myristoyl-sn-glycero-3-phospho- choline. At physiological temperatures, there is evidence of filipin-induced cholesterol immobilization in the membrane. The 2H NMR spectra of cholesterol show two domains in which ordering and dynamics are very different. In one of these, cholesterol is static on the 2H NMR time scale, whereas in the other it undergoes rapid axially symmetric motions similar to those it exhibits in the drug-free membrane; this indicates that the jumping frequency of cholesterol between the labile and immobilized domains is less than 10(5) s-1. The distribution of cholesterol between these two sites is temperature dependent; at 0 degrees C all sterol molecules are immobilized, whereas at 60 degrees C they are almost totally in the labile site. In contrast to cholesterol, the phospholipids sense only one type of environment, at both the top and center of the bilayer, indicating that cholesterol acts as a screen, preventing the lipids from direct interaction with the antibiotic. At low temperature, the ordering of the lipid in the presence of cholesterol does not change upon filipin addition, whereas at elevated temperatures the local ordering of both the lipid and the labile cholesterol is significantly lower than that in the absence of the drug. Moreover, there is a very important difference between the degree of local ordering as measured by the lipids and by cholesterol at high temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential modulation of membrane structure and fluctuations by plant sterols and cholesterol

We have studied the concentration and temperature dependent influence of cholesterol, stigmasterol, and sitosterol on the global structure and the bending fluctuations of fluid dimyristoyl phosphatidylcholine and palmitoyl oleoyl phosphatidylcholine bilayers applying small-angle x-ray scattering, as well as dilatometry and ultrasound velocimetry. Independent of the lipid matrix, cholesterol was found to be most efficient in modulating bilayer thickness and elasticity, followed by sitosterol and stigmasterol. This can be attributed to the additional ethyl groups and double bond at the C₁₇ alkyl side-chain of the two plant sterols. Hence, it seems that some flexibility of the sterol hydrocarbon chain is needed to accommodate within the lipid bilayer. In addition, we did not observe two populations of membranes within the putative liquid-ordered/liquid-disordered phase coexistence regime of binary sterol/lipid mixtures. Instead, the diffraction patterns could be interpreted in terms of a uniform phase. This lends further support to the idea of compositional fluctuations of unstable sterol rich domains recently brought up by fluorescence microscopy experiments, which contrasts the formation of stable domains within the miscibility gap of binary lipid/sterol mixtures.     

Specificity of cholesterol and analogs to modulate BK channels points to direct sterol-channel protein interactions

The activity (Po) of large-conductance voltage/Ca(2+)-gated K(+) (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel-forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol's mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the β configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>epicholesterol) did not follow molecular area rank (coprostanol>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself.     

Domain-formation in DOPC/SM bilayers studied by pfg-NMR: effect of sterol structure

Pulsed field gradient (pfg)-NMR spectroscopy was utilized to determine lipid lateral diffusion coefficients in oriented bilayers composed of 25 mol % sterol and equimolar amounts of dioleoylphosphatidylcholine and sphingomyelin. The occurrence of two lipid diffusion coefficients in a bilayer was used as evidence of lateral phase separation into liquid ordered and liquid disordered domains. It was found that cholesterol, ergosterol, sitosterol, and lathosterol induced domains, whereas lanosterol, stigmasterol, and stigmastanol resided in homogeneous membranes in the temperature interval of 24-70 degrees C. Among the domain-forming sterols, differences in the upper miscibility temperature indicated that the stability of the liquid ordered phase could be modified by small changes in the sterol structure. The domain-forming capacity for the different sterols is discussed in terms of the ordering effect of the sterols on the lipids, and it is proposed that the driving force for the lateral phase separation is the reduced solubility of the unsaturated lipid in the highly ordered phase.     

Inhibition of cholesterol absorption in rats by plant sterols

The extent and site(s) of inhibition of cholesterol absorption by plant sterols, sitosterol and fucosterol, were studied in rats. The intragastric administration of a single emulsified lipid meal containing 25 mg [3H]cholesterol and 25 mg of either sitosterol or fucosterol inhibited the lymphatic absorption of cholesterol by 57% and 41%, respectively, in 24 hr. Less than 2% of each plant sterol was absorbed in the 24-hr period. In contrast, neither plant sterol (50 microM) inhibited cholesterol absorption when co-administered with equimolar amounts of cholesterol in phospholipid-bile salt micelles nor was either absorbed from the micellar solution. A series of in vitro studies was conducted to identify the site(s) of plant sterol inhibition of cholesterol absorption and to account for the difference in inhibitory effectiveness of sitosterol and fucosterol. A comparison of the micellar solubility of each sterol alone and in equimolar binary mixtures (to 2.0 mM) revealed that the solubility of individual sterols decreased in the following order: cholesterol, fucosterol, sitosterol, and that in binary mixtures cholesterol solubility was decreased by sitosterol and, to a lesser extent, by fucosterol relative to its solubility alone. A comparison between micellar-solubilized cholesterol and either sitosterol or fucosterol for binding to isolated brush border membranes, intestinal mucin, or for esterification by either cholesterol esterase or acyl coenzyme A:cholesterol acyltransferase revealed moderate to no competition. The data suggest that plant sterols displace cholesterol from bile salt (taurocholate) micelles and that sitosterol is more effective than fucosterol in this capacity.     

Discrimination between cholesterol and sitosterol for absorption in rats

The intestinal absorption of cholesterol and sitosterol was compared in rats. The intragastric administration of a single emulsified lipid meal containing either 50 mg of [4-14C]cholesterol or [4-14C]sitosterol resulted in the lymphatic absorption of 18.2% and 0.42% of each sterol, respectively, in 6 hr. This difference was unaltered when the mucosal sterol load was equalized by reducing the cholesterol to 1 mg in the emulsified lipid meal while maintaining the same sitosterol load or when the physical state in the lumen was equalized by infusion of a micellar solution containing both sterols into bile-diverted intestine. Lymphatic cholesterol was 90% esterified compared to 12% for sitosterol. Both sterols were associated predominantly (greater than 70%) with the chylomicron fraction. Eighty percent of the chylomicron cholesterol was recovered as ester with the core lipids, while 77% of the sitosterol was recovered as free sterol with the chylomicron coat. In mucosal homogenates at 6 hr, sitosterol recovery was one-eleventh that of cholesterol. When [3H]cholesterol (10 mg) and [14C]sitosterol (10 mg) were co-administered in an emulsified intragastric lipid meal, sitosterol associated with the brush border isolated 2 hr later was one-fifth that of cholesterol. Similar differences were seen when brush border membranes were incubated in vitro with micellar solutions containing either 50 microM [3H]cholesterol or [14C]sitosterol and the relative uptake of each sterol was unaffected by micellar phospholipid type (egg yolk phospholipids, phosphatidylcholine, or phosphatidylethanolamine).(ABSTRACT TRUNCATED AT 250 WORDS)