AT414211B - Determining functionality of surfactants, useful e.g. for diagnosis of respiratory disease when applied to pulmonary surfactant, comprises measuring electrokinetic properties - Google Patents

Abstract

Determining the quality and functional capacity of a surfactant (I) comprising measuring the electrokinetic potential (EP) or zeta potential (ZP) of a polymeric surface after adsorption of (I), is new.

Description

2

AT 414 211 B

The present invention relates to a method for determining the quality of pulmonary surfactant.

From WO 86/00707 a method for determining the zeta potential of solids 5 is known. Furthermore, publications are known in connection with the determination or measurement of electrokinetic properties or the zeta potential of different suspensions, wherein in each case an additive is used for changing the surface properties of a suspension to be measured (see, for example, http: //www.dwi.rwth- aachen.de/lb/87.html, "zeta potential measurements according to the flow potential method"; io http://www.colloidal-dynamics.com/ NewZPIntrorev.pdf, "Zeta Probe Applications").

The determination of the quality or ability to function of pulmonary surfactant is, for example, in connection with the treatment of so-called shock lung or the so-called respiratory distress syndrome of significant interest, since it is already before the onset of the full clinical picture of ARDS (Acute Respiratory Distress Syndrome, Respiratory distress syndrome) or ALi, it is possible to take therapeutic measures from the condition or condition of the pulmonary surfactant and thus to start therapy before the complete appearance of the clinical picture of the ARDS. ARDS / ALI or respiratory distress syndrome is the rapid onset of progressive pulmonary dysfunction and is essentially characterized by diffuse microvascular lung injury resulting in increased permeability of the small vessels and alveolar membrane leading to non-cardiogenic pulmonary edema. It is currently known, with the help of a so-called bronchoalveolar lavage (BAL) pulmonary surfactant to remove the lung of a patient, whereupon, for example, in a complex method for determining the surface tension, the quality or the state of the pulmonary surfactant is determined. These currently known methods are not only extremely time consuming and require a complicated apparatus, but also partly require a comparatively large amount of surfactant to be tested and, in particular due to their time complexity, the danger that therapy proposals are derived too late from such measurements. Therefore, these methods are not used in the routine.

The present invention therefore aims to provide a method for determining the quality or operability of surfactant, in particular pulmonary surfactant, with which a rapid and accurate determination of the state of the surfactant is mög-35 lent. In connection with the investigation of pulmonary surfactant therapy should be possible as early as possible in particular for the treatment of ARDS and / or ALi, for example by determining a dose of exogenous surfactant.

To solve these problems, the inventive method for determining the quality 40 and operability of Surfaktant essentially characterized in that the electrokinetic potential or zeta potential of a polymer surface after adsorption of pulmonary surfactant by a determination of the flow potential or flow, by a method chosen from electroosmosis, electrophoresis or microelectrophoresis is determined. Since pulmonary surfactant is capable of absorbing, for example, on polymer surfaces 45 and thus altering their surface properties, it is proposed according to the invention to determine the electron kinetics potential or, derived therefrom, the zeta potential of a polymer surface after adsorption of the surfactant, in order to draw conclusions regarding the condition or condition to enable the quality or functionality of a surfactant to be tested. It can thus be determined with a simple and short method so even with small amounts of available surfactant whose state or properties, so that, for example, in a change in the state of the pulmonary surfactant immediately appropriate measures or therapies, for example by administering an individual co-administered administration of exogenous surfactant to a patient or in a sample can be initiated. In addition to a rapid determination 55 of the state or the quality of Surfaktant resulting from the invention proposed 3

AT 414 211 B gene method of determining the electrokinetic potential or the zeta potential and the ability to fine-tune an optionally required surfaktantgabe so that a cost optimization when using exogenous surfactant is possible.

According to a preferred embodiment of the present invention, it is proposed that natural, synthetic and / or recombinant surfactant be used as the pulmonary surfactant. In addition to the abovementioned possibilities for determining the quality or functionality of natural surfactant, in particular for supporting therapies, for example for the treatment of ARDS and / or ALI, it is also possible with the method according to the invention to synthesize synthetic and / or recombinant pulmonary surfactant To check its functionality and quality to what extent, for example, newly developed Surfaktants are actually suitable for certain, predetermined uses. Thus, it is not necessary to carry out expensive and complex investigation methods, but it is possible to directly check or determine the quality or usefulness of, for example, a newly developed synthetic surfactant by determining the electrokinetic potential or zeta potential. Furthermore, it is possible, for example, after determination or determination of calibration or calibration curves or measured values for the electrokinetic potential or zeta potential of healthy, pulmonary surfactant, to find a way to check that more natural, synthetic and / or or recombinant pulmonary surfactant is compared with such calibrated readings and that a relative quality or functionality is derived therefrom, whereas possibly more difficult to determine absolute values for this are not absolutely necessary. The repetition of these measurements can also be used to check the therapeutic success.

According to a particularly preferred procedure, it is proposed that the electrokinetic potential be determined. Depending on the application or intended use, the electrokinetic potential or, derived therefrom, the zeta potential of the pulmonary surfactant can thus be determined in different ways when adsorbed on a polymer surface.

In this connection, it is proposed according to a particularly preferred embodiment that the surfactant is introduced into a measuring cell equipped with a macroscopic polymer surface and with two electrodes and the flow potential is determined, thereby making it possible with a simple device reliable results to determine the quality or functional ability of Surfaktant to obtain. The flow potential of a liquid, for example containing the surfactant, with respect to a measuring cell equipped with a polymer surface is determined hiebei by applying or applying different pressures on the liquid with simultaneous determination of the potential or the current at the electrodes of the measuring cell. To further improve or simplify the process management, the liquid volume received in the measuring cell can be subjected to an oscillating or periodically changing pressurization.

To achieve a correspondingly meaningful result concerning the quality or functionality of surfactant, in particular pulmonary surfactant, it is proposed according to a further preferred embodiment of the method according to the invention that the macroscopic polymer surface of fibers, films or membranes, in particular of cellulose acetate or cellulose nitrate, is formed , Such a macroscopic polymer surface has well-defined or definable properties, so that it is possible to draw conclusions about the state of the surfactant in knowledge of the electrokinetic potential or zeta potential, knowing the nature of the polymer surface.

To avoid a falsification of the result in the determination of the determination of the quality or functionality of natural, pulmonary surfactant is proposed according to a further preferred embodiment that natural, pulmonary surfactant vorgere- 4

AT 414 211 B nigt, in particular physically prepurified, wherein, for example, cells, cell components or the like, are removed. Such a pre-cleaning, in particular physical cleaning, is also simple and quick to carry out with known methods and enables a more reliable determination of the electrokinetic potential or zeta potential of the pulmonary surfactant seeking to -5 without possibly required, complex corrections.

To further facilitate the determination of the quality or operability of pulmonary surfactant is proposed according to a further preferred embodiment, that in the io measuring cell, a buffer, in particular a phosphate buffer having a pH of about 7, is used, which also requires such a buffering correction process can be avoided.

Instead of determining the flow potential for determining the zeta potential, it is also possible, in particular, to introduce diluted surfactant into a measuring cell which contains latex or pigment particles, wherein an electric field is generated in these measuring cells by two electrodes, which causes the migration of the latex - or pigmented article caused. In the case of the application of this method of so-called microelectrophoresis, the migration speed of the particles is determined and, in turn, the zeta potential derived therefrom in order to determine the quality or functional capability of, in particular, pulmonary surfactant. In this connection, it is proposed according to a particularly preferred embodiment that the surfactant is immobilized on latex or pigment particles and the migration or movement of the latex or pigment particles in the electric field of the measuring cell is determined. 25

The invention will be explained in more detail below with reference to an accompanying drawing of an apparatus for carrying out the method according to the invention and the results derived therefrom and on the basis of the attached exemplary embodiments. 1 shows a schematic view of an apparatus for carrying out the method according to the invention for determining the electrokinetic potential or zeta potential of, in particular, pulmonary surfactant;

FIG. 2 shows a diagram of the determined potential for a non-functional surfactant in comparison to the theoretical results for a functional, in particular pulmonary surfactant; FIG. and

3 shows a diagram of the determined zeta potential as a function of the pH for different adsorption materials.

In Fig. 1 is generally designated 1 a measuring cell, which consists for example of Plexiglas 40, wherein moreover two electrodes 2 and 3 are provided, which limit the volume of the measuring cell 1. Via a supply line 4, a supply of the liquid to be examined, containing the pulmonary surfactant to be tested, in the region of the measuring cell 1, wherein it can be seen that the lower electrode 3 shown in FIG. 1 via an eccentric 5 and a thus connected piston 6 is drivable to a reciprocating movement, whereby the volume in the measuring cell 1 is subjected to an oscillating or periodically changing pressurization.

In the piston 6, a pressure sensor can be integrated directly, as indicated by 7, or it can from the known movement of the eccentric 5 and thus the piston 6 in relation to the known volume of the measuring cell 1 of the prevailing in the measuring cell 1, itself periodically changing or oscillating pressure can be determined directly.

The pressure determined by the pressure sensor 7 is supplied via a schematic line 8 to an evaluation and recording unit 9, wherein this evaluation and recording unit 9 55 beyond at least the present at the electrodes 2 and 3 potential or the current fifth

AT 414 211 B is supplied via a line 10.

In the device shown in FIG. 1, for example, a potential which changes over time can be determined, from which the zeta potential can be directly derived, as shown in FIG. 2.

In Fig. 2, in addition to the results for a non-functional, in particular pulmonary surfactant also a curve of a theoretical calculation of the expected change in potential as a function of pH value is shown, wherein it can be seen that with the apparatus shown in Fig. 1 io rapidly and in a reliable way, a determination of the quality or the ability to function of particular pulmonary surfactant is possible because of the periodically changing potential directly the electrokinetic potential and the zeta potential of the pulmonary surfactant can be determined. The pulmonary surfactant which was subjected to the examination shown in FIG. 2 was precleaned, for example cells, cell components and the like having been removed in order to be able to largely rule out an influence on the measurement result. To simplify the evaluation, a buffer, for example a phosphate buffer, with a pH of about 7 is further used in the measuring cell 1. 20

To determine the electrokinetic potential or zeta potential of the particular pulmonary surfactant, the measuring cell 1 moreover has a macroscopic polymer surface which is formed, for example, from cellulose acetate or cellulose nitrate. 25 Instead of determining the electrokinetic potential or zeta potential of a Surfaktants using the device shown in Fig. 1, the potential can also be carried out while passing the Surfaktants to be examined at different pressure by a comparable with the measuring cell 1 measuring cell, again the potential or, where appropriate, the current at electrodes 2 and 3 is detected or determined, and from this the electrical kinetic potential and the zeta potential are derived.

In addition to such potential determination, the surfactant can be immobilized on latex or pigment particles, wherein the migration or movement of the latex or pigment article in an electric field, for example, again between two electrodes is determined in a measuring cell, wherein from the migration speed of the particles turn Determining the electrokinetic potential or zeta potential, the quality or operability of surfactant can be determined.

Embodiment 1 40

A patient is bled using a bronchoalveolar lavage (BAL) with a volume of 30 ml taken from the lungs using 50 ml of physiological saline followed by filtration through four layers of surgical gauze followed by centrifugation (5 min x 200 xg) for the removal of cells, cell components and 45.

The material obtained after the purification, which contains inter alia the pulmonary surfactant to be tested, is introduced into the measuring cell, whereupon in the apparatus according to FIG. 1, using a macroscopic polymer surface consisting of latex, a meso result is obtained, from which the zeta potential shown in Fig. 2 can be determined.

From the determined zeta potential and the difference from the calibration or calibration curve likewise shown in FIG. 2, it can be deduced that the functionality of the pulmonary surfactant tested is reduced by 70%. 55

Claims (8)

6 AT 414 211 B In view of this malfunction or deficiency of the pulmonary surfactant, the administration of exogenous surfactant is suggested for immediate treatment. In FIG. 3, similar to the diagram of FIG. 2, the zeta potential is shown as a function of the pH-5 value for different adsorption materials, from which, in particular when different surfactants are used, a pH corresponding to the zeta potential of the particular surfactant to be investigated is determined Value can be adjusted. Furthermore, it can be seen from FIG. 3 that when a cationic membrane is used, in each case a positive zeta potential in a wide pH range is obtainable, while when using an anionic membrane for different pH values, in part a positive and partly a negative zeta potential is obtained. Potential is determined, so that in knowledge of the change in the zeta potential of a surfactant to be examined in each case a corresponding membrane and a corresponding pH value is selected to obtain the most significant possible signal. 15 It can be assumed that, for example, pulmonary surfactant, as shown in FIG. 2, each has a negative zeta potential, while on the other surfactants exist which give a positive zeta potential, so that for determining the quality thereof, for example, a cationic Membrane to choose between a intact and inhibited surfactant according to different signals. 20 Patentansprüche 1. A method for determining the quality or operability of pulmonary surfactant, 25 characterized in that the electrokinetic potential or zeta potential of a Po lymeroberfläche after an adsorption of pulmonary surfactant by a determination of the flow potential or flow, by a Method chosen is determined from electro-osmosis, electrophoresis or microelectrophoresis. 2. The method according to claim 1, characterized in that is used as a pulmonary surfactant natural Licher, synthetic and / or recombinant surfactant. 3. The method according to claim 1 or 2, characterized in that the electrokinetic potential is determined. 35 4. The method of claim 1, 2 or 3, characterized in that the surfactant is placed in a equipped with a macroscopic polymer surface and two electrodes measuring cell and the flow potential is determined. 5. The method according to claim 4, characterized in that the macroscopic polymer surface of fibers, films or membranes, in particular of cellulose acetate or cellulose nitrate, is formed. 6. The method according to claim 5, characterized in that natural, pulmonary surfacant 45 pre-cleaned, in particular physically prepurified, being, for example, protein, hemoglobin, fibrin or the like., Are removed. 7. The method according to any one of claims 1 to 6, characterized in that in the measuring cell, a buffer, in particular a phosphate buffer having a pH of about 7, is used. 50 8. The method according to any one of claims 1 to 7, characterized in that the surfactant is immobilized on latex or pigment particles and the migration or movement of the latex or pigment particles in the electric field of the measuring cell is determined. 55 5 7 AT 414 211 B For 2 sheets Drawings 10 15 20 25 30 35 40 45 50 55