International Ophthalmology 16: 16%176,1992. 9 1992KluwerAcademic Publishers. Printedin the Netherlands.
Biophysical characteristics of the Meibomian lipid layer under in vitro conditions Thomas Kaercher, 1 Dietmar MObius2 & Rfidiger Welt 1 1Augenklinik Ludwigshafen, Bremserstr. 79, D-6700 Ludwigshafen, Germany 2Max-Planck-Institutfgtr Biophysikalische Chemie, Arbeitsgruppe Molekular organisierte Systeme, D-3400 GOttingen-Nikolausberg, Germany
Key words: Meibomian gland secretion, surface potential, surface pressure Abstract
The biophysical behaviour of the Meibomian gland secretion was tested under in vitro conditions. Thereby, simultaneous recording of surface pressure and surface potential was performed. The Meibomian lipid layer was compared with other surface-active components like polyvinylalcohol and polyvinylpyrrolidone. On the other hand, Eledoisin was tested as an example for a surface-inactive substance. An attempt was made to describe the biophysical interaction between a given artificial tear substitute and the Meibomian lipid layer. With respect to the surface potential Dipalmitoyl-phosphatidyl-choline was established as an analogue for Meibomian gland secretion. Fluorescence measurements in the presence of a cyanine dye (1 N,N'dioctadecyloxacarbocyanine) were used as a method to localize the site of the characteristic potential change. From the fluorescence spectra under compression we conclude that the molecular change takes place at the lipid-subphase interface of the Meibomian lipid layer.
Abbreviations: DPPC - dipalmitoyl-phosphatidyl-choline, MGS - Meibomian gland secretion, PVA polyvinyl-alcohol, PVP - polyvinyl-pyrrolidone
Introduction
One of the classical methods for investigating thin layers at air-water interfaces is the Langmuir-Blodgett-Technique. Thereby, a thin layer is spread over an aqueous subphase in a Teflon-coated trough. By the action of a movable barrier the surface layer is compressed or decompressed. This device provides a useful analogue for the action during blinking. Thus, by means of this analogue, the behaviour of the tear film can be tested in vitro during compression and decompression, with the surface pressure and surface potential being recorded simultaneously. The relevance of the surface pressure as a bio-
physical factor in blinking has been established in a number of studies [1, 2]. However, since the information, gleaned from the surface pressure, is rather limited, further cirteria for biophysical characterisation are required. Own previous research showed that further information is conveyed by the surface potential [3]. It was a purpose of the present study to record the surface potential of Meibomian gland secretion (MGS) and artificial tears. The thin lipid layer of MGS forms the outermost layer of the preocular tear film. Underneath, this layer is in contact with the aqueous layer. The artificial tears presently in use are aqueous solutions. Upon topical application they first get in contact with the lipid layer. After a few blinks the
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Fig. 1. Teflontroughwiththe movablebarrier in the background,The Wilhelmybalancewiththe immersedfilterpaper on the lefthand
side. The vibratingcondenserand the platinumcounter-electrodeon the righthandside. aqueous fluid of the drop mixes with the aqueous layer of the tear film. It is generally assumed that artificial tears are compatible with the preocular tear film, due to the surface activity of both components. However, the quality of the interaction has hardly been described in the literature. It would be of interest to know if this interaction supports the integrity of the surface lipid layer. The lipid layer of the preocular tear film mainly consists of waxes and cholesteryl esters. Thickness measurements indicate the lipid layer to be a monolayer [3]. The molecular arrangement of this layer depends on the adjacent layers. Thus the hydrophilic head groups of the lipids should be so arranged as to point towards the monolayer-waterinterface, whereas the hydrophobic chains of the lipids should be positioned at the monolayer-airinterface.
The present study aimed at determining the exact molecular site of the change of the thin MGS layer and at describing the molecular interaction with the artificial tear solution. Through fluorescence measurements and with the aid of especially designed molecules, an attempt was made to answer these questions.
Materials and methods Materials
MGS was taken from volunteers who did not show any pathological changes of the lids or the conjunctiva. It was ~obtained by gently squeezing the lid margins. The lipid samples were stored on a silicone strip either at + 2 to + 5~ or by freezing
Biophysics of MGS
169
Fig. 2. Arrangement for the determination of surface pressure and surface potential using a glass trough.
them with stepwise reduction of the temperature to - 70 ~C. The secretion was solved in chloroform (1 mg/ ml) acting as a spreading solvent. Purified chloroform (Baker Chemicals, p.a.) was run through a one-meter-column filled with alumina (alumina, Woelm, B-Super I) and used after addition of one volume percent of absolute ethanol (Merck). Water was purified by a Milli-QSystem (Millipore). Eledoisin (Farmitalia) was used in a concentration of 0.1 mg/ml. Polyvinylalcohol (PVA, Allergan) has a molecular weight between 28000 and 40000; the concentration in our tests was 14mg/ml. Polyvinylpyrrolidone (PVP, Thilo) has a molecular weight of 27000; the concentration used in our tests was 20 mg/ml. The cyanine dye 1 N,N'dioctadecyloxacarbocyanine was synthesized according to Sondermann [4]. The mixing ratio between the cyanine dye and MGS was 1 : 100.
Dipalmitoyl-phosphatidyl-choline (DPPC, Larodan) was tested in combination with PVA (concentration 1 x 10-3 M) and in combination with PVP (concentration 1 x 10-3M). MGS and Eledoisin were spread on a water surface in a trough made of Teflon (total area 350cm 2, depth 0.8 cm). The movable barrier (constant speed of 20 ram/ rain) consisted of Teflon as well (Fig. 1). All experiments involving PVA and PVP were performed in a trough made of glass (total area 225 cruZ), which was used after silanisation (Fig. 2). The movable barrier was, again, Teflon. Control experiments were performed with a Dynal barrier. The experiments were carried out at room temperature. The pH of the aqueous subphase was 5.5. The system was tested by using arachidic acid as a control (Merck, p.a., recrystallized from ethanol, concentration 5 x 10-3M).
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Methods
Results
After spreading a surface layer on the water subphase of the trough the surface layer was compressed by the movable barrier. Surface pressure and surface potential were recorded continuously and simultaneously during compression. Decompression of the surface film was achieved by moving the barrier in the opposite direction, again while monitoring surface pressure and surface potential. The surface pressure was determined with a Wilhelmy type film balance for a given surface area. Here, the force acting on a thin suspended filter paper, partially immersed, was measured (mN/m). The immersed filter paper moves upward when the surface pressure is high, downward when it is low. The surface potential was determined with the vibrating plate method. The air electrode oscillates with respect to the water surface, thus yielding an alternating current flow in the external circuit due to capacity change (mV). The interfacial electrical potential is changed when a surface layer is spread on it. Fluorescence spectra were recorded at different stages of compression of the surface layer. The excitation was at 450 nm. The spectra were recorded between 470 nm and 600 nm. The instruments for recording the spectra are described by Kuhn et al. [5].
1. Behaviour of normal MGS
The MGS of patients who did not show any disease of the anterior segment of the eye was tested. After spreading 100 t~l of MGS dissolved in CHC13 compression was started. The lower section of Fig. 3 shows the course of the surface pressure. After an initial plateau-phase a sudden increase of the surface pressure up to 12.4 mN/m was observed with increasing compression. This value is in excellent agreement with the equilibrium filter pressure of normal MGS reported by Holly [8]. The reverse picture was obtained upon decompression of the film. The upper section of Fig. 3 displays the surface potential under compression. After an initial phase a sudden potential jump of more than 200 mV was recorded, followed by a steady increase of the potential under further compression. The potential jump was fully reversible under decompression. Repeated hysteresis of the lipid layer was possible without any change in the course taken by the surface pressure and the surface potential. Figure 4 represents a continuous recording of the same experiment. The two biophysical parameters are plotted against time. Normal MGS showed identi-
Biophysics of MGS
171
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cal biophysical behaviour under repeated compression and decompression.
2. Biophysical behaviour of Eledoisin Eledoisin, the endekapeptide of the salivary glands of Eledona moschata and aldrovandi, is known to act as a stimulator of the lacrimal glands [6]. We
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wanted to know if its therapeutic effect is not only due to the above mentioned mechanism but also to any surface-activity. The compression of Eledoisin, which was spread on water as a subphase, did not result in an increase of the surface pressure. The surface pressure remained near zero. Recording of the surface potential did not show any significant change except for some fluctuations. These data prove that Eledoisin does not act as a
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under compression and decompression. The graphs were recorded from right to left. The terminal, inverse potential peak was shown to be an electrostatic effect of the Teflon barrier.
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Fig. 9. Surface pressure (1) and surface potential (2) of the combination of PVA and MGS, plotted against time.
surface-active component. Its therapeutic effect is bound to be due to stimulation of the lacrimal glands, as was reported previously.
decompression (Fig. 7). The surface potential was characterized by a slight increase of 30 mV, too. The final inverse peak at the end of compression was proven to be an effect of the Teflon barrier. It was absent when a Dynal barrier was used. PVA and PVP showed all characteristics of surface-activity (rising isotherm under compression).
3. Characteristics of artificial substances with surface-activity PVA and PVP are two surface-active components which are already introduced as therapeutic concepts in ophthalmology. We tested the biophysical behaviour of PVA dissolved in the aqueous subphase, using a glass trough (Fig. 5). Under compression a steady increase of the surface pressure was seen, which proved reversible under decompression. The surface potential showed a slight increase under compression, ending in a high peak of 200 mV at the end of compression (Fig. 6), Varying the concentration of PVA in the subphase did not alter the position of this peak as it should do. When replacing the Teflon barrier by a barrier made of Dynal the terminal peak was not recorded any more. The steady slight increase of the surface potential remained unchanged. The above effect was probably due to an electrostatic influence of the Teflon barrier. Testing PVP resulted in an increase of the surface pressure under compression, reversible under
4. Interaction of two surface-active components Using PVA in the subphase and spreading normal MGS on top we determined the surface pressure and the surface potential under compression and decompression (Fig. 8). The surface pressure showed a characteristic increase up to 14.4 mN/m under compression, fully reversible under decompression. Following the surface potential we only saw fluctuations of some 10 mV. Continuous monitoring of both parameters plotted against time demonstrated repeatable behaviour of the surface pressure and the surface potential (Fig. 9).
5. Combination of DPPC with a surface-active component Dipalmitoyl-phosphatidyl-choline is an amphiphile
Biophysics of MGS
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well known from biological membranes. DPPC shows a characteristic two-step increase of the surface potential under compression (Fig. 10). Because of its high potential peak we used it as an analogue for MGS. Using a PVA-solution as a subphase and spreading DPPC on top, the isotherm increased slightly (Fig. 11). The surface potential showed an initial peak of almost 100 mV, remaining constant afterwards. The terminal peak was only detectable in the presence of the Teflon barrier, which obviously exerted electrostatic effects. The characteristic potential of DPPC was changed in the presence of PVA. Using a PVP-solution as a subphase and spreading DPPC on top the surface pressure increased tremendously up to 43 mN/m (Fig. 12), at which point it collapsed. The surface potential showed a one-step increase by 150 inV. The terminal, inverse peak, again, was due to electrostatic effects. In conclusion, the characteristic potential of DPPC was changed in the presence of PVP.
6. Fluorescence spectra of MGS spread on water After spreading 100 txl MGS on a water surface we tested the fluorescence intensity in the presence of 1 N,N'dioctadecyloxacarbocyanine (Fig. 13). This
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dye molecule is attached to the inner interface of the surface layer. The fluorescence spectra were recorded at different stages of compression (0.3 - 5.0 - 10.0 15.0 mN/m, Fig. 14a-d), invariably showing a peak of fluorescence intensity near 500 nm. This peak was lowered from 30 A U to 13 AU under further compression. In other words: the higher the surface potential, the lower the fluorescence intensity.
Discussion
The biophysical data found in the literature usually refer to surface pressure monitoring only. Meanwhile there are reliable methods for the detection of electric phenomena at the surface of thin layers. The recording of surface potentials has become a routine procedure when monolayer assemblies are to be characterized. Two methods are available: the vibrating plate method used in our series of experiments and, alternatively, the ionizing-electrode-method. Recently, efforts were made to specify the surface potential of monolayers [7]. A monolayer has two interfaces: the surface of the hydrophobic end groups and the interface of the hydrophilic head groups towards the aqueous subphase. Both interfaces contribute to the interracial potential. The surface potential of the monolayer is defined as
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being the difference AV between the potential of the monolayer-covered surface and that of the clean water surface. The difference AV can be separated into two components: the component of the monolayer-air-interface may be assumed to be an absolute potential, whereas the second component represents the joint contributions of the electric double layer as well as the influence of the aqueous subphase on the monolayer. Recording the surface potential of MGS in persons with unaffected lids and conjunctivae showed that this material was characterized by a surprisingly high potential peak under compression. This peak was fully reversible under decompression. In synthetic lipids a potential peak of 200 mV or even more is seen occasionally (e.g. DPPC, Dipalmitoyl-phosphatidyl-ethanolamine, 7). Is MGS able to form a monolayer under certain conditions? The literature suggests that only multilayers are observed in vitro and in vivo [8]. From our thickness measurements on glass plates, which indicated surprisingly thin layers of 80 to 100 A, we inferred that at least on glass plates MGS is capable of forming monolayers. An estimate of the amount of material spread on the aqueous subphase and calculation of the surface pressure made clear that the thickness of the surface layer on water was comparable. Therefore we conclude that the formation of a MGS monolayer occurs not only on glass plates but also on water.
MGS is a highly surface-active mixture. By way of contrast, we demonstrated an instance of a surface-inactive component: Eledoisin. This substance is characterized by a lack of surface pressure. Other than that, there are practically no alterations of the surface potential. PVA and PVP are two instances of surface-active therapeutics for dry eyes. Indeed, both polymers are characterized by an increasing isotherm, but fail to undergo marked potential changes under compression. In sum, the present data clearly indicate that the characteristics of the surface potential of a surfaceactive component provide more detailed information than the surface pressure. Previous experiments showed that the surface potential of siccapatients had no identical potential courses under repeated compression and decompression, whereas the surface pressure remained unchanged [9]. If we administer artificial tears to the eyes of a patient suffering from keratoconjunctivitis sicca we are likely to observe an interaction between what is left of the patient's own tears and the artificial tears. Under these conditions, the interaction of two surface-active components does have clinical relevance. Artificial tears are found in the aqueous layer of the physiological tear film. Accordingly, in our experiments the surface-active component of the artificial tears was dissolved in the aqueous subphase. MGS was spread over the surface of such solutions. We used the combination of PVA and MGS, the former for the subphase, the latter for the surface layer. Despite the presence of MGS we could not detect the typical potential peak of 200 mV under compression. What are the possible explanations? PVA from the subphase may fill up holes in the MGS layer. This would explain why the typical potential peak of MGS is no longer detectable. Alternatively, the two surface-active compo-
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nents may be arranged at the water surface so as to form a multilayer. Due to the high concentration of PVA we only find the potential course typical of PVA with the typical MGS potential being vanished. The MGS is characterized by a high jump of the surface potential under compression. Some synthetic lipids show a similar course of the potential (e.g. DPPC, Dipalmitoyl-phosphatidyl-ethanolamine). Therefore they may act as a useful analogue for MGS. We tested DPPC, which is an amphiphile, in the presence of PVA and PVP. In both series of experiments we noticed that the typical potential peaks of DPPC were altered in combination with PVA or PVP. Only one of the two potential peaks of DPPC was recorded. Here again, the surface-active component of the aqueous subphase caused a characteristic potential change of the thin surface layer. DPPC proved to be an analogue for MGS with respect to the surface potential and to
the interaction with other surface-active components. Up to now we have studied the surface potential as a useful biophysical parameter without understanding the molecular basis of the potential change under compression and without knowing, where precisely these molecular alterations take place. Fluorescence measurements constitute a method for localizing the site of the potential changes. Cyanine dyes (e.g. 1 N,N'dioctadecyloxacarbocyanine) align with the inner surface of the monolayer. If we detect alterations of the characteristic fluorescence spectra of this cyanine dye under compression, we can take those as hints on a molecular alteration at the inner interface. In fact, a characteristic reduction of the fluorescence intensity at 500 nm was found under stepwise compression. Compression of the film entailed an increase in potential, which invariably goes along
176
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with a decrease in fluorescence intensity [10], According to the specific quality of the above cyanine dye the molecular change takes place at the monolayer-water-interface. An ideal tear substitute is not yet available. The present study established some new biophysical criteria for artificial tears. The surface potential was confirmed as the essential parameter. The interaction between artificial tears and MGS is important for therapy, and the behaviour at the lipidwater-interface is of critical relevance. Future studies should take into account these findings. The search for new therapeutic concepts needs a fresh impetus. It may be promising to take MGS of infants as a model. They are known to stare without blinking for long periods of time, hence their MGS must possess the excellent stability any designer of artificial tears would wish for.
Acknowledgements We express our thanks to Mr W. Zeiss for the expert technical assistance. We wish to thank Mr G. Overbeck and Mr M. Budach for their helpful discussion and assistance. The co-operation of the University Eye Hospital, G6ttingen, (Director: Prof Dr M. Vogel) is gratefully acknowledged.
References 1. Holly F, Letup M. Wettability and wetting of corneal epithelium. Exp Eye Res 1971; 11: 239-50. 2. Holly F. Surface chemistry of tear film component analogues. J Colloid Interface Sci 1974; 49: 221-31. 3. Jaeger W, M6bius D, Kaercher T. Biophysical experiments with lipid-layers formed with Meibomian gland secretion. In: Holly F (ed) The preocular tear film in health, disease and contact lens wear. Lubbock, 1986: 609-21. 4. Sondermann J. Darstellung oberflfichenaktiver Polymethincyanin-Farbstoffe mit langen N-Alkyl-Ketten. Liebigs Ann Chem 1971; 749: 183-97. 5. Kuhn H, M6bius D, Biicher H. Spectroscopy of monolayer assemblies. In: Weissberger A, Rossiter B (eds) Physical methods of chemistry. Wiley and Sons, 1972: 577-702. 6. Bertaccini G, de Caro G. The effect of Physalaemin and related polypeptides on salivary secretion. J Physiol 1965; 181: 68-81. 7. Vogel V, M6bius D. Local surface potentials and electric dipole moments of lipid monolayers: Contributions of the water-lipid and the lipid-air interfaces. J Colloid Interface Sci 1988; 126: 408-20. 8. Holly F. Surface chemical evaluation of artificial tears and their ingredients. Interaction with a superficial lipid layer. Contact and Intraocular Lens Med J 1978; 4: 52-65. 9. Kaercher T, M6bius D. Stabilitfit und Flexibilitfit der Lipidschicht des Trfinenfilms sowie deren pathologische Ver/~nderungen im biophysikalischen Experiment. Fortschr Ophthalmol 1989; 86: 245-8. 10. M6bius D, Cordroch W, Loschek R, Chi L, Dhathathreyan A, Vogel V. Control of interracial equilibria by local charge distribution and average surface potential. Thin Solid Films 1989; 178: 53-60.
Biophysical characteristics of the Meibomian lipid layer under in vitro conditions Thomas Kaercher, 1 Dietmar MObius2 & Rfidiger Welt 1 1Augenklinik Ludwigshafen, Bremserstr. 79, D-6700 Ludwigshafen, Germany 2Max-Planck-Institutfgtr Biophysikalische Chemie, Arbeitsgruppe Molekular organisierte Systeme, D-3400 GOttingen-Nikolausberg, Germany
Key words: Meibomian gland secretion, surface potential, surface pressure Abstract
The biophysical behaviour of the Meibomian gland secretion was tested under in vitro conditions. Thereby, simultaneous recording of surface pressure and surface potential was performed. The Meibomian lipid layer was compared with other surface-active components like polyvinylalcohol and polyvinylpyrrolidone. On the other hand, Eledoisin was tested as an example for a surface-inactive substance. An attempt was made to describe the biophysical interaction between a given artificial tear substitute and the Meibomian lipid layer. With respect to the surface potential Dipalmitoyl-phosphatidyl-choline was established as an analogue for Meibomian gland secretion. Fluorescence measurements in the presence of a cyanine dye (1 N,N'dioctadecyloxacarbocyanine) were used as a method to localize the site of the characteristic potential change. From the fluorescence spectra under compression we conclude that the molecular change takes place at the lipid-subphase interface of the Meibomian lipid layer.
Abbreviations: DPPC - dipalmitoyl-phosphatidyl-choline, MGS - Meibomian gland secretion, PVA polyvinyl-alcohol, PVP - polyvinyl-pyrrolidone
Introduction
One of the classical methods for investigating thin layers at air-water interfaces is the Langmuir-Blodgett-Technique. Thereby, a thin layer is spread over an aqueous subphase in a Teflon-coated trough. By the action of a movable barrier the surface layer is compressed or decompressed. This device provides a useful analogue for the action during blinking. Thus, by means of this analogue, the behaviour of the tear film can be tested in vitro during compression and decompression, with the surface pressure and surface potential being recorded simultaneously. The relevance of the surface pressure as a bio-
physical factor in blinking has been established in a number of studies [1, 2]. However, since the information, gleaned from the surface pressure, is rather limited, further cirteria for biophysical characterisation are required. Own previous research showed that further information is conveyed by the surface potential [3]. It was a purpose of the present study to record the surface potential of Meibomian gland secretion (MGS) and artificial tears. The thin lipid layer of MGS forms the outermost layer of the preocular tear film. Underneath, this layer is in contact with the aqueous layer. The artificial tears presently in use are aqueous solutions. Upon topical application they first get in contact with the lipid layer. After a few blinks the
168
T. Kaercher et al.
Fig. 1. Teflontroughwiththe movablebarrier in the background,The Wilhelmybalancewiththe immersedfilterpaper on the lefthand
side. The vibratingcondenserand the platinumcounter-electrodeon the righthandside. aqueous fluid of the drop mixes with the aqueous layer of the tear film. It is generally assumed that artificial tears are compatible with the preocular tear film, due to the surface activity of both components. However, the quality of the interaction has hardly been described in the literature. It would be of interest to know if this interaction supports the integrity of the surface lipid layer. The lipid layer of the preocular tear film mainly consists of waxes and cholesteryl esters. Thickness measurements indicate the lipid layer to be a monolayer [3]. The molecular arrangement of this layer depends on the adjacent layers. Thus the hydrophilic head groups of the lipids should be so arranged as to point towards the monolayer-waterinterface, whereas the hydrophobic chains of the lipids should be positioned at the monolayer-airinterface.
The present study aimed at determining the exact molecular site of the change of the thin MGS layer and at describing the molecular interaction with the artificial tear solution. Through fluorescence measurements and with the aid of especially designed molecules, an attempt was made to answer these questions.
Materials and methods Materials
MGS was taken from volunteers who did not show any pathological changes of the lids or the conjunctiva. It was ~obtained by gently squeezing the lid margins. The lipid samples were stored on a silicone strip either at + 2 to + 5~ or by freezing
Biophysics of MGS
169
Fig. 2. Arrangement for the determination of surface pressure and surface potential using a glass trough.
them with stepwise reduction of the temperature to - 70 ~C. The secretion was solved in chloroform (1 mg/ ml) acting as a spreading solvent. Purified chloroform (Baker Chemicals, p.a.) was run through a one-meter-column filled with alumina (alumina, Woelm, B-Super I) and used after addition of one volume percent of absolute ethanol (Merck). Water was purified by a Milli-QSystem (Millipore). Eledoisin (Farmitalia) was used in a concentration of 0.1 mg/ml. Polyvinylalcohol (PVA, Allergan) has a molecular weight between 28000 and 40000; the concentration in our tests was 14mg/ml. Polyvinylpyrrolidone (PVP, Thilo) has a molecular weight of 27000; the concentration used in our tests was 20 mg/ml. The cyanine dye 1 N,N'dioctadecyloxacarbocyanine was synthesized according to Sondermann [4]. The mixing ratio between the cyanine dye and MGS was 1 : 100.
Dipalmitoyl-phosphatidyl-choline (DPPC, Larodan) was tested in combination with PVA (concentration 1 x 10-3 M) and in combination with PVP (concentration 1 x 10-3M). MGS and Eledoisin were spread on a water surface in a trough made of Teflon (total area 350cm 2, depth 0.8 cm). The movable barrier (constant speed of 20 ram/ rain) consisted of Teflon as well (Fig. 1). All experiments involving PVA and PVP were performed in a trough made of glass (total area 225 cruZ), which was used after silanisation (Fig. 2). The movable barrier was, again, Teflon. Control experiments were performed with a Dynal barrier. The experiments were carried out at room temperature. The pH of the aqueous subphase was 5.5. The system was tested by using arachidic acid as a control (Merck, p.a., recrystallized from ethanol, concentration 5 x 10-3M).
170
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Fig. 4. Surface pressure (1) and surface potential (2) of normal MGS, plotted against time.
Methods
Results
After spreading a surface layer on the water subphase of the trough the surface layer was compressed by the movable barrier. Surface pressure and surface potential were recorded continuously and simultaneously during compression. Decompression of the surface film was achieved by moving the barrier in the opposite direction, again while monitoring surface pressure and surface potential. The surface pressure was determined with a Wilhelmy type film balance for a given surface area. Here, the force acting on a thin suspended filter paper, partially immersed, was measured (mN/m). The immersed filter paper moves upward when the surface pressure is high, downward when it is low. The surface potential was determined with the vibrating plate method. The air electrode oscillates with respect to the water surface, thus yielding an alternating current flow in the external circuit due to capacity change (mV). The interfacial electrical potential is changed when a surface layer is spread on it. Fluorescence spectra were recorded at different stages of compression of the surface layer. The excitation was at 450 nm. The spectra were recorded between 470 nm and 600 nm. The instruments for recording the spectra are described by Kuhn et al. [5].
1. Behaviour of normal MGS
The MGS of patients who did not show any disease of the anterior segment of the eye was tested. After spreading 100 t~l of MGS dissolved in CHC13 compression was started. The lower section of Fig. 3 shows the course of the surface pressure. After an initial plateau-phase a sudden increase of the surface pressure up to 12.4 mN/m was observed with increasing compression. This value is in excellent agreement with the equilibrium filter pressure of normal MGS reported by Holly [8]. The reverse picture was obtained upon decompression of the film. The upper section of Fig. 3 displays the surface potential under compression. After an initial phase a sudden potential jump of more than 200 mV was recorded, followed by a steady increase of the potential under further compression. The potential jump was fully reversible under decompression. Repeated hysteresis of the lipid layer was possible without any change in the course taken by the surface pressure and the surface potential. Figure 4 represents a continuous recording of the same experiment. The two biophysical parameters are plotted against time. Normal MGS showed identi-
Biophysics of MGS
171
Fig. 5. Glass trough inside of a portable frame.
cal biophysical behaviour under repeated compression and decompression.
2. Biophysical behaviour of Eledoisin Eledoisin, the endekapeptide of the salivary glands of Eledona moschata and aldrovandi, is known to act as a stimulator of the lacrimal glands [6]. We
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wanted to know if its therapeutic effect is not only due to the above mentioned mechanism but also to any surface-activity. The compression of Eledoisin, which was spread on water as a subphase, did not result in an increase of the surface pressure. The surface pressure remained near zero. Recording of the surface potential did not show any significant change except for some fluctuations. These data prove that Eledoisin does not act as a
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Fig. 7. Surface pressure (1) and surface potential (2) of PVP
under compression and decompression. The graphs were recorded from right to left. The terminal potential peak was shown to be an electrostatic effect of the Teflon barrier.
under compression and decompression. The graphs were recorded from right to left. The terminal, inverse potential peak was shown to be an electrostatic effect of the Teflon barrier.
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Fig. 9. Surface pressure (1) and surface potential (2) of the combination of PVA and MGS, plotted against time.
surface-active component. Its therapeutic effect is bound to be due to stimulation of the lacrimal glands, as was reported previously.
decompression (Fig. 7). The surface potential was characterized by a slight increase of 30 mV, too. The final inverse peak at the end of compression was proven to be an effect of the Teflon barrier. It was absent when a Dynal barrier was used. PVA and PVP showed all characteristics of surface-activity (rising isotherm under compression).
3. Characteristics of artificial substances with surface-activity PVA and PVP are two surface-active components which are already introduced as therapeutic concepts in ophthalmology. We tested the biophysical behaviour of PVA dissolved in the aqueous subphase, using a glass trough (Fig. 5). Under compression a steady increase of the surface pressure was seen, which proved reversible under decompression. The surface potential showed a slight increase under compression, ending in a high peak of 200 mV at the end of compression (Fig. 6), Varying the concentration of PVA in the subphase did not alter the position of this peak as it should do. When replacing the Teflon barrier by a barrier made of Dynal the terminal peak was not recorded any more. The steady slight increase of the surface potential remained unchanged. The above effect was probably due to an electrostatic influence of the Teflon barrier. Testing PVP resulted in an increase of the surface pressure under compression, reversible under
4. Interaction of two surface-active components Using PVA in the subphase and spreading normal MGS on top we determined the surface pressure and the surface potential under compression and decompression (Fig. 8). The surface pressure showed a characteristic increase up to 14.4 mN/m under compression, fully reversible under decompression. Following the surface potential we only saw fluctuations of some 10 mV. Continuous monitoring of both parameters plotted against time demonstrated repeatable behaviour of the surface pressure and the surface potential (Fig. 9).
5. Combination of DPPC with a surface-active component Dipalmitoyl-phosphatidyl-choline is an amphiphile
Biophysics of MGS
173
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well known from biological membranes. DPPC shows a characteristic two-step increase of the surface potential under compression (Fig. 10). Because of its high potential peak we used it as an analogue for MGS. Using a PVA-solution as a subphase and spreading DPPC on top, the isotherm increased slightly (Fig. 11). The surface potential showed an initial peak of almost 100 mV, remaining constant afterwards. The terminal peak was only detectable in the presence of the Teflon barrier, which obviously exerted electrostatic effects. The characteristic potential of DPPC was changed in the presence of PVA. Using a PVP-solution as a subphase and spreading DPPC on top the surface pressure increased tremendously up to 43 mN/m (Fig. 12), at which point it collapsed. The surface potential showed a one-step increase by 150 inV. The terminal, inverse peak, again, was due to electrostatic effects. In conclusion, the characteristic potential of DPPC was changed in the presence of PVP.
6. Fluorescence spectra of MGS spread on water After spreading 100 txl MGS on a water surface we tested the fluorescence intensity in the presence of 1 N,N'dioctadecyloxacarbocyanine (Fig. 13). This
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dye molecule is attached to the inner interface of the surface layer. The fluorescence spectra were recorded at different stages of compression (0.3 - 5.0 - 10.0 15.0 mN/m, Fig. 14a-d), invariably showing a peak of fluorescence intensity near 500 nm. This peak was lowered from 30 A U to 13 AU under further compression. In other words: the higher the surface potential, the lower the fluorescence intensity.
Discussion
The biophysical data found in the literature usually refer to surface pressure monitoring only. Meanwhile there are reliable methods for the detection of electric phenomena at the surface of thin layers. The recording of surface potentials has become a routine procedure when monolayer assemblies are to be characterized. Two methods are available: the vibrating plate method used in our series of experiments and, alternatively, the ionizing-electrode-method. Recently, efforts were made to specify the surface potential of monolayers [7]. A monolayer has two interfaces: the surface of the hydrophobic end groups and the interface of the hydrophilic head groups towards the aqueous subphase. Both interfaces contribute to the interracial potential. The surface potential of the monolayer is defined as
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being the difference AV between the potential of the monolayer-covered surface and that of the clean water surface. The difference AV can be separated into two components: the component of the monolayer-air-interface may be assumed to be an absolute potential, whereas the second component represents the joint contributions of the electric double layer as well as the influence of the aqueous subphase on the monolayer. Recording the surface potential of MGS in persons with unaffected lids and conjunctivae showed that this material was characterized by a surprisingly high potential peak under compression. This peak was fully reversible under decompression. In synthetic lipids a potential peak of 200 mV or even more is seen occasionally (e.g. DPPC, Dipalmitoyl-phosphatidyl-ethanolamine, 7). Is MGS able to form a monolayer under certain conditions? The literature suggests that only multilayers are observed in vitro and in vivo [8]. From our thickness measurements on glass plates, which indicated surprisingly thin layers of 80 to 100 A, we inferred that at least on glass plates MGS is capable of forming monolayers. An estimate of the amount of material spread on the aqueous subphase and calculation of the surface pressure made clear that the thickness of the surface layer on water was comparable. Therefore we conclude that the formation of a MGS monolayer occurs not only on glass plates but also on water.
MGS is a highly surface-active mixture. By way of contrast, we demonstrated an instance of a surface-inactive component: Eledoisin. This substance is characterized by a lack of surface pressure. Other than that, there are practically no alterations of the surface potential. PVA and PVP are two instances of surface-active therapeutics for dry eyes. Indeed, both polymers are characterized by an increasing isotherm, but fail to undergo marked potential changes under compression. In sum, the present data clearly indicate that the characteristics of the surface potential of a surfaceactive component provide more detailed information than the surface pressure. Previous experiments showed that the surface potential of siccapatients had no identical potential courses under repeated compression and decompression, whereas the surface pressure remained unchanged [9]. If we administer artificial tears to the eyes of a patient suffering from keratoconjunctivitis sicca we are likely to observe an interaction between what is left of the patient's own tears and the artificial tears. Under these conditions, the interaction of two surface-active components does have clinical relevance. Artificial tears are found in the aqueous layer of the physiological tear film. Accordingly, in our experiments the surface-active component of the artificial tears was dissolved in the aqueous subphase. MGS was spread over the surface of such solutions. We used the combination of PVA and MGS, the former for the subphase, the latter for the surface layer. Despite the presence of MGS we could not detect the typical potential peak of 200 mV under compression. What are the possible explanations? PVA from the subphase may fill up holes in the MGS layer. This would explain why the typical potential peak of MGS is no longer detectable. Alternatively, the two surface-active compo-
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nents may be arranged at the water surface so as to form a multilayer. Due to the high concentration of PVA we only find the potential course typical of PVA with the typical MGS potential being vanished. The MGS is characterized by a high jump of the surface potential under compression. Some synthetic lipids show a similar course of the potential (e.g. DPPC, Dipalmitoyl-phosphatidyl-ethanolamine). Therefore they may act as a useful analogue for MGS. We tested DPPC, which is an amphiphile, in the presence of PVA and PVP. In both series of experiments we noticed that the typical potential peaks of DPPC were altered in combination with PVA or PVP. Only one of the two potential peaks of DPPC was recorded. Here again, the surface-active component of the aqueous subphase caused a characteristic potential change of the thin surface layer. DPPC proved to be an analogue for MGS with respect to the surface potential and to
the interaction with other surface-active components. Up to now we have studied the surface potential as a useful biophysical parameter without understanding the molecular basis of the potential change under compression and without knowing, where precisely these molecular alterations take place. Fluorescence measurements constitute a method for localizing the site of the potential changes. Cyanine dyes (e.g. 1 N,N'dioctadecyloxacarbocyanine) align with the inner surface of the monolayer. If we detect alterations of the characteristic fluorescence spectra of this cyanine dye under compression, we can take those as hints on a molecular alteration at the inner interface. In fact, a characteristic reduction of the fluorescence intensity at 500 nm was found under stepwise compression. Compression of the film entailed an increase in potential, which invariably goes along
176
T. Kaercher et al.
with a decrease in fluorescence intensity [10], According to the specific quality of the above cyanine dye the molecular change takes place at the monolayer-water-interface. An ideal tear substitute is not yet available. The present study established some new biophysical criteria for artificial tears. The surface potential was confirmed as the essential parameter. The interaction between artificial tears and MGS is important for therapy, and the behaviour at the lipidwater-interface is of critical relevance. Future studies should take into account these findings. The search for new therapeutic concepts needs a fresh impetus. It may be promising to take MGS of infants as a model. They are known to stare without blinking for long periods of time, hence their MGS must possess the excellent stability any designer of artificial tears would wish for.
Acknowledgements We express our thanks to Mr W. Zeiss for the expert technical assistance. We wish to thank Mr G. Overbeck and Mr M. Budach for their helpful discussion and assistance. The co-operation of the University Eye Hospital, G6ttingen, (Director: Prof Dr M. Vogel) is gratefully acknowledged.
References 1. Holly F, Letup M. Wettability and wetting of corneal epithelium. Exp Eye Res 1971; 11: 239-50. 2. Holly F. Surface chemistry of tear film component analogues. J Colloid Interface Sci 1974; 49: 221-31. 3. Jaeger W, M6bius D, Kaercher T. Biophysical experiments with lipid-layers formed with Meibomian gland secretion. In: Holly F (ed) The preocular tear film in health, disease and contact lens wear. Lubbock, 1986: 609-21. 4. Sondermann J. Darstellung oberflfichenaktiver Polymethincyanin-Farbstoffe mit langen N-Alkyl-Ketten. Liebigs Ann Chem 1971; 749: 183-97. 5. Kuhn H, M6bius D, Biicher H. Spectroscopy of monolayer assemblies. In: Weissberger A, Rossiter B (eds) Physical methods of chemistry. Wiley and Sons, 1972: 577-702. 6. Bertaccini G, de Caro G. The effect of Physalaemin and related polypeptides on salivary secretion. J Physiol 1965; 181: 68-81. 7. Vogel V, M6bius D. Local surface potentials and electric dipole moments of lipid monolayers: Contributions of the water-lipid and the lipid-air interfaces. J Colloid Interface Sci 1988; 126: 408-20. 8. Holly F. Surface chemical evaluation of artificial tears and their ingredients. Interaction with a superficial lipid layer. Contact and Intraocular Lens Med J 1978; 4: 52-65. 9. Kaercher T, M6bius D. Stabilitfit und Flexibilitfit der Lipidschicht des Trfinenfilms sowie deren pathologische Ver/~nderungen im biophysikalischen Experiment. Fortschr Ophthalmol 1989; 86: 245-8. 10. M6bius D, Cordroch W, Loschek R, Chi L, Dhathathreyan A, Vogel V. Control of interracial equilibria by local charge distribution and average surface potential. Thin Solid Films 1989; 178: 53-60.
