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Foam Bilayers from Amniotic Fluid: Formation and Phase State A. Nikolova and D. Exerowa* Institute of Physical Chemistry, Bulgarian Academy of Sciences, Sofia 1040, Bulgaria Received May 12, 1995. In Final Form: December 11, 1995X The temperature effect on the threshold dilution (e.g., threshold concentration) for formation of foam bilayers (Newton black films) from amniotic fluid is investigated. Samples of amniotic fluid are collected during the 39th gestation week. Microscopic foam films are formed from diluted amniotic fluid and studied interferometrically by the method of Scheludko and Exerowa within the temperature range 10-30 °C. The threshold dilution is determined from the experimental dependence of the probability for observation of foam bilayer on the dilution for each sample at different temperatures. It is shown that the threshold dilution is inversely proportional to the threshold concentration for formation of foam bilayer. This parameter is crucial for assessment of the risk for respiratory distress syndrome in newborns. The binding energy Q of a lipid molecule in the foam bilayer is calculated from the Arrhenius dependence of the threshold dilution applying the hole-nucleation theory of bilayer stability. The so-obtained values of Q for the various samples are very close to each other and vary from 5.3 × 10-20 to 9.4 × 10-20 J (about 13-22 kT), which indicates that in all cases the foam bilayers are in the liquid-crystalline state. Comparison is made with the phase states of dipalmitoylphosphatidylcholine foam bilayers.
Introduction Recently a new approach was introduced for clarification of amphiphile bilayer formation and stability. A holenucleation theory which explains the amphiphile bilayer stability and permeability on the basis of short-range molecular interactions was developed.1-3 This theory was confirmed experimentally for foam4-6 and emulsion bilayers.7 The parameters (for example the critical concentration Cc for formation of foam bilayer) and relations set up in the theory are convenient for investigation of the formation and phase state of foam bilayers from various synthetic and natural surfactants. They present a method of obtaining some molecular characteristics of foam bilayers such as the binding energy of a phospholipid molecule in the bilayer and the specific edge free energy of the nucleus hole for different phase states of the bilayers. This new approach was applied successfully for studying foam bilayers from amniotic fluid (AF), as well as from some of its components8 in relation to the physicochemical clinical method for diagnostics of lung maturity of newborns, proposed by us.9 This method includes the detection of the threshold bulk concentration of phosphatidylcholines necessary for foam bilayer formation. This threshold concentration is determined from the experimental dependence of the probability for observation of foam bilayer from AF on the concentration.9 Numerous methods for prediction of lung maturity by examination of amniotic fluid10-16 and lung surfactant17,18 Abstract published in Advance ACS Abstracts, February 15, 1996. X
(1) Kashchiev, D.; Exerowa, D. J. Colloid Interface Sci. 1980, 77, 501. (2) Exerowa, D.; Kashchiev, D. Contemp. Phys. 1986, 27, 429. (3) Kashchiev, D.; Exerowa, D. Biochim. Biophys. Acta 1983, 732, 133. (4) Exerowa, D.; Balinov, B.; Kashchiev, D. J. Colloid Interface Sci. 1983, 94, 45. (5) Nikolova, A.; Kashchiev, D.; Exerowa, D. Colloids Surf. 1989, 36, 339. (6) Exerowa, D.; Kashchiev, D.; Platikanov, D. Adv. Colloid Interface Sci. 1992, 40, 201. (7) Muller, H. J.; Balinov, B.; Exerowa, D. Colloid Polym. Sci. 1988, 266, 921. (8) Exerowa, D.; Lalchev, Z.; Kashchiev, D. Colloids Surf. 1984, 10, 113. (9) Exerowa, D.; Lalchev, Z.; Marinov, B.; Ognyanov, K. Langmuir 1986, 2, 664.
have been developed. The air/solution interface is essential in the methods related to monolayer,17 bubble,18 and foam.11 Each of these methods exploits a certain surface parameter for determination of the properties of amniotic fluid. The microscopic foam film is convenient for investigation of microheterogeneous systems. It enables the formation of foam bilayers at very low amphiphile concentrations and the investigation of fluctuation phenomena. The physical parameters related to formation and stability of microscopic foam bilayers are very sensitive to amphiphile concentration6 which makes these bilayers very useful for assessment of fetal lung maturity.9 The microscopic foam bilayer proved to be an appropriate model for investigation of alveolar surface and alveolar stability also.19 This approach is in agreement with the findings of Scarpelli that the lung surfactant takes the form of intraalveolar bubbles with formation of foam films at birth.20,21 As is well-known22,23 the natural lipid-protein mixtures (such as amniotic fluid) can undergo different phase transitions due to variation in temperature or composition. Of special importance for the natural bilayer lipid membranes is the so-called main phase transition between the liquid crystalline and gel state at which a melting of the hydrocarbon tails of lipid molecules occurs. For example, existence of an upper limit of the gel phase (10) Gluck, L.; Kulovich, M.; Borer, R.; Brenner, P.; Anderson, G.; Spellacy, N. Am. J. Obstet. Gynecol. 1971, 109, 440. (11) Clements, J. A.; Platzker, G. G.; Tierney, D. F.; Hobel, C. J.; Creasy, R. K.; Margolis, A. J. M.; Thiebault, D. W.; Tooley, W. H. N. Engl. J. Med. 1972, 286, 1077. (12) Tiwary, C. M.; Goldkrand, J. M. Obstet. Gynecol. 1976, 48, 191. (13) Goldkrand, J. W.; Varki, A.; McClurg, S. E. Am. J. Obstet. Gynecol. 1977, 128, 591. (14) Golde, S.; Mosley, G. Am. J. Obstet. Gynecol. 1980, 136, 222. (15) Aberg, A.; Gislen, L. Am. J. Obstet. Gynecol. 1986, 154, 68. (16) Garite, T. J. Clin. Obstet. Gynecol. 1987, 30, 985. (17) Schurch, S.; Goerke, J.; Clements, J. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 4698. (18) Enhorning, G. J. Appl. Physiol. 1977, 43, 198. (19) Exerowa, D.; Lalchev, Z. Langmuir 1986, 2, 668. (20) Scarpelli, E. M. Pediat. Res. 1978, 12, 1070. (21) Scarpelli, E. M. Surfactant and the lining of the lung; The Johns Hopkins University Press: Baltimore, MD, 1988. (22) Jones, M. N. Stud. Mod. Thermodyn. 1979, 1, 185. (23) Yeagle, P. The membranes of cells; Academic Press: New York, 1987.
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content in membranes has been demonstrated22 above which the membrane morphology and permeability change dramatically, which makes the execution of physiological functions of membranes impossible. The aim of the present work is to find regularities for the parameters related to formation of foam bilayers from mature amniotic fluid and to study the phase state of these bilayers. For that purpose we employed the microinterferometric method for investigation of foam films developed by Scheludko and Exerowa,24 applied in its contemporary variant.6 An appropriate experimental parameter was usedsthe threshold dilution of AF (e.g., the maximum dilution of amniotic fluid at which formation of foam bilayer is observed). The application of the holenucleation theory for stability of bilayers allowed us to determine the phase state of foam bilayers from AF and the value of the binding energy of a lipid molecule in the foam bilayer from the experimentally obtained temperature dependence of the threshold dilution.
Langmuir, Vol. 12, No. 7, 1996 1847 composition was not possible. For that reason we accepted as a main parameter of our study the threshold dilution dc,6 i.e., the maximum dilution of amniotic fluid samples at which foam bilayer was formed. The determination of d was carried out on the basis of the final state which the foam film reaches at its drainage. At high dilution d of AF (d > dc) rupture of the films at critical thickness was observed24,30 without formation of black spots and foam bilayer. At low dilution (d < dc) formation of black spots6 was observed which occupied all the film, turning it into foam bilayer (Newton black film). The electrolyte concentration of the solutions from which the foam bilayers are formed is very important when physicochemical characteristics of foam bilayers are determined. That is why we regulated the electrolyte concentration for each solution by addition of NaCl. The calculations were carried out by assuming the case of a 1-1 valent electrolyte. The electrolyte concentration of each sample was calculated from the data for specific conductivity, obtained by measuring the specific resistance. The addition of NaCl in the solutions used ensured the necessary electrolyte concentration at all dilutions studied. The foam bilayers were examined within the temperature range 10-30 °C, which guaranteed a precise determination of dc.
Materials and Methods Materials. The amniotic fluid samples were provided from the Institute of Obstetrics and Gynecology of the Medical Academy in Sofia (Bulgaria). Five samples of about 10 mL of amniotic fluid (39 gestation week, normal pregnancy) were taken by using amniocentesis and amnioscopy and were centrifuged for elimination of cell fragments.8 The chemical composition of amniotic fluid has been studied by numerous authors in detail, e.g., refs 25 and 26. Saturated and unsaturated phosphatidylcholines (about 80% of the total lipid content) and mostly dipalmitoylphosphatidylcholine predominate in amniotic fluid. The other important components are phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, cholesterol, and some proteins (albumin, transferrin, immunoglobulin, etc.). Doubly distilled water was used for the dilution of amniotic fluid. The solutions were obtained by addition of electrolyte (NaCl, Merck, supra pure), which was heated prior to use at 500 °C for removal of surface active impurities. The electrolyte concentrations were chosen8 to ensure the formation of black foam films of minimal thickness (about 8 nm at capillary pressure 29 N m-1). These thinnest black foam films, called foam bilayers, have a constant thickness which is not altered by increase in electrolyte concentration. For them the short-range interactions have a decisive role. The solutions of AF used for foam bilayer formation contained also 47.5 vol % ethanol. It increases the solubility of phosphatidylcholines and the critical micelle concentration and ensures sufficiently high concentration of monomer amphiphile in the solution, necessary for formation of foam bilayers.8,27 The ethanol concentration chosen guarantees predominant adsorption of phospholipids at the air/solution interface with respect to the proteins present in the amniotic fluid.11 Experimental Equipment. The investigation of foam films from amniotic fluid was carried out by the microinterferometric method of Scheludko and Exerowa,24 largely applied for investigation of kinetic and thermodynamic properties of foam films from different surfactants. This method was applied in its latest variant, described elsewhere.6,28 The microscopic horizontal foam films of radius 0.1 mm were formed in a special measuring cell6,9,29 in the middle of a biconcave drop. The measuring cell was fixed in a thermostating device, attached to a microscope, which was provided with a special optical and electronic system for observation of foam films and for detection of the reflected light from the film.28 Experimental Procedure. Due to the small amount of amniotic fluid samples, a parallel chemical analysis of their (24) Sheludko, A. Adv. Colloid Interface Sci. 1967, 1, 391. (25) Freer, D.; Statland, B. Clin. Chem. 1981, 27, 1629. (26) Lalchev, Z. Thesis; Bulgarian Academy of Sciences, Sofia, 1984. (27) Exerowa, D.; Nikolova, A. Langmuir 1992, 8, 3102. (28) Exerowa, D.; Kolarov, T.; Khristov, K. Colloids Surf. 1987, 22, 171. (29) Exerowa, D.; Zacharieva, M.; Cohen, R.; Platikanov, D. Colloid Polym. Sci. 1979, 257, 1089.
Results The main results on formation of foam bilayers from AF were obtained by determination of the parameter threshold dilution dc. The value of dc for a certain sample was found from the dependence of the probability W of observation of foam bilayer on dilution d at different temperatures. Such a dependence is shown in Figure 1a for one of the samples (sample no. 1) at 15 °C. A sharp drop in the W value at dc is seen in the figure. In the same way totally 24 values of dc for the five AF samples at different temperatures were determined, the accuracy being about 5%. The equivalent dependence calculated with respect to the total concentration C of phosphatidylcholines in the AF solutions is shown in Figure 1b. This concentration is inversely proportional to dilution d
C ) Cs/d
(1)
where Cs is the total concentration of phosphatidylcholines in the sample of amniotic fluid before dilution. The value of Cs was determined for each sample from the linear dependence of the total phosphatidylcholine concentration of AF samples on dc at 25 °C (Cs ) 13.3dc,25°C) given in ref 26. The values of threshold dilution dc for different temperatures are presented in Figure 2 in Arrhenius coordinates for the five samples investigated. As seen, linear dependencies of similar slope were obtained within the temperature interval 10-30 °C. The temperature dependencies of dc were determined at concentrations higher than the critical electrolyte concentration Cel,cr for formation of foam bilayers from amniotic fluid, 0.0534 mol dm-3 NaCl.8 The experimental data for dc were determined at an electrolyte concentration corresponding to the electroconductivity of 0.07 mol dm-3 NaCl for samples 1, 3, and 5 and to 0.14 mol dm-3 NaCl for samples 2 and 4. The full square (sample 2) denotes the threshold dilution dc determined at electrolyte concentration corresponding to a conductivity of 0.07 mol dm-3 NaCl and the half-full square (sample 2) denotes a threshold dilution determined at electrolyte concentration corresponding to a conductivity 0.254 mol dm-3 NaCl. The data for sample 2 show that at electrolyte concentrations higher than Cel,cr the (30) Krugljakov, P.; Exerowa, D. Foam and foam films; Himija: Moskwa, 1990.
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Figure 2. Arrhenius dependence of the threshold dilution dc for AF foam bilayers (samples 1-5): symbols, experimental data; straight lines, theoretical dependencies according to eq 3.
minimum bulk concentration of amphiphile molecules at which thermodynamically stable foam bilayers are formed)
Ce ) C0 exp[-Q/2kT]
Figure 1. Probability W for observation of AF foam bilayer (sample 1 at 15 °C): (a) experimental dependence on the dilution d; (b) experimental dependence on the phosphatidylcholine concentration C.
threshold dilution dc does not change with increase in electrolyte concentration. Discussion The experimental curves W(d) and W(C), shown in Figure 1 are very informative with respect to formation of foam bilayers from amniotic fluid because these dependencies are very steep. This fact allows precise determination of dc and Cc. The clearly pronounced value of Cc practically coincides with the threshold concentration Ct for observation of foam bilayer with W ) 1, which was defined in the method for assessment of lung maturity of newborns.9 The phase state of foam bilayers from amniotic fluid was determined on the basis of experimental results for the temperature dependence of threshold dilution dc. For interpretation of these data we applied the holenucleation theory of Kashchiev and Exerowa1-3,6 for bilayer stability. This theory regards the foam bilayer as a two-dimensional ordered system in which the shortrange molecular interactions are of major importance. The theoretical considerations1-3,6 enable determination of the binding energy Q of an amphiphile molecule in the foam bilayer from the Arrhenius dependence of the equilibrium bulk concentration Ce of amphiphile molecules (i.e., the
(2)
where T is the absolute temperature, C0 is a reference concentration of amphiphile molecules, and k is the Boltzmann constant. If one accounts for the first neighbor interactions only, Q ) z + z00, where and 0 are respectively the energy of lateral and normal interaction between two neighboring amphiphile molecules in the foam bilayer; z and z0 are respectively the number of first neighbors in lateral and normal direction with respect to the bilayer surface. The systematic study of foam bilayers from phospholipids8,27,31,32 shows that they do not rupture spontaneously at any concentration allowing their formation. That is why in the case of phospholipid foam bilayers the dependence of their mean lifetime on the bulk amphiphile concentration cannot be measured in contrast to foam bilayers from common surfactants.4,5 This infinite stability of phospholipid foam bilayers is the cause for the steep W(d) and W(C) dependencies. In the case of foam bilayers from amniotic fluid, this high stability was confirmed by a very sensitive method, proposed by us6,33 consisting of R-particle irradiation of foam bilayers. As is wellknown,6,33 the R-particle irradiation substantially shortens the mean lifetime of foam bilayers. Our experiments showed that at all temperatures and dilutions studied (even at dc), the foam bilayers from AF did not rupture even at the highest intensity of irradiation applied, 700 µCi. This extreme stability of foam bilayers from AF allowed us to assume that Cc ) Ce and to use the data for the temperature dependence of threshold dilution (shown in Figure 2) for determination of Q for each sample of amniotic fluid. For that reason we used the following eq 3, which results from eqs 1 and 2 under the assumption that Cc ) Ce (31) Nikolova, A.; Exerowa, D.; Lalchev, Z.; Tsonev, L. Eur. Biophys. J. 1994, 23, 145. (32) Nikolova, A.; Exerowa, D. J. Stat. Phys. 1995, 78, 147. (33) Penev, I.; Exerowa, D.; Kashchiev, D. Colloids Surf. 1987, 25, 67.
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ln dc ) K + Q/2kT
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(3)
where K ) ln(Cs/C0). The above mentioned threshold dilution dc and critical concentration for formation of foam bilayer Cc are measures for the bilayer stability,6 which is determined by the first neighbor lateral and normal interactions in the foam bilayer. This is the difference of the parameter dc from the change in the free surface energy which is usually used as a measure of the surface activity. Thus the parameters dc and Cc are proposed as new characteristics of the surface activity of an amphiphile molecule, which are determined with high accuracy from the sharp W(d) and W(C) dependencies respectively. The results for the binding energy Q of an amphiphile molecule in AF foam bilayer, obtained from the slopes of the straight lines in Figure 2 by using eq 3, are shown in Table 1 for the five AF samples studied. One should bear in mind that these values of Q are effective ones as far as they hold for foam bilayers from a complex natural mixture. These values are physically relevant (in all cases Q > 8kT) and in agreement with the hole nucleation theory provided that the condensed state of the monolayers composing the foam bilayer is accepted, which as is wellknown is a necessary condition for foam bilayer formation.34 It is seen from Table 1 that the Q-values vary from 5.3 × 10-20 to 9.4 × 10-20 J, which is explicable if one recognizes the different lipid-protein composition of AF samples. The mean value of Q is 7.6 × 10-20 J (about 19 kT at 20 °C), the standard deviation being 1.8 × 10-20 J. As is well-known,10,25 phosphatidylcholines are of the highest content in AF, the main component being dipalmitoylphosphatidylcholine (DPPC). That is why it is important to compare the data for the temperature dependence of the critical concentration Cc for formation of foam bilayer from AF and from DPPC.32 These data are shown in Figure 3 by full circles and squares, respectively. The Cc data for AF foam bilayers are calculated from the data of the temperature dependence of dc by using eq 1. The total concentration Cs of phosphatidylcholines in the AF samples before dilution was determined from the linear dependence26 of Cs on dc at 25 °C. The full circles in Figure 3 present the mean value of Cc for the five AF samples at the corresponding temperatures and the standard deviation is marked also. The Q value for AF foam bilayers is determined from the slope of Arrhenius dependence by using eq 2. This value coincides precisely with the mean Q value cited above (7.6 × 10-20 J) determined from the ln dc vs (1/T) dependencies. It is seen from Figure 3 that the slope of the linear dependence ln Cc vs (1/T) for the liquid crystalline DPPC foam bilayers (curve 1) is very close to that for AF foam bilayers (curve 3), which corresponds to similar Q values for AF and liquid crystalline DPPC foam bilayers. The Q value for AF foam bilayers is also very close to the Q values for the liquid crystalline sodium dodecyl sulfate5 and dimyristoylphosphatidylcholine27,31 foam bilayers. Hence, the interaction between lipid molecules is very similar in these foam bilayers and we can suppose that the AF foam bilayers are in the liquid crystalline state within the temperature range studied. This assumption is in agreement with the fact that amniotic fluid contains a substantial amount of unsaturated phospholipids, which, as known,35 lower considerably the temperature of the chain-melting phase transition. Bearing in mind the similarity of the phase behavior of phosphatidylcholine (34) Exerowa, D.; Cohen, R.; Nikolova, A. Colloids Surf. 1987, 24, 43. (35) Lee, A. Biochim. Biophys. Acta 1977, 472, 285.
Figure 3. Arrhenius dependence of the critical concentration Cc for formation of AF foam bilayer (Cc in µg cm-3): 9, experimental data for DPPC foam bilayers; b, experimental data for AF foam bilayers; O, Cc value for AF foam bilayers at 37 °C, calculated by extrapolation according to eq 2 under assumption Cc ) Ce; curves, best fit of eq 2 to experimental data under assumption Cc ) Ce (curve 1, DPPC liquid crystalline foam bilayers; curve 2, DPPC gel foam bilayers; curve 3, AF foam bilayers). Table 1. Values of the Binding Energy Q of an Amphiphile Molecule in Foam Bilayers from Amniotic Fluid (Samples 1-5), Obtained from the Best Fit of Equation 3 to the Data for Temperature Dependence of Threshold Dilution dc J Q× Q, kT (20 °C) 10-20,
1
2
3
4
5
9.4 23
7.2 18
5.3 13
9.4 23
6.9 17
aqueous dispersions and foam bilayers,27,31,32 it can be supposed that at the temperatures which are important for in vivo systems, the AF foam bilayers are in the liquid crystalline state. This assumption allows us to determine the critical concentration of phosphatidylcholines in amniotic fluid, which is necessary for formation of foam bilayer by extrapolation of the Arrhenius dependence of Cc for AF foam bilayers to 37 °C. From this extrapolation we obtain that at 37 °C Cc ) 19.9 µg cm-3 and dc ) 1.47. This value of Cc at 37 °C corresponds to the lower limit (found by independent methods, refs 25 and 36) of phosphatidylcholine concentration which permits us to define a sample of amniotic fluid as a mature one. The above value should be regarded as a tentative one as far as it is determined for foam bilayers obtained from a complex lipid-protein mixture and its accuracy is the one of determination of the threshold dilution for samples of both normal pregnancy and pathological cases. The present investigations expand the use of the method for assessment of fetal lung maturity with the aid of microscopic foam bilayers.9 It is important to make a clear distinction between this method9 and the foam test.11 The disperse system “foam” is not a simple sum of single foam films. A series of investigations30,37,38 shows the role (36) Amenta, J.; Silverman, J. Am. J. Clin. Pathol. 1983, 79, 52. (37) Bikerman, J. J. Foams; Springer-Verlag: New York, 1973. (38) Kruglyakov, P. M.; Exerowa, D.; Khristov, Khr. Adv. Colloid Interface Sci. 1992, 40, 257.
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of the different types of the foam films (thick, common black, and bilayer) on the formation and stability of foams. The difference between thick and bilayer foam films6,30 results from the transition from long- to short-range molecular interactions. The type of the foam film depends considerably also on the capillary pressure of the liquid phase of the foam.38,39 That is why the stability of a foam consisting of thick films and of a foam consisting of foam bilayers (Newton black films) is different and the physical parameters related to this stability are also different. Furthermore if the structure properties (e.g., syneresis, polydispersity) of the disperse system “foam” are accounted for, it becomes clear that the foam and the foam film are different physical objects and their stability is described by different physical parameters. The practical application of the method for assessment of lung maturity by determination of the threshold dilution has proved a number of advantages:9 high accuracy (about 95%), short operating time (about 30 min), and small amount of the amniotic fluid used (about 0.5 mL). The good correlation of the threshold dilution with the fetal lung maturity9 indicates that the single microscopic foam (39) Khristov, Khr.; Exerowa, D.; Kruglyakov, P. M. Colloid Polym. Sci. 1983, 261, 265.
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bilayer can be used successfully as an in vitro model for investigation of the alveolar surface and stability. Conclusion The present study enlarges the knowledge of the state of foam bilayers (Newton black films) from amniotic fluid at the molecular level. It is established that the phase state of these bilayers is liquid crystalline and the mean value of the binding energy Q of an amphiphile molecule in the bilayer is 7.6 × 10-20 J within the temperature range studied. The proposed parameters threshold dilution dc and critical concentration Cc for observation of foam bilayer are very informative. Their temperature dependence clarifies the formation and phase state of AF foam bilayers. Amniotic fluid is an essential natural lipidprotein mixture and knowledge about its behavior at the air/solution interface is very important. These results are valuable also in relation to the application of foam bilayers for assessment of lung maturity in clinical practice. Acknowledgment. We are indebted to the Bulgarian Ministry of Education, Science and Technologies for financial support. LA950372Z
