the impact of sterol structure on the interactions with sphingomyelin in mixed langmuir monolayers

The Impact of Sterol Structure on the Interactions with Sphingomyelin in Mixed LangmuirMonolayers

Katarzyna Hac-Wydro* and Patrycja Dynarowicz-ŁatkaFaculty of Chemistry, Jagiellonian UniVersity, Ingardena 3, 30-060 Krakow, Poland

ReceiVed: April 13, 2008; ReVised Manuscript ReceiVed: June 20, 2008

In this work, the Langmuir monolayer technique was applied to study the interactions between sphingomyelinand various sterols differing in the structure of the side chain (cholesterol, �-sitosterol, stigmasterol). Themean area per molecule and the excess free energy of mixing values were analyzed in the context of sterol-induced condensing effect and interactions between molecules in the mixed monolayers. Moreover, thecompression modulus values were calculated and widely discussed from the point of view of the orderingeffect of sterols. It was found that all of the sterols investigated form the most stable monolayers withsphingomyelin at 2:1 sphingomyelin:sterol proportion and the strongest interactions exist between moleculesin cholesterol-containing films. Moreover, cholesterol provokes the strongest area condensation and revealsthe highest ordering properties, while plant sterols were found to differ only slightly with regards to theirordering properties. Additionally, the ordering effect of the sterols on dipalmitoylphosphatidylcholine (DPPC)films was analyzed and compared to that on sphingomyelin films.

1. Introduction

The asymmetrical distribution of lipids between the inner(cytosolic) and the outer (exoplasmic) leaflets is the attributeof biological membranes. In human membranes, the outermonolayer is dominated by phosphatidylcholines (PCs) andsphingomyelins (Sph), while the remaining phospholipids,phosphatidylethanolamines (PEs), phosphatidylserines (PSs), andphosphatidylinositol (PIs), are concentrated mainly in the innerleaflet of the membrane.1,2 Apart from the foregoing lipids, avery important group of membrane components are sterols. Theyare found in human (cholesterol), fungi (ergosterol), and plant(campesterol, stigmasterol, �-sitosterol) membranes. Cholesterolmolecules are present in cytosolic as well as exoplasmicmembrane layers; however, there are controversies regardingits exact percentage distribution between both leaflets.2-6

Because various lipids constitute cellular membranes, numer-ous studies were devoted to examining their mutual interactions.Especially, the influence of cholesterol on the organization ofmembranes was intensively studied due to the fundamental roleof this lipid in regulating membrane physicochemical properties[e.g., refs 6-14].

It was found that the incorporation of cholesterol moleculesinto phospholipid bilayer provokes ordering of the phospholipidsacyl chains (“an ordering effect”) and decrease of the area perlipid (“condensing effect”).15,16 The bilayer devoid of sterolscan exist either in the gel-solid ordered (“so”) phase or in thefluid-liquid disordered (“ld”) state. Upon addition of a sterol,the above-mentioned effects lead to the formation of the liquid-ordered (“lo”) phase.17-21 This phase describes, for example,the state of the system containing cholesterol and saturatedlipids. In these mixtures, the ordering effect of sterol and stronginteractions between membrane lipids provoke the formationof tightly packed microdomains in membranes called “rafts”.1,22-24

The lipid rafts, composed of cholesterol, sphingolipids, andsaturated lipids, are more ordered and constitute less fluid region

than the rest of the membrane.1,24 The lipid rafts were found tobe formed also between phytosterols and sphingolipids in plantmembranes.25

Interestingly, it was found26 in fluorescence experimentsperformed on mixtures of various sterols and dipalmitoylphos-phatidylcholine (DPPC) that some sterols strongly inhibitformation of saturated lipids-enriched domains. These sterolsbehave as “anti-cholesterols”26 because, in contrast to cholesterolthat is able to pack closely with saturated lipids, they causelooser lipid packing and lower their ordering, preventing in thisway domain formation. Therefore, it is evident that theeffectiveness of sterol ordering effect and thereby the strengthof sterol/membrane lipid interactions are strongly dependent onthe kind of a sterol.

In this work, we report on the studies of interactions betweensphingomyelin and sterols in mixed Langmuir monolayers,which can be treated as a model of the outer layer of cellularmembrane. Sphingomyelin is the sphingolipid that constitutes,percentage-wise, a significant amount of membrane lipids,1 and,as a component of membrane and lipid rafts,1,23,27-29 wassubjected to numerous experiments, which were undertaken tounderstand the properties of sphingomyelin in model phospho-lipid bilayers. On the basis of these results, the structural featuresof the sphingomyelin molecule (e.g., its hydrocarbon chainslength and their saturation degree) determining the behavior ofthis sphingolipid in membranes were discussed.30-32 Moreover,the properties of cholesterol/sphingomyelin model membranesare being systematically analyzed in the context of domainsformation.33-41

It should be noted that the investigations of the sterol/sphingomyelin systems have been focused mainly on thecholesterol-containing mixtures. Because the sterol structure wasfound to be an important factor affecting the condensing andordering properties and the strength of its interactions withsaturated lipids [e.g., refs 26, 43], in this work the interactionsbetween sphingomyelin and sterols differing in the structure ofthe side chain were studied. For our investigations, both humanmembrane sterol (cholesterol) and two phytosterols, stigmasterol

* Corresponding author. Phone: +48 0-12 633-20-79. Fax: +48 0-12-634-05-15. E-mail: [email protected].

J. Phys. Chem. B 2008, 112, 11324–1133211324

10.1021/jp803193s CCC: $40.75 2008 American Chemical SocietyPublished on Web 08/14/2008

and �-sitosterol, were chosen. Stigmasterol and �-sitosterol arethe components of plant membranes and lipid rafts; however,they can also be incorporated into human cellular membranesby absorption from food.42-45 Although the concentration ofphytosterols in human membranes is rather low, it was provedthat these compounds strongly affect the properties of humanmembranes and can partially replace cholesterol, for example,in human erythrocytes.44,46-48 In this context, the study on theinteractions between sphingomyelin and human as well as plantsterols is of great importance from the point of view of theproperties of sphingomyelin/sterol domains.

The results of the Langmuir monolayer studies on mixed filmsof sphingomyelin and sterols presented herein provided infor-mation on the influence of the side chain of sterols moleculeon its interactions with sphingomyelin. Moreover, on the basisof these results, the impact of sterol tail on its condensing andordering properties was analyzed. Additionally, the comparisonof the obtained results with similar experiments performedpreviously for dipalmitoylphosphatidylcholine-containing mono-layers allowed one to discuss the differences in the affinity ofthe investigated sterols toward saturated phospholipid versussphingomyelin.

2. Experimental Section

All of the investigated sterols were synthetic compounds ofhigh purity (>99%) purchased from Sigma, whereas eggsphingomyelin (g98%) was purchased from BioChemica. Thespreading solutions were prepared by dissolving the investigatedcompounds in freshly distilled chloroform p.a. (POCh). Mixedsolutions were prepared from the respective stock solutions.Spreading solutions were deposited onto the water subphase withthe Hamilton micro syringe, precise to 2.0 µL. After beingspread, the monolayers were left to equilibrate for 10 min beforethe compression was initiated with the barrier speed of 20 cm2/min. π-A isotherms were recorded with a NIMA (U.K.)Langmuir trough (total area ) 300 cm2, width 10 cm, length30 cm) placed on an antivibration table. Surface pressure wasmeasured with the accuracy of (0.1 mN/m using a Wilhelmyplate made of filter paper (ashless Whatman Chr1) connectedto an electrobalance. The subphase temperature (20 °C) wascontrolled thermostatically to within 0.1 °C by a circulatingwater system.

3. Results

3.1. The π-A Isotherms. The surface pressure (π)-area (A)isotherms for the investigated mixed systems of sphingomyelin(Sph) and cholesterol, �-sitosterol, and stigmasterol, respectively,are presented in Figure 1a-c. As concerns the π-A curve foregg sphingomyelin monolayer, the surface pressure increasesat area ≈ 78 Å2/molecule and the film collapses at π ≈ 60 mN/m. In the isotherm at π ≈ 17 mN/m, a phase transition betweenliquid expanded (LE) and liquid condensed (LC) state can beobserved. The existence of LE-LC phase transition for eggsphingomyelin monolayer was postulated also by other authors.39

The addition of sterols into sphingomyelin film stronglyinfluences both the shape and the position of the π-A curves.With the increase of the sterol concentration in the mixed film,the isotherm for the mixed monolayers becomes steeper andstarts to resemble the isotherm for the respective sterol.Moreover, with the increase of sterol content in the monolayer,the shift of π-A curves for sphingomyelin/sterol monolayerstoward the isotherm for a one-component sterol film can beobserved. It is worth noting that the shift of the isotherm formonolayer of Xsterol ) 0.3 is more pronounced than that observed

for a mixture of Xsterol ) 0.1, whereas further addition of sterolinto sphingolipid film causes systematic changes of the positionof π-A curves. The incorporation of sterols into sphingomyelinfilm affects also the collapse surface pressure (πcoll) of the mixedmonolayer. Interestingly, a low amount of sterol in the mixedsystem (Xsterol ) 0.1) raises the collapse surface pressure tohigher values as compared to that observed for pure sphingo-myelin film. Further addition of sterol causes a systematicdecrease of the πcoll to that characteristic for a sterol film. InFigure 2, the variation of the collapse surface pressure with themonolayer composition was presented.

3.2. Miscibility and Interactions in the Mixed Films. Toanalyze the miscibility of the components of the mixedmonolayers and to verify the magnitude of area condensationdue to sterol addition, the plots of the mean area per molecule(A12) and the excess area per molecule (AExc) versus filmcomposition (Xsterol) were considered. It is known that A12)f(X1,2) shows deviations from ideality if the mixed monolayercomponents are miscible and a mixed film shows a nonidealbehavior resulting from the molecular interactions.49-51 If the

Figure 1. The surface pressure-area (π-A) isotherms of mixedmonolayers formed at the air/water interface by egg sphingomyelinand sterols: (a) sphingomyelin/cholesterol monolayers, (b) sphingo-myelin/�-sitosterol monolayers, and (c) sphingomyelin/stigmasterolmonolayers.

Impact of Sterol Structure on Interactions with Sph J. Phys. Chem. B, Vol. 112, No. 36, 2008 11325

components of monolayer are immiscible or ideally miscible,A12 is a linear function of the composition49-51 according toeq 1:

A12id )A1X1 +A2X2 (1)

wherein A12id is the mean area per molecule for ideal mixing,

A1, A2 are molecular areas of the respective component in theirpure films at a given surface pressure, and X1, X2 are the molefractions of components 1 and 2 in the mixed film. The excessareas per molecule were calculated on the basis of eq 2.50

AExc )A12 -A12id (2)

The mean area per molecule (A12) versus film composition plotsfor the investigated mixtures are presented in Figure 3, whileAExc versus composition plots are shown in Figure 4. The A12

values were determined from the isotherms at five surfacepressures (π ) 5, 10, 20, 30, and 35 mN/m) and are shown asa function of the sterol mole fraction in the respective mixture(solid lines in Figure 3). The plots for ideal mixing are presentedas dashed lines. For all of the investigated mixtures in a wholerange of the monolayer composition, the deviations from idealbehavior can be observed. This proves mixing of the investigatedcompound in the monolayer. The fact that these deviations arenegative nearly in the whole range of the monolayer composition(except for cholesterol- and stigmasterol-containing mixedsystems of Xsterol ) 0.9 at higher surface pressure where thedeviations are positive; see Figure 3 and 4) suggests that theinteractions between sterols and sphingomyelin are moreattractive (or less repulsive) than the sphingomyelin/sphingo-myelin and sterol/sterol intermolecular forces in the respectiveone-component films. The strongest deviations appear for allof the studied mixed systems for monolayers of Xsterol ) 0.3.Comparing the excess area per molecule values obtained forthe mixed monolayers, it can be concluded that the strongestcondensation of area is provoked by the addition of cholesterolmolecules (see Figure 4).

To examine the interactions between the components of themixed monolayers quantitatively, the values of the excess freeenergy of mixing (∆GExc) were calculated according to eq 3:49,50

∆GExc )N∫0

π(A12 -X1A1 -X2A2) dπ (3)

The ∆GExc values were calculated directly from the isothermsat the same surface pressures as the A12 and are presented in afunction of the monolayer composition in Figure 5. The valuesof the excess free energy of mixing for the studied mixedsystems are negative in the whole range of the monolayers

composition. This proves that in the mixed monolayer sphin-gomyelin and sterol molecules attract stronger (or the interac-tions are less repulsive) than in one-component films formedby the investigated lipids. The minimum of ∆GExc appears forall of the investigated mixed systems at Xsterol ) 0.3. However,the values of the excess free energy of mixing are lower (morenegative) for the mixed films containing cholesterol (∆GExc )-1700 J/mol at Xcholesterol ) 0.3 and π ) 35 mN/m) than formonolayers formed by sphingomyelin and plant sterol (forsphingomyelin/�-sitosterol mixtures ∆GExc ) -1320 J/mol andfor sphingomyelin/stigmasterol mixtures ∆GExc ) -1200 J/molat Xsterol ) 0.3 and π ) 35 mN/m).

To conclude on the mixed monolayers stability, the freeenergy of mixing values were calculated according to eqs 4 and549,50 and are presented as a function of the films compositionin Figure 6.

∆GM )∆GExc +∆Gid (4)

where

∆Gid)RT(X1 ln X1+ X2 ln X2) (5)

R is the universal gas constant and T is temperature.

Figure 2. The dependence of collapse surface pressure (πcoll) vscomposition (Xsterol) for mixed monolayers of sphingomyelin and sterols.

Figure 3. The mean area per molecule (A12) vs composition plots(Xsterol) for mixed monolayers of sphingomyelin and sterols at differentconstant surface pressures.

11326 J. Phys. Chem. B, Vol. 112, No. 36, 2008 Hac-Wydro and Dynarowicz-Łatka

As can be observed in Figure 6, the free energy of mixingvalues are negative for all of the mixed systems studied in thewhole monolayer composition with a minimum at Xsterol ) 0.3.This indicates that the 2D mixed state is thermodynamicallymore stable (especially at Xsterol ) 0.3) and more favorable thanthe corresponding unmixed state.

3.3. Analysis of the Compression Modulus Values. Toverify the influence of the respective sterols on the state ofsphingomyelin monolayer, the compression modulus valueswere calculated from the isotherm data points according toeq 6:52

CS-1 )-A(dπ/dA) (6)

It is suggested that the properties of the lipids Langmuir filmmimicking one of membrane leaflets correlate with the propertiesof a bilayer within the range of surface pressure 30-35 mN/m.53 Therefore, in Figure 7 the values of the compressionmodulus at π ) 32 mN/m are presented. The sterols are known

to form tightly packed monolayers, which is reflected in highcompression modulus values for sterol films (e.g., CS

-1max for

cholesterol is about 900 mN/m6). However, it should be pointedout that although CS

-1max values for all of the sterols films are

very high and their monolayers are densely packed, the structuraldifferences between sterols (see Discussion for details) causethe compression modulus values to differ significantly. Amongthe investigated sterols, cholesterol forms monolayer of thehighest (the highest CS

-1max) packing, while stigmasterol forms

monolayer of the lowest (the lowest CS-1

max) packing.6,54 Ascan be seen in Figure 7, the addition of respective sterol intosphingomyelin film provokes a systematic increase of thecompression modulus values. However, at a lower proportionof sterol in the mixed monolayer (Xsterol ) 0.1), the drop ofCS

-1 occurs for all of the mixed systems studied. This decreaseof the CS

-1 values is caused by disordering of the orderedsphingomyelin hydrocarbon chains due to the presence of sterolmolecules. Similar behavior was found also for the mixedmonolayer of sphingomyelin containing a low proportion ofandrosterol and 6-ketocholestanol.55 Moreover, the disorderingof both phospholipid and sterols hydrocarbon tails was also

Figure 4. The excess area per molecule (AExc) vs composition plots(Xsterol) for mixed monolayers of sphingomyelin and sterols at differentconstant surface pressures.

Figure 5. The excess free energy of mixing (∆GExc) vs compositionplots (Xsterol) for mixed monolayers of sphingomyelin and sterols atdifferent constant surface pressures.


proved in molecular dynamic simulation studies on DPPCbilayers containing 11% of cholesterol.56 For bilayers of DPPC/

cholesterol richer in cholesterol, the phospholipids hydrocarbontails are more ordered and become oriented more parallel tothe normal of a bilayer.56 It is also evident (Figure 7) that theaddition of cholesterol into sphingomyelin film increases theCS

-1 to values higher than those attained upon phytosterolsincorporation; thus it is evident that the former films are moredensely packed and more ordered than the latter. Comparingthe compression modulus values for sphingomyelin/phytosterolmixed systems, it can be concluded that �-sitosterol-containingmonolayers are of a slightly higher packing than those containingstigmasterol. A similar effect of these sterols can be observedfor dipalmitoylphosphatidylcholine (DPPC)-sterol monolayers(Figure 8a). The CS

-1 values presented in Figure 8a for mixedsystems of DPPC and cholesterol, �-sitosterol, and stigmasterolwere calculated on the basis of previously published results6,54

at π ) 32 mN/m. The results compiled in Figure 8a prove thatDPPC/cholesterol films reveal the highest values of the com-pression modulus, and therefore they are the most denselypacked, while the effect of �-sitosterol is a little bit stronger ascompared to that of stigmasterol. To compare the effect of theinvestigated sterols on sphingomyelin versus DPPC film, theincrease of the compression modulus values caused by therespective sterol addition was calculated at π ) 32 mN/m(Figure 8b-d). As can be observed in Figure 8b-d, the increaseof the CS

-1 values upon sterol addition is almost identical inthe cases of both sphingomyelin and DPPC.

4. Discussion

Both cholesterol and the investigated plant sterols belong tothe group of so-called “membrane active sterols”, whichincorporate into lipid bilayer and modulate its physicochemicalproperties. The specific effect of these sterols on membraneparameters results from the structure of these compounds.Membrane active sterols possess a hydrophobic steroid ringsystem with a flexible side tail and a �-hydroxyl polar head.19

In a bilayer, the polar group of a sterol molecule is localizedclose to the hydrophilic group of phospho- and sphingolipids,while the hydrophobic part is oriented parallel to the hydro-carbon chains of phospho- and sphingolipids.16 In the presenceof membrane active sterol, the surface density of membraneincreases (condensing effect) and the lipid acyl chains (especiallythose fully saturated) become more ordered and stretched.1,15,16

It was proved that the magnitude of these effects dependsstrongly on the sterol structure.26,47,55,57

The influence of sterols on membrane lipids and the stabilityof membrane are closely related to the sterol/lipids interactions.These interactions involve the forces between sterol hydroxylgroup and polar heads of phospho- and sphingolipids as wellas the van der Waals interactions between the hydrophobic partof sterol molecule and lipids chains.

The results of experiments presented herein allow us todiscuss the influence of the side chain structure on theinteractions between sterols and sphingomyelin in mixedmonolayers. As it was found, the addition of sterol intosphingomyelin film decreases the surface area occupied bysphingomyelin molecule in the monolayer (Figures 1, 3, and4). This phenomenon, known as a “condensing effect”, isstronger for cholesterol than for phytosterols-containing films.

The values of the excess free energy of mixing calculatedfor the investigated systems were negative in a whole range ofmonolayer composition. This fact proves that the interactionsbetween sterols and sphingomyelin in their mixed films are morefavorable than those between sterol and sphingomyelin mol-ecules in their respective one-component monolayers. This is a

Figure 6. The free energy of mixing values (∆GM) vs compositionplots (Xsterol) for mixed monolayers of sphingomyelin and sterols atdifferent constant surface pressures.

Figure 7. The compression modulus (CS-1) values at π ) 32 mN/m

vs the composition (Xsterol) of the mixed sphingomyelin/sterol monolayers.


consequence of a beneficial separation of sphingomyelinmolecules by sterols causing a decrease of the electrostatic forcesexisting between polar groups of sphingolipid molecules.

The minimum of ∆GExc and ∆GM appears for monolayerscontaining 30% of sterol (Xsterol ) 0.3); thus the sphingomyelin/sterol interactions are the strongest and the monolayers are ofthe highest stability when these lipids are in the proportion of

2:1. The stoichiometry of the most stable complexes formedwith sphingomyelin is the same for all of the investigated sterols;however, the values of the excess free energy of mixing varydepending on the sterol type. The general conclusion is thatthe human sterol (cholesterol) interacts with sphingomyelin morestrongly than do the plant sterols (the values of ∆GExc are morenegative for cholesterol-containing monolayers than for mono-layers containing �-sitosterol and stigmasterol). As regardsphytosterols, only slightly lower values of the excess free energyof mixing were found for �-sitosterol than for stigmasterol. Toexplain these differences in the ∆GExc values achieved for therespective mixed systems, first the structure of the investigatedsterols must be considered. The factor diversifying the sterolsmolecules is their side chain (see Scheme 1). The tails inphytosterols molecules are less flexible and bulkier than that ofthe cholesterol, due to the presence of a supplementary ethylgroup in both of the investigated plant sterols molecules ascompared to human sterol and, in stigmasterol chain, anadditional double bond. The foregoing factors determine thestructural geometry of the respective sterols. The branched�-sitosterol side chain protrudes out of the rigid ring systemmore strongly than does the side chain of cholesterol, however,slightly weaker than in stigmasterol molecule.58 The structureof the side chain makes phytosterols tails larger and less flexiblethan in the cholesterol molecule. In consequence, as it was foundfrom molecular modeling studies,58 the cholesterol moleculepossesses smaller cross-sectional area and is longer than theplant sterols. Such a structure of cholesterol ensures strongervan der Waals forces between this lipid and sphingomyelinhydrocarbon chains versus phytosterols, which is reflected inmore negative values of the excess free energy of mixing forcholesterol/sphingomyelin monolayers as compared to phy-tosterols-containing films.

As far as the plant sterols are concerned, the factor diversify-ing the molecules is the double bond in the side chain ofstigmasterol. Although such a double bond influences thegeometry of the sterol molecule, in fact stigmasterol has only alittle bit greater diameter and it is slightly shorter than the�-sitosterol molecule.58 Thus, it is not surprising that the excessfree energy of mixing values calculated for both plant sterols/sphingomyelin mixed monolayers differ not too much (at Xsterol

) 0.3 and π ) 35 mN/m for sphingomyelin/�-sitosterol mixtures

Figure 8. (a) The compression modulus (CS-1) values at π ) 32 mN/m

vs the composition (Xsterol) of the mixed DPPC/sterol monolayers. (b-d)The increase of the compression modulus values at π ) 32 mN/m dueto sterol addition into sphingomyelin monolayer vs DPPC film.

SCHEME 1: The Chemical Structures of Cholesterol,�-Sitosterol, and Stigmasterol


∆GExc ) -1320 J/mol and for sphingomyelin/stigmasterolmixtures ∆GExc )-1200 J/mol). Slightly lower (more negative)values of ∆GExc for �-sitosterol/sphingomyelin than for stig-masterol/sphingomyelin films allow us to draw the conclusionthat the interactions between molecules in the former systemsare slightly stronger than those in the latter mixtures.

The incorporation of sterols into sphingomyelin film causesthe systematic increase of the compression modulus values forthe mixed monolayers. This proves a closer packing ofmolecules in the monolayer and increase of the order ofsphingolipid hydrocarbon chains due to the presence of sterols.It was found (see Figure 7) that the compression modulus valuesachieved for mixtures containing cholesterol are higher and themonolayers are more ordered than the phytosterols-containingfilms. Thus, it can be concluded that cholesterol is significantlymore effective in ordering of the sphingolipid chains than theplant sterols. As regards the investigated phytosterols afteraddition of �-sitosterol into sphingomyelin film, the compressionmodulus reaches slightly higher values than in the presence ofstigmasterol.

Although there is an agreement that the human sterol(cholesterol) provokes stronger area condensation and higherordering of the lipids hydrocarbon chains than phytosterols, thereare controversies regarding the condensing and ordering proper-ties of �-sitosterol versus stigmasterol.43,47,59-61 The results ofdifferential scanning calorimetry (DSC) and resonance energytransfer (RET) experiments43 and a steady-state fluorescencepolarization technique59 for DPPC/plant sterol mixed systemssuggest that stigmasterol has a slightly higher ordering effecton DPPC in the range of the sterol concentration up to 50%.On the other hand, in the Langmuir monolayer experiments aswell as in a bilayer investigation, �-sitosterol was found to beslightly more effective than stigmasterol as an ordering agent.60,61

To take the floor in the discussion on the ordering properties ofboth plant sterols, we compared the compression modulus valuesfor the mixed monolayer of DPPC and cholesterol, �-sitosterol,and stigmasterol, respectively, calculated at π ) 32 mN/m basedon previously published results6,54 (Figure 8a). The observations(Figure 8a) were similar to those for sphingomyelin/sterolsystems (Figure 7). The strongest condensation and orderingproperties show cholesterol molecules, while the effect of theplant sterols is significantly weaker. The compression modulusvalues for both plant sterols/sphingomyelin mixed systems arevery similar, and only slightly higher values are observed for�-sitosterol. Our results are therefore consistent with thosesuggesting that the �-sitosterol ordering effect is a little bit higherthan the effect of stigmasterol.60,61 However, in fact, thedifferences between the ordering properties of �-sitosterol andstigmasterol found both in this work and by other authors aresmall, and therefore it seems to be reasonable to conclude thatboth of the investigated plant sterols are of similar orderingproperties toward DPPC and egg sphingomyelin.

Finally, the ordering effect of sterols on sphingomyelinmonolayers versus DPPC films was analyzed. The propertiesof egg sphingomyelin are often compared to those of DPPCbecausetheC16:0hydrocarbonchainsprevailinthissphingolipid.39,62

At a high surface pressure (herein π ) 32 mN/m), puresphingomyelin monolayer is less ordered than DPPC film, whichis reflected in lower values of the compression modulus for theformer monolayers than for the DPPC films. Lower packing ofegg sphingomyelin film in this surface pressure region was alsofound by other authors.62 Comparing the values of the CS

-1 forparticular mixtures (Figures 7 and 8a), it can be noticed thatDPPC/sterol films achieved higher values of the compression

modulus than did sphingomyelin-containing mixtures of thesame composition. For chain-matched sphingomyelins andphosphatidylcholines (C16:0 sphingomyelin vs di-C16:0 PC),at high pressure region, the compression modulus values werefound to be similar.62 However, taking into account that eggsphingomyelin, although dominated by C16:0 chains, containsalso the residues of unsaturated chains, the lower values of theCS

-1 for sphingomyelin versus DPPC and for particularsphingomyelin-containing mixtures are not surprising.39,62 How-ever, herein the increase of the compression modulus valuesupon respective sterol addition was calculated (Figure 8b-d).It can be observed that the respective investigated sterols inducedalmost identical growth of the compression modulus values ofboth egg sphingomyelin and DPPC monolayer (Figure 8b-d),and, in this sense, the ordering effect of sterols on eggspingomyelin and DPPC is similar. It was also found that thecondensing effect of cholesterol on both of these lipids issimilar.39

The results of studies performed for sphingomyelins differingin the structure of the chain prove that such structural featuresas the saturation degree of the chains and their length modulatethe behavior of sphingolipids in mono- and bilayers andinfluence their properties (e.g., in plane elasticity, phasetransition temperature, phase structure, mixing with othermembrane components) and their interactions with membranelipids.32,35 The phase state of sphingomyelin (phospholipid) waspostulated as a key factor affecting the interactions withcholesterol.39 Although it was found39 that mixing of eggsphingomyelin with cholesterol is nearly identical to mixingbetween cholesterol and N-palmitoylsphingomyelin, it shouldbe pointed out that egg sphingomyelin is dominated by C16:0chains and the residues of unsaturated chains are in a very lowproportion. However, comparing the results obtained for sph-ingomyelins differing in their chain length structure, significantvariations in their interactions with cholesterol were proved.38

It was found that the sphingomyelin/cholesterol interactionsbecome stronger with the increase of the sphingomyelin chainlength and are stronger for sphingomylin with saturated versusunsaturated chains.

The results presented in this work prove a nonideal behaviorof sphingomyelin/sterol mixed systems manifested, for example,in deviations from additivity of molecular areas of the monolayercomponents, a condensing effect of sterols on sphingomyelinfilm, and the existence of interactions between film components.Negative deviations observed in A12 and AExc versus monolayercomposition plots together with negative values of the excessfree energy of mixing prove that interactions between filmcomponents are more attractive than those occurring in respec-tive one-component films. The behavior of sphingomyelin/sterolmixtures, for example, a nonideal behavior, is similar to that ofphospholipid/sterol mixed systems, that is, DPPC/cholesterolmixtures. The condensing effect of cholesterol on phospholipidfilms is attributed to the formation of stable complexes ofspecific stoichiometry between interacting molecules.63 Becausemixtures of phospho- and sphingolipids and sterols are of greatimportance due to their relevance to biological systems, intensivestudies have been performed to describe the properties of thesemixtures and the formation process of condensed complexes.The application of epifluorescence microscopy allowed one tovisualize the domains and to observe the liquid-liquid im-miscibility between complexes and phases rich in phospholipidor rich in cholesterol. These experiments allowed the authorsto prepare the phase diagrams for the investigated mixedsystems. The foregoing diagrams were determined for various


phospholipid/sterol mixtures63-67 as well as for sphingomyelin/cholesterol monolayers.68,69 Generally it was postulated that inthe mixed monolayer, at lower surface pressure, two coexistingliquid phases are present: the complex is immiscible withcholesterol-rich liquid or phospholipid-rich liquid. In the phasediagram for sphingomyelin/cholesterol monolayer, two two-phase coexistence regions (R and �) are observed.68,69 The Rtwo-phase region corresponds to a low mole fraction ofcholesterol and denotes the immiscibility between complex andsphingomyelin-rich phase, while the � two-phase region indi-cates immiscibility between complex and cholesterol-richphase.68,69 The minimum between R and � phases correspondsto the stoichiometry of the formed complex and for sphingo-myelin/cholesterol monolayers appears at Xsterol ≈ 0.3. The datapoints in the diagram determine the boundary between themonolayer composition and surface pressure region of two-phasecoexistence and the region in which the two liquid phases formone homogeneous liquid phase. The properties of sphingomyelin(phospholipids) and cholesterol mixtures and formation of thecondensed complexes have been studied both in monolayersand in bilayers with various experimental techniques, forexample, differential scanning calorimetry (DSC), X-ray dif-fraction (XRD), electron spin resonance spectroscopy (ESR),and grazing incidence X-ray diffraction (GIXD).70-73 Interestingresults concerning the structures existing in mixed sphingomy-elin/cholesterol monolayers at higher pressure region wereobtained, for example, from GIXD measurements.73 It was foundthat the Bragg peaks observed for the foregoing mixed mono-layers at a pressure range of 25-40 mN/m lie between thosefor respective one-component films. This proves the formationof the structures of new type, which were found to be dynamicin nature.73

5. Summary

The studied sphingolipid/sterol mixed systems are of greatimportance due to their relevance to biological systems. To studythe interactions between egg sphingomyelin and sterols and toanalyze the differences in the ordering effect induced bycholesterol versus the plant sterols, the Langmuir monolayertechnique was applied. It was found that the investigated mixedsystems show nonideal behavior. The addition of a sterol intosphingomyelin film provokes the decreases of the surface areaoccupied by molecules in the monolayer (“condensing effect”),and this effect is stronger for cholesterol than for phytosterols-containing films. The sphingomyelin/sterol interactions for allof the systems studied are the strongest when these lipids arein the proportion of 2:1; however, the human sterol (cholesterol)interacts with sphingomyelin more strongly than do the plantsterols. The differences in the interactions between sphingo-myelin and the respective sterols are a consequence of thestructure of the side chain in sterol molecules. On the basis ofthe analysis of the compression modulus values, the orderingeffect of sterol was examined. The incorporation of sterols intosphingomyelin film increases the packing of molecules in themonolayer and increases the order of sphingolipid hydrocarbonchains. However, cholesterol was found to be significantly moreeffective in ordering of the sphingolipid chains than were theplant sterols. Because, in fact, differences between orderingproperties of �-sitosterol and stigmasterol are found to be small,it was concluded that both of the investigated plant sterols areof similar ordering properties toward egg sphingomyelin.Finally, the ordering effect of the sterols on sphingomyelinversus DPPC was compared. It can be observed that therespective investigated sterols induced almost an identical

increase of the compression modulus values of both eggsphingomyelin and DPPC monolayer, and, in this sense, theordering effect of sterols on egg spingomyelin and DPPC issimilar.

Acknowledgment. Katarzyna Hac-Wydro wishes to thankThe Foundation for Polish Science for financial support.

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the impact of sterol structure on the interactions with sphingomyelin in mixed langmuir monolayers

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