www.elsevier.com/locate/apsusc

Applied Surface Science 253 (2006) 2425–2431

Ethanol and methanol induced changes in phospholipid monolayer

M. Weis a, M. Kopani b,*, J. Jakubovsky b, L’. Danihel b

a Slovak University of Technology, Faculty of Electrical Engineering and Information Technology, Department of Physics, Bratislava, Slovakiab Comenius University, School of Medicine, Institute of Pathology, Sasinkova 4, 811 08 Bratislava, Slovakia

Received 21 April 2006; received in revised form 28 April 2006; accepted 28 April 2006

Available online 30 June 2006

Abstract

The main components of cell membranes are phospholipids and proteins. The aim of our study was to examine structural changes of

dipalmitoyl-phosphatidylcholine (DPPC) monolayer as a simple model system of a cell membrane in different environments. Pure water, ethanol

and methanol solutions were used as subphases of Langmuir films as a membrane models. For detection of changes in charge states of the

molecules as well as relation with structural and conformational changes, a contactless method Maxwell’s displacement currents (MDC) was used.

Behaviour of DPPC molecules on two different subphases is substantialy different. In DPPC monolayer on the subphase of methanol–water, a

gradual absorption (incorporation, penetration) of methanol molecules into the layer can appear. In DPPC monolayer on the subphase of ethanol–

water adsorption of ethanol molecules on the layer can be observed. The membrane permeability might change. At both subphases (ethanol–water

and methanol–water) the elasticity modulus of the monolayer decreases leading to the loss of membrane elasticity.

# 2006 Elsevier B.V. All rights reserved.

PACS: 68.08.�p Liquid–solid interfaces; 68.18.�g Langmuir–Blodgett films on liquids

Keywords: Maxwell displacement current; Conformation; Membrane; Ethanol; Methanol; Permeability

1. Introduction

Cell membrane is an important component of all cells. It

separates intracellular from extracelluar spaces. It forms a

selective permeabile border providing dynamic balance

between a cell and an outer space. It contains enzymes,

receptors, transport and signaling systems and antigens. The

main components of cell membranes are phospholipids and

proteins. They are incorporated into the membranes or proteins

and can be electrostatically bounded to the surface. In addition

to phospholipids and proteins, membranes contain water and

different chemical compounds. Most important are cholestorol

and glycolipids. Cholesterol provides firmness and fluidity of

the membrane.

Membranes seem to be altered by the presence of ethanol

[1–4]. The mechanism by which ethanol affects function of a

membrane at the molecular level is still not known. It is likely

that ethanol acts in a hydrophobic environment. Alcohols cause

the inhibition of molecule transport through the cell membrane.

* Corresponding author. Tel.: +421 2 59357273; fax: +421 2 59357592.

E-mail address: martin.kopani@fmed.uniba.sk (M. Kopani).

0169-4332/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.apsusc.2006.04.053

Hutchinson et al. [5] compared the relative potencies of the

inhibition caused by the different alcohols. They found that

potencies of the inhibition increased with the alcohol chain

length. Alcohols can induce a disordering of the acyl chains of

the fluid phospholipids, as well as the formation of an

interdigitated gel phase [6–8]. From NMR studies it can be

concluded that ethanol binding to lipid membrane near lipid–

water interface [9–12].

The exact nature of the interactions among ethanol,

membrane proteins, and the lipid framework remains obscure.

It is known that ethanol fluidizes the bulk lipid of membranes

and may alter cell function. The changes of cell membrane

involve direct effect of ethanol on proteins, other membrane

acting drugs, temperature effects, effects of ethanol on aged

membranes and inconsistent effects of chronic ethanol

consumption on lipid content [13]. Interaction between ethanol

and cell membrane results in a disorder or ‘‘fluidize’’

membranes, or act as a local anesthetic. In some kinds of

membranes low concentrations of drugs have an ordering effect

[14,15]. Also influence of the alcohol (with various chain

lengths) on disorder and rigidity of surfactants was investigated

[16]. In the simplest picture, the molecular entropy varies with

the area per molecule A as S = kBT ln A (kB and T denotes the

M. Weis et al. / Applied Surface Science 253 (2006) 2425–24312426

Boltzmann constant and temperature, respectively). Therefore,

incorporation of cosurfactant into the phospholipid leads to

disorder due to increasing of area per molecule.

Luzzati et al. [17] investigated the structure of toad sciatic

nerves exposed to either low temperature or influence of

tetracaine. Their results suggest that membranes were

thickened and stiffened. These phenomena are typical for

lipid-containing systems with disordered chains.

Some evidences indicate that interaction between ethanol

and membranes have a membrane-fluidizing effect. The

chronic response to this effect is not the change of the

membrane bulk lipid composition [18].

Properties of the membrane model system may be

investigated by examining the dipole moment projection of

the monolayer during compression and analysis of the

elasticity. Dipole moment projection analysis by the Maxwell

displacement current (MDC) [19] is very sensitive method for

evaluation of molecular orientation (so-called order parameter)

as well as electric state of the molecule. In this way is possible

to investigate phospholipid phase transition and influence of

alcohol in the subphase. In contrast with surface pressure–area

isotherm analysis is MDC measurement is extremely sensitive

also in the low surface pressure–area, where these methods are

useless.

The aim of our study was to examine structural changes of

dipalmitoyl-phosphatidylcholine (DPPC) monolayer as a

simple model system of a cell membrane in different

environments and to compare effects of ethanol and methanol

on this system.

2. Material and methods

2.1. Chemicals and preparation of monolayers

The material used in this study as model phospholipid was

1,2-dipalmitoyl-sn-glycero-3-phosphocholine monohydrate

(DPPC) purchased from Sigma–Aldrich. Lipid was dissolved

in chloroform at the stock concentration 0.5 mg/ml and spread

on the subphase using microsyringe (Hamilton, USA). Pure

water (bidistilled deionized water, 15 MV cm) and 20%

ethanol and methanol solutions were used as subphases. For

alcohol solutions ethyl alcohol and methyl alcohol were used

(spectrophotometric grade purity) from Sigma–Aldrich. Sub-

phase was thermostated to the temperature 17 8C. Monolayer

Fig. 1. Schematic view of the experimental setup for displacement current measure

water interface (right) with maximal tilt angle QA (A and m stand for the area per mo

the top electrode is not drawn.

was allowed to equilibrate and solvent to evaporate for 15 min.

This time was sufficient for chloroform to evaporate and

monolayer to stabilize.

2.2. Experimental methods

Various experimental techniques have been designed for the

observation of structure properties and order parameter in

organic monolayers situated onto the air–water interface [20].

However, the orientational parameter measurement in the time

domain is possible only with some of them. For detection of

changes in charge states of the molecules as well as relation

with structural and conformational changes, a contactless

method was developed based on analysis of Maxwell’s

displacement currents. This method was originally introduced

by Iwamoto and Majima [21,22] and improved by other authors

[23–26]. Proposal of MDC experiment application for

biological membrane phantom measurement was presented

for the first time in our previous work [19].

The basic components of the Maxwell’s displacement

current—experimental setup attached to the computer-con-

trolled model Langmuir trough (model 611, Nima Technology,

UK) are schematically shown in Fig. 1. The top electrode was

suspended in air, parallel to the interface, without a direct

mechanical or electrical contact with a floating monolayer on

the water surface. The air gap between the top electrode and the

water surface was regulated to a certain spacing (approximately

1 mm) by measuring the capacitance of the electrode system.

The displacement current was detected with a Keithley 617

electrometer (Keithley Instruments, Cleveland, Ohio, USA).

The sensitivity of measuring the current was 0.1 fA, the

background noise was suppressed by a multiple electrical

shielding of electrode as well as whole measuring system to

2 fA. Langmuir through was situated in a laminar-flow box on

an antivibrating stand to avoid any mechanical stress.

The total working area of the trough was 600 cm2 and the

compression rate was 50 cm2/min, which correspond to

0.17 A2/s per one molecule. The area of top electrode was

AE = 20 cm2.

Due to dynamic processes in the monolayer associated with

the change in charge distribution caused by its compression, the

induced charge in the top electrode varies with time and this

generates a current flowing through the outer circuit via the

electrometer.

ment (left). Rod-like polar molecules execute precessional motion on the air–

lecule and the dipole moment of molecule, respectively). Electrical shielding of

M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431 2427

Fig. 2. Record of the displacement current–area isotherm measurement of

DPPC monolayer onto the pure water subphase.

The level of the displacement current detected is very low

and therefore the problem of background was carefully

considered. The noise signal, known as the Johnson–Nyquist

thermal noise, is at the level of 1–3 fA the value being at least

one order of magnitude less than the signal of the monolayer.

The MDC technique is sensitive only to dynamic charge

processes, which in this experimental setup are caused by

lateral compression of the monolayer. Therefore any time-

independent charge (mainly structured water layer and

additional substances in subphase) distributed near/at the

interface has no effect. In comparison with conventional

electrical measurements of surface potential (by the Kelvin

method) it provides big advantage in time-depended signals.

The surface pressure–area isotherms were measured by the

Willhelmy plate method with accuracy �0.05 mN/m. Conse-

quently various mechanic and thermodynamic properties as

elastic modulus of Langmuir films or thermodynamic proper-

ties (Gibbs free energy, entropy and enthalpy) can be evaluated.

Membrane curvation due to thermal fluctuations is essential

for shape and/or conformations of membranes as well as for

cracks and defects generation [27]. The elastic modulus

characterizes the elasticity of the monolayer and is in analogy

with bulk materials defined as

jEj ¼ �A

�@p

@A

�T

(1)

where p is the surface pressure, A the area per molecule and T is

the temperature. The elastic modulus expresses the elasticity of

the Langmuir film under influence of the compression force.

The stability of the mixed monolayer can be determined by

evaluation of excess Gibbs free energy of mixture following the

Goodrich method [28] by integration of surface pressure–area

isotherm up to selected surface pressure p

DG ¼Z p

0

ðA12 � x1A1 � x2A2Þ dp (2)

where A12 is the molecular area in the mixed monolayer, A1 and

A2 the molecular areas in the pure component monolayer and x1

and x2 are the molar ratios of pure components in the mixture

(x2 = 1 � x1). In our case surfactant is DPPC and cosurfactant is

adsorbed alcohol, x2 is time-depending parameter.

2.3. Theoretical background of MDC

The analysis of MDC experiment is based on the assumption

that each molecule behaves like a weak dipole moment with a

negative pole bound to the water surface. Individual molecules

have random directions within a certain solid angle and execute

a random precessional motion with a maximal possible tilt QA

from the vertical axis. Generally, we consider the molecule as a

rod-like rigid body without a possibility of bending.

If we consider the organic film as a system of electric dipole

moments then it is possible to calculate the induces charge on

the upper electrode with the method of images.

Qi ¼ hmziNG ¼ mhcos QiNG (3)

where m is the dipole moment of one molecule (mz is projection

of m to the normal), N the number of molecules under the top

electrode and G is the geometrical factor depending only on the

distance between the top electrode and the top plane of the

monolayer and on the shape and area of the upper electrode.

The hcos Qi stands for the statistical mean value cos Q where

Q is the angle between the vector of dipole moment and the

normal. Detailed analysis of dipole moment projection of

simple fatty acid was described in Ref. [29].

As we show in our previous studies [23] the current flowing

in the outer circuit can be expressed as a time change of the

induced charge

I ¼ @Qi

@t¼ mNG

@hcos Qi@t

þ mhcos QiG @N

@t(4)

By integrating the displacement current with respect to time,

the induced charge Q can be obtained and in this way we also

evaluated the vertical component of the molecular dipole

moment. Thus, the dipole moment projection to the normal mz

should be calculated as

mz ¼ mhcos Qi ¼ 1

G

ZI

Ndt (5)

3. Results

3.1. MDC measurements

During the measurement of the displacement current of the

DPPC monolayer situated onto the pure water in relation to area

per molecule we can notice a sharp maximum at 110 A2

(Fig. 2).

Relation between displacement current of the monolayer

DPPC on the subphase methanol–water and area per molecule

is depicted in Fig. 3. Our results indicate maximum at about

90 A2. In addition, we can observe time-shift of a maximum of

the monolayer displacement current to higher values of area per

molecule.

M. Weis et al. / Applied Surface Science 253 (2006) 2425–24312428

Fig. 3. Records of the displacement current–area isotherm measurements of

DPPC monolayer on subphase methanol–water.Fig. 5. The dipole moment projection to the normal calculated from MDC

measurement of monolayer on pure water. Rapid growth of dipole moment

projection at 110 A2 indicates ordering of molecules during the gas–liquid

phase transition.

Relation between displacement current of the monolayer

DPPC on the subphase ethanol–water and area per molecule is

depicted in Fig. 4. We can notice maximum at about 50 A2. In

addition, we can observe a mild time-shift of the monolayer

displacement current maximum to hihger values of area per

molecule. It is notable, that in all gaseous phase of the

monolayer negative displacement current is observed.

3.2. Dipole moment projection analysis

By analysis of records of the Maxwell diplacement current

projection, we can calculate dependence of the dipole

moment on the area per molecule of the monolayer. On

the surface of pure water at DPPC monolayer we can see a

rapid change of the dipole moment projection at value around

110 A2 (Fig. 5).

At DPPC monolayer, on the surface of subphase methanol–

water, no significant change of the dipole moment projection of

DPPC molecule is observed related to the change of the dipole

moment projection of DPPC molecule on water, even though

the effect of methanol–water subphase is obvious. Rapid

growth of the dipole moment projection is observed at around

area 90 A2 per molecule (Fig. 6).

Fig. 4. Records of the displacement current–area isotherm measurements of

DPPC monolayer on subphase ethanol–water.

Measurement of the dipole moment projection of DPPC

monolayer on the surface of subphase ethanol–water in

dependence of area per molecule indicates the rapid phase

transition from liquid phase to solid phase at 50 A2 (Fig. 7).

Moreover, our recordings show a time shift to lower values of

the dipole moment projection of DPPC molecule on the surface

of subphase ethanol–water. We can observe that dipole moment

projection reaches negative values of the polar head, even

though the change of the tail dipole moment can occur.

3.3. Surface pressure–area isotherms

Fig. 8 depicts relationship between surface pressure and area

per molecule of the DPPC layer on the surface of subphase

methanol–water.

We can observe the time shift of p–A isotherm to higher

values of the surface pressure.

Fig. 6. The dipole moment projections to the normal calculated from MDC

measurements on subphase methanol–water. Rapid growth of dipole moment

projection at 90 A2 indicates ordering of molecules during the gas–liquid phase

transition.

M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431 2429

Fig. 7. The dipole moment projection to the normal calculated from MDC

measurement on subphase ethanol–water. Rapid growth of dipole moment

projection at 50 A2 indicates ordering of molecules during the liquid–solid

phase transition.

Fig. 8. p–A Isotherms of DPPC monolayer on subphase methanol–water.

Fig. 9. p–A Isotherms of DPPC monolayer on subphase ethanol–water.

Fig. 10. Isothermal elastic modulus curves of DPPC monolayer on subphase:

(top) ethanol–water, (bottom) methanol–water. Elastic modulus of DPPC situated

onto the pure water subphase is presented for a comparison (grey curve).

At subphase ethanol–water, a significant change in the

dipole moment projection of DPPC molecules forming the

monolayer appeared (Fig. 9). Monolayer exhibits very sharp

phase transition, phase transition from gaseous to liquid phase

at area about 80 A2 (observable only for first compression) and

from liquid phase to solid phase at 50 A2.

Similarly, as at DPPC monolayer on the subphase methanol–

water we can observe a time shift of p–A isotherm to higher

values. However, this shift is not as obvious as it was at DPPC

monolayer on the subphase methanol–water (Fig. 10).

From the graph representing the relation between elasticity

modules and area per molecule we found out that at DPPC

monolayer on the surface of both subphases (ethanol–water and

methanol–water) elasticity modulus of the monolayer

decreases. Our results suggest a gradual increase of membrane

rigidity (decrease of elasticity).

Time dependence of Gibbs energy and maximum of dipole

moment projection is shown in Fig. 11. The dipole moment

projection maximum represents the phase transition area;

therefore its change is directly proportional to adsorbed

alcohol. In both cases is observable continuous adsorption. The

Gibbs free energy characterizes the stability of the phospho-

lipid–alcohol mixture. Methanol molecules destabilize the

monolayer in contrast with ethanol, which incorporation into

the monolayer carry to lower free energy.

4. Discussion

4.1. Dipole moment and electric properties

4.1.1. Water

Results of experiments performed on pure water [20]

suggest that at the value around 100–110 A2 area per molecule,

a phase transition occurs from gaseous phase to liquid phase.

From results of the measurement of the surface potential by

Kelvin probe [30] the value of the dipole moment of DPPC

molecule was determined to be 820 mD.

M. Weis et al. / Applied Surface Science 253 (2006) 2425–24312430

Fig. 11. Time dependence of Gibbs free energy of DPPC monolayer onto the ethanol–water (up) and methanol–water (down) subphase for various values of surface

pressure (left view). Time dependence of dipole moment maximum position DPPC onto ethanol–water and methanol-water subphase (right view).

Our recordings show the value around 815–825 mD, which

is in according with values obtained by independent measure-

ments.

4.1.2. Methanol

By the analysis of the measurement of the Maxwell

diplacement current in relation with area per molecule, we

calculated the dependence of the dipole moment projection on

the area per molecule of the DPPC monolayer on the subphase

methanol–water [19]. At DPPC monolayer on the surface of

subphase methanol–water no significant changes of the dipole

moment projection of the DPPC molecule appear when

compared to the dipole moment projection of the DPPC

molecule on water, even though the effect of subphase

methanol–water is obvious.

We can observe the time shift of the area per DPPC molecule

to higher values. We assume that DPPC molecules move away

from each other leading to incorporation (penetration) of

methanol molecules into the air–liquid interface [16,31–34].

During incorporation (penetration) into DPPC molecules layer,

no significant influence on to DPPC is observed, as we do not

observe any changes of the dipole moment projection.

Incorporation (penetration) of the methanol molecules into

the layer is manifested by the shift of p–A isotherm to higher area

values. From the shape of p–A isotherm we can assume a smooth

transition from liquid expanded to liquid condensed phase [20].

DPPC monolayer on subphase methanol–water has no

distinct electrical properties in comparison to the DPPC

monolayer on water. We suppose that incorporation (penetra-

tion) of methanol molecules into DPPC layer causes no changes

in electrical properties of this layer when compared to electrical

properties of DPPC on water [35]. Consequently, we assume no

electrical interactions between ions of the polar head and

methane ions. Due to moving molecules away from each other,

increased membrane permeability can occur [36,37].

4.1.3. Ethanol

Measurement of the dipole moment projections of the DPPC

monolayer on the surface of subphase ethanol–water in relation

to area per molecule show two order transitions. As we observe

no time shift of the dipole moment projection, we assume that

the distance between molecules does not increase. Unlike

DPPC monolayer on the subphase methanol–water, adsorption

of ethanol to this layer might occur. Yamamoto et al. [38]

investigated interaction with DPPC and dihexadecyl phosphate

with ethanol. They found different behaviour of ethanol–water

solution depending on its concentration. Based on their results,

it can be concluded that at low concentration of ethanol,

hydrates adsorb on the monolayer–water interface and saturate

on the interface. The increase of ethanol concentration causes

multilayer formation of hydrates and/or penetration of hydrates

into the monolayer core.

From NMR results Barry and Gawrisch [6] showed ethanol

binding in the lipid–water interface. The interaction of ethanol

in the lipid–water interface changed order parameter. The

amount of cholesterol influenced the phase transition to the

liquid-ordered phase and ethanol binding decreased with

increasing amounts of cholesterol.

Moreover, measurements show the time shift of the dipole

moment projection of the DPPC molecule to lower values.

Values of the dipole moment projection reach negative values.

Major part of the dipole moment of DPPC molecule consists of

the phosphatocholin (PC) group in the polar head. Interaction of

DPPC–ethanol influenced bonds in this group. Due to the

adsorption of ethane molecules to monolayer consisting of

DPPC molecules, reversion of the dipole moment projection of

the DPPC molecule is observed. We suppose that either change

of bond orientation or disruption of a bond occured [39]

resulting in significant change of electric properties of PC

group. The change of electric charge on the membrane surface

can appears which will influence the diffusion of ions. The

change of electric charge on the membrane surface (in natural

state the membrane surface is weakly negatively charged due to

the PC group) will change the ion diffusion through the

membrane (reduction of the negative charge might occur

resulting in easier transition of negative ions).

Measurements of MDC in dependence of area per molecule

on the subphase ethanol–water suggest, that no time-dependent

M. Weis et al. / Applied Surface Science 253 (2006) 2425–2431 2431

significant changes occur as it was in case of DPPC monolayer

on the subphase methanol–water. We suppose that electric

properties of this monolayer have not changed significantly.

4.2. Membrane rigidity

From graph representing the relation between elasticity

modulus and area per molecule of DPPC monolayer on the

surface of both subphases (ethanol–water and methanol–water)

we found that elasticity modulus of the monolayer decreases.

Based on our results, we assume a gradual ‘‘solidification’’ of

the membrane and loss of its elasticity [40,41]. Increase of

rigidity is proportional to alcohol length, what is in agreement

with other studies [16]. Inequality of influence of alcohol

molecules on phospholipid monolayer is caused by the different

alcohol chain length. Bending rigidity depends on chain length

and adsorption rate in complex form. The measurements of the

area compressibility modulus, bending modulus, lysis tension,

lysis strain, and area expansion of fluid phase 1-stearoyl, 2-

oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to

aqueous solutions of short-chain alcohols revealed that the

order in decreasing mechanical properties was butanol > pro-

propanol > ethanol > methanol [42].

Goldstein [43] desribed that animals after chronical treatment

of ethanol showed stiffer membranes. This stiffer effect of

ethanol may be reduced by cholesterol or saturated fatty acids.

Goldstein and Chin [44] examined the influence of ethanol

on a cell membrane. The mice were treated with ethanol for 8

days. It was found that ethanol disorders mouse cell

membranes, making the lipid matrix more fluid. The

consequent disruption of the function of integral membrane

proteins may be the cause of ethanol’s central actions. The

tolerance to the disordering effect of ethanol was accompanied

by an increased proportion of cholesterol in the membranes.

5. Conclusion

Behaviour of DPPC molecules on two different subphases is

substantialy different. In DPPC monolayer on the subphase of

methanol–water, a gradual absorption (incorporation, penetra-

tion) of methanol molecules into the layer can appear leading to

‘‘dilution’’ of the layer and thus to the change of monolayer

permeability. In DPPC monolayer on the subphase of ethanol–

water adsorption of ethanol molecules to the layer can be

observed leading to the change of electric properties of the layer

surface. Consequently, the membrane permeability might

change.

At both subphases (ethanol–water and methanol–water) the

elasticity modulus of the monolayer decreases leading to the

loss of membrane elasticity.

Acknowledgement

This work was supported by grant of Science and

Technology Assistance Agency nos. APVT-20-003104 and

APVT-51-013904.

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