INACTIVATION OF PULMONARY SURFACTANT BY LYSOPHOSPHATIDYLCHOLINE S. Alexandrov1, R. Todorov2, A. Jordanova3, Z. Lalchev4 and D. Exerowa2 Department of Medical Physics and Biophysics, Medical Faculty, Medical University of Sofia, 1431 Sofia, Bulgaria 2 Institute of Physical Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria 3 Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria 4 Department of Biochemistry, Biological Faculty, Sofia University “St. Kliment Ohridski”, 1164 Sofia, Bulgaria Correspondence to: Svobodan Alexandrov E-mail:
[email protected]
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ABSTRACT The aim of the study is to examine the influence of the lysophosphatidylcholine (lysoPC) to surface properties of INFASURF, a replacement lung surfactant, using the methods of thin foam films and Wilhelmy method. By the latter it is shown the change of surface tension (γ, mN/m) during time of the pure INFASURF and their mixture with lysoPC. Foam films are an efficient and useful model for the study of many surface phenomena especially for investigating the surface properties of pulmonary surfactant. Formation and stability of common black foam films (CBF) of pure INFASURF and of mixtures with various concentrations of lysoPC indicate the influence of low concentrations of lysoPC for the stability of CBF from INFASURF. It is proved that the curve W/C (where W is probability and C – concentration) is shifted to the lower concentrations. Keyword: INFASURF, lysophosphatidylcholine (lysoPC), pulmonary surfactant (PS), black foam films
Introduction Pulmonary surfactant (PS) is a multicomponent complex of lipids and proteins which covers the alveolar surface. The main functions of PS are reducing the collapsing force in the alveolus by reducing alveolar surface tension and conferring mechanical stability to the alveoli. Phospholipids (PL) comprise 80–90% of the surfactant lipids, with phosphatidylcholine (PC) being the most abundant PL (70– 85%), about half of which is dipalmitoylphosphatydilcholine (DPPC). DPPC is traditionally considered to be the major contributor to surfactant surface activity (6). The lack, deficiency or inactivation of PS is the cause of severe respiratory disorders, sometimes lethal, such as the Neonatal Respiratory Distress Syndrome (NRDS), or the pulmonary dysfunction associated with the Acute Respiratory Distress Syndrome (ARDS), a disease related with different processes as injury, inflammation or sepsis (5). The pathogenesis of ARDS is still not fully understood but surfactant inhibition, due to action of a variety of inhibitory substances (e.g., plasma proteins, lyso- and unsaturated phospholipids, free fatty acids, and etc.), is believed to be an XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY
operative cause (3). In inflammatory lung diseases, hydrolysis of surfactant PL by phospholipase generates lysophospholipids. Such degradation not only can deplete active surfactant lipids, but also releases products like lysophosphatidylcholines (lysoPC) and free fatty acids that are severe biophysical inhibitors of surfactant activity (2). If such compounds are present in a mixture with PS, their intrinsic surface active behavior can reduce the adsorption of surfactant constituents into the interface, or alter film properties during dynamic cycling, or both. Several techniques and models have been largely used to investigate interfacial behavior of PS in vitro (7). Among them are the most valuable model systems, which form the same air–water interface as in the alveoli, e.g. Langmuir monolayers, captive bubbles, etc. Black foam films (BFF), as an experimental model, are formed at air–water interface too and recently have been successfully involved also to the model investigations of PS (1, 4). These studies showed a direct correlation between the formation of stable BFF and the concentration of phospholipids. The technique is easy to handle and has the advantage of possibility to work at very low surfactant concentrations, high sensitivity; evaluation of film dynamics and the direct visualization of surface film architecture.
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In this paper we examine the effect of a lysoPC on pulmonary surfactant as studied by the black foam film method and Langmuir monolayers. The therapeutic lung surfactant preparation INFASURF is used to model pulmonary surfactant and effect of lysoPC concentration are studied.
wire probe attached to the microbalance sensor head. The dynamic characteristics of the monolayers were studied by measuring the values of surface tension during compression/decompression between 100% (γmax) and 20% (γmin) of the initial monolayer area. The speed of compression/decompression cycle was 140 mm/min.
Materials and methods
Results and Discussion
Materials The INFASURF (Calfactant - ONY Pharmaceuticals, Amherst, NY) is an extract of natural surfactant from calf lungs which includes lipids and hydrophobic surfactantassociated proteins B and C (SP-B and SP-C). Each milliliter of suspension (in 0.9% aqueous sodium chloride solution) contains 35 mg total phospholipids (including 26 mg PC of which 16 mg is disaturated phosphatidylcholine) and 0.65 mg proteins including 0.26 mg of SP-B. Investigated surfactant concentrations are calculated from the total phospholipid content in INFASURF. Lysophosphatidylcholine (>99%) purity was obtained from Avanti Polar Lipids (Alabaster, AL) and used without further purification. NaCl was a Merck product (Suprapur 99.99%). All solutions were prepared by ultra pure deionized water (specific conductivity of about 1 μS cm−1). Foam Films The experimental investigation of black foam films has been carried out by the microinterferometric method of Scheludko–Exerowa (1). A microscopic horizontal foam film of radius r ≈ 100 μm is formed in the middle of double concave drop in a glass tube and was observed by inverted light microscope. Stable common black films (CBF) are obtained at sufficiently high phospholipid concentrations C ≥ Ct. This case corresponds to 100% probability (W) for CBF formation. W is determined experimentally after 30 min waiting time for saturation of the adsorption layers of both biconcave drop surfaces. W is calculated for each surfactant concentration as the ratio between the number of the black films formed to the number of all formed films. Surface tension measurements
Foam Films The dependence of probability (W) for observation of a black film in the foam film on surfactant concentration (C) is most informative for the black foam film formation. Fig. 1 shows W(C) dependence referred to a black foam film obtained from INFASURF solution in the presence of 0.15 M NaCl at 30 min waiting times. As it is seen the W(C) curve is very steep and concentration Ct (concentration at which 100% black film is obtained) is clearly distinguished. The obtained value of Ct=75 μg/ml we used in further experiments with mixture of INFASURF and LysoPC. The dependence W(C) of lysoPC has been obtained at the same experimental conditions (Fig. 2, filled triangles). As seen, the slope of curves is steep, which is typical for phospholipid foam films. Besides, it is shifted to much lower concentrations compared to the dependence obtained for pure INFASURF. LysoPC formed stable black foam films at Ct=8 μg/ml. Below that concentration the probability of formation of black films is less than 100% and tend to 0% with the decrease of the amount of the lysolipid. We did not observed black foam films of pure lysoPC at 6 μg/ml after 30 minutes. The same figure (Fig. 2) shows also the curve (open triangles) obtained for mixtures of INFASURF with lysoPC added. The INFASURF concentration in the experiments was constant and equal to the sufficient concentration for stable black film formation (Ct). Addition of small amount of lysoPC (2 µg/ml) leads to black foam film destabilization (W=0%). The destabilization effect is observed between 2 and 8 µg/ml lysoPC. For concentrations of lysoPC above 8 µg/ml, stable black films were obtained probably due to formation of mixed adsorption layers. Surface tension measurements The study of surface tension of pure INFASURF (75 μg/ml) during the time at air/water interface is shown on Fig. 3 (red line). As seen, the surface tension decreases with the time. The comparison of the results for INFASURF with these for its mixtures with lysoPC (Fig. 3, dot line - 2 µg/ml and solid.
The surface activity of the INFASURF and their mixture with lysoPC was characterized by measuring the decrease of the surface tension γ (mN/m) with time. The γ/time dependence of the formed Langmuir monolayers was measured in teflon coated Kibron Microtrough -X (Kibron, Inc., Helsinki, Finland) with area of 236 cm2. The total volume was 40 ml. The probe with surfactants was dissolved in 0.15 M NaCl. The instrument uses the Wilhelmy method with platinum BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE SPECIAL EDITION/ON-LINE
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Fig. 1. The dependence of the probability for black foam film formation on phospholipid concentration of INFASURF. Experiments are performed at 0.15 M NaCl, T = 22ºC after 30 min.
Fig. 2. The dependence of the probability for black foam film formation from lysoPC (filled triangles) and its mixtures with INFASURF (open triangles) on concentration of lysoPC. Experiments are performed at 0.15 M NaCl, T = 22ºC after 30 min
line- 8 µg/ml) evidences the influence of lysoPC on monolayer formation It shows that low concentraions of lysoPC (2 µg/ml) significantly inhibits INFASURF adsorption. That is the same concentration at which we
observed destabilization of foam films. When 8 µg/ml lysoPC was added, no significant increase in γ was seen. These results suggest formation of mixed monolayer from INFASURF components and lysoPC.
Fig. 3. Surface tension (γ) as a function of time for adsorption of INFASURF Fig. 4. Surface tension-area isotherms of adsorbed monolayers from (75 µg/ml) and for its mixture with lysoPC. Experiments are performed at INFASURF (75 µg/ml) and its mixture with lysoPC. Representative curves of the 3rd cycles of compression/decompression between 100% and 20% of the 0.15 M NaCl, T = 22ºC. initial area are shown. Experiments are performed at 0.15MNaCl,T=22ºC.
The dependence of minimal surface tension (γmin) on compression/decompression 3rd cycle for monolayers of INFASURF and its mixtures with lysoPC on air/water interface is shown on Fig.4. When lysoPC was added to XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY
INFASURF, significant decrease in γmin was seen even at the low concentration (2 μg/ml). Accordingly, these results indicate that INFASURF and lysoPC form at least partially miscible films in which the components interact to affect
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surface pressure-area behavior. In conclusion, lysoPC was found to change surface activity in a content-dependent pattern. Prominent in the actions of lysoPC was its ability to penetrate and interact in interfacial films with surfactant phospholipids, interfering with surface tension lowering during dynamic compression. The results show the applicability of the black foam film model for investigations of lysophospholipids inhibitory effect on pulmonary surfactant as well as the role of lysophospholipids on physiological activity of therapeutic surfactants due to alteration in their composition
Acknowledgements
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This work was supported by Bulgarian Ministry of Education and Science, project N NT-1-02/04, and partially D002-107/08
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Exerowa D., Kruglyakov P.M. (1998) Foam and foam films (D. Mobius, R. Miller Eds) Elsevier, Amsterdam. Hite R.D., Seeds M.C., Jacinto R.B., Grier B.L., Waite B.M., Bass D.A. (2005) Biochim. Biophys. Acta, 1720, 14-21. Holm B.A., Wang Z., Notter R.H. (1999) Pediatric Research., 46, 85-93. Lalchev Z., Todorov R., Exerowa D. (2008) Curr. Opin. Colloid Interface Sci., 13, 183-193. Notter R.H.. (2000) Lung surfactants. Basic science and clinical applications, Marcel Dekker, New York. Possmayer F. (2004) Physiochemical aspects of pulmonary surfactant In: Fetal and Neonatal Physiology (R.A. Polin, W.W. Fox, S.H. Abman, Eds.) W. B. Saunders, Philadelphia, p. 1014–1034. Wustneck R., Perez-Gil J., Wustneck N., Cruz A., Fainerman V.B., Pison U. (2005) Adv. Colloid Interface Sci., 117, 33–58.
XI ANNIVERSARY SCIENTIFIC CONFERENCE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 45 YEARS FACULTY OF BIOLOGY
