WO1994023280A1

WO1994023280A1 - Method for the measurement of the surface tension of biological fluids

Info

Publication number: WO1994023280A1
Application number: PCT/HU1994/000009
Authority: WO
Inventors: Domokos Boda
Original assignee: Formarket Vállalkozói Kereskedo^'ház Rt.
Priority date: 1993-04-07
Filing date: 1994-04-06
Publication date: 1994-10-13
Also published as: HUT71363A; AU6580094A; HU9301008D0

Abstract

A method for the measurement of the surface tension of biological fluids, especially for the determination of the maximum and minimum surface tension of biological fluids having aqueous phase, during which the maximum surface tension of the test fluid is measured, then the fluid is periodically moved, after which the minimum surface tension is measured. In the sense of the invention a capillary tube is inserted in the fluid tank containing the standard fluid having a known surface tension, the capillary is fixed at an angle of 0 < α « ∏/2 in relation to the plane of the surface of the standard fluid in the fluid tank, a scale is made on the basis of the known surface tension of the standard fluid in such a manner that the height identical with the fluid level formed in the capillary the value of the surface tension, while at the plane of the fluid surface in the fluid tank the 0 point of the scale are marked, after which the test fluid is poured over from the fluid tank into a capillary of the same volume, the capillary is set to the fluid level in the fluid tank at the α angle given above; the fluid is pulsated in the capillary and both before and after pulsation the values of the surface tension are read off the scale.

Description

Method for the Measurement of the Surface Tension of

Biological Fluids

Field of the Invention

The subject matter of the invention is a method for the measurement of the surface tension of biological fluids, mainly for the measurement of the maximum and minimum surface tension of the biological fluid with aqueous phase. During the application of the method the maximum surface tension of the test fluid is measured, then the fluid is periodically made to move and finally the minimum surface tension is measured.

Prior Art

As a result of the discoveries made during the past one or two decades it is known that the lungs produce certain phospholipids which cover their alveolar surface in a thin layer, reducing the surface tension of the fluid of aqueous phase being there to almost zero. The lack of these surfactant substances in premature infants or the insufficiency thereof in other pulmonary diseases of the adult age leads to grave respiratory disturbances. Therefore, the examination of the pulmonary surfactant has become an important task from diagnostic, disease follow-up and research points of view. A new phase had started in research when it was discovered that the pulmonary surfactant can be taken into the pulmonary airways and thereby the atelectasis disease can effectively be overcome. The verification of the surfactant's presence and its quantitative measurement have become important tasks. The surfactant is now examined in everyday practice to judge the maturity of the lung of the foetus and to prevent the respiratory problem of the child to be born. The methods developed so far for the examination of the pulmonary surfactant - an essential part of which is the measurement of the surface tension of the fluid - are of the physical and chemical nature.

The procedures applied in theoretical physics can be used as physical methods. Such a method is, for example, the Wilhelmy-balance. It is an expensive procedure requiring special professional knowledge.

According to another method used in biological research the shape of air bubble in the test fluid is measured, which, with the reduction of the surface tension, takes up an increasingly elongated disc-shape instead of a sphere's shape. (Schruch, S., Bachofen, H., Goerke, J., Green, F.: „Surface properties of rat pulmonary surfactant studied with the captive bubble method: adsorption, hypertensis, stability", Biochim, Biophys. Acta 1103. p.127-136. , 1992) A disadvantage of the method is that it is rather difficult to use it for precision measurements. There are several simpler solutions offered, such as the measuring of the speed at which fluids flow out of tubes, of the extent of the spreading of fluids on blotting paper, the estimation of the simple effect of capillarity, etc. None of these methods have proved to be usable in practice. However, the Clements bubble stability test is used relatively widely. In the case of this method the disappearing time of the bubbles floating on the surface of the shaken up fluid is measured, but this method can only be regarded as a test giving rough results.

In biological research, exclusively for precision measurements, the Enhorning pulsating bubble method is an internationally accepted reference method (Enhorning, G. _^Pulsating bubble technique for evoluating pulmonary surfactant" , J. Appl.Physiol. : Respirat. Environ. 43., p.198-203., 1977) which is based on the so-called Gibbs- Thomson principle, according to which in biological fluids - and only in these - the lipids finely dispersed in an aqueous medium are streaming towards the surface as a result of movement and thereby their surface tension reducing effect is tremendously increasing. The essence of the Enhorning method is that a plastic capillary is reaching into a fluid of controlled temperature. Through this capillary a small amount of air is sucked in and thus at the other end of the capillary reaching into the fluid a bubble is formed. The diameter of the bubble is measured using an optical method and by means of a sensitive manometer the suction is also measured. Using these measurement figures the maximum surface tension is calculated. Thereafter, the intensity of suction is changed in several cycles, which means that the bubble is made to pulsate. In the meantime, the measurement is repeated after 1, and then after 5 minutes. As a result of pulsation, in accordance with the Gibbs-Thomson law, the

* surface tension is reduced to a minimum value. The drawback of this method is that

' the determination of the bubble's dimension using an optical technique is difficult, a very sensitive manometer is required for the measurement of small changes in

5 pressure and that the test can be performed only under laboratory conditions and thus it is used only for research purposes. Its wide-spread use is also prevented by the fact that the method requires an expensive equipment.

Chemical techniques analyse the lipid components of biological fluids using classical methods, most often by means of thin-layer chromatography. The

10 fluorometric-polarization measurement which measures the viscosity of the fluid is also widely spread. (Blumenfeld, T.A.: _^Determination of fetal lung maturity by fluorescence polarizatio of amniotic fluid", Am.J.Obstet.Gynecol. 130., p.782-785, 1976) This method is an indirect one, because it does not measure the surface tension directly, but the measured values are in correlation with that. However, these

15 procedures are rather expensive, they need special devices and knowledge and the measurement take a long time.

In general, the above methods measure the surface tension in an indirect way and/or they are only more or less of the quantitative type. The direct method requires expensive equipment and devices and a very special knowledge and skill are needed

20 to perform it. In spite of this, the determination of the surface tension by means of the direct method may become all the more necessary, because under pathologic conditions substances suppressing the effect of the phospholipids reducing the surface tension may appear in the fluid covering the surface of the lungs, when, even despite a normal phospholipid content, the surface tension is abnormally high.

25 Based on observations it is also known that the free surface of a fluid behaves differently from that which could be expected on the basis of hydrostatic laws. For example, at the wall of the containing vessel fluids do not have a horizontal plain surface, but a curved one, and if one of the legs of a U tube is a small-diameter capillary tube, the level of e.g. water in this leg will be considerably higher than in

30 the other, larger-diameter leg. This characteristic is related to the surface tension of fluids and to the wetting effect between a fluid and a solid body being in contact with each other. It is also known that in a capillary tube the height of the fluid level can be calculated using the following formula:

h= — ^-cosϋ ,

where h - is the perpendicular height of fluid column in the capillary, as compared to the level of fluid in the fluid tank, γ - is the surface tension of the fluid, ρ - is the density of the fluid, g - is the constant of gravity, r - is the radius of the capillary tube, and t? - is the wetting or contact angle.

Summary of the Invention

In the elaboration of our invention it was our purpose, in addition to eliminating the drawbacks of the other well-known methods, to develop a method and a device that can be used easily in everyday, diagnostic medical practice, to ensure that the measurement can be performed quickly and without the need of special knowledge and to meet the criteria of the "methods that can be used at the bed side" , i.e. of performing the measurement immediately on the spot after taking the test substance.

During working out the details of the method incorporated in the invention it was found that the measurement of the fluid's surface tension can be performed in the simplest manner by using the capillary feature of fluids in such a way that the extent and measurement of the surface tension are traced back to the extent of the fluid's rising in the capillary tube. It was realized that if a scale division corresponding to the rising of a standard fluid, with a known surface tension, in the capillary is made and the capillary containing the test fluid is placed next to this scale in the same position, the value of the surface tension can directly be read off the scale. Furthermore, it was also found that in addition to the above measurement method the Gibbs-Thomson principle, which says that as a result of pulsation the surface tension of biological fluids is reduced to minimum, and which is typical of the behaviour of such fluids, could be realised in our method most simply in such a way that in the capillary the test fluid is pulsated by means of a pump for a pre-determined period of time and with a certain frequency.

Furthermore, it was also our recognition that the value of the surface tension can be read off a mm scale if the capillary containing the standard fluid used for making the scale is kept at such an a angle related to the plane of the fluid surface in the fluid tank that the length of the fluid column forming in the capillary be the value of the standard fluid's surface tension in the unit of mm. Accordingly, the angle or looked for will be as follows:

_α=sin-¹^^ ,

-^ stand

where α - is the angle between the longitudinal axis of the capillary and the fluid surface in the fluid tank,

and

¹βtaαd -^ιπ . --Ystand l S cmH i

It was found that the standard fluid most suitable for our purposes was distilled water which can be obtained very easily in everyday medical practice. Distilled water behaves as a perfect wetting fluid, thus COSU=l

and

2γ viz ^lviz~

Qvizffr

where

¹ cm

Furthermore it was found that the pulsation of the fluid can effectively be achieved by the periodic movement of the air cushion above the fluid surface in the capillary, using a device specially designed for this purpose.

The main task to be solved by a method for the measurement of the surface tension of biological fluids, for the determination of the maximum and minimum surface tension of mainly biological fluids with aqueous phase; during the procedure according to the method the maximum surface tension of the test fluid is measured, then the fluid is periodically moved and then the minimum surface tension is measured. In the sens of the invention that a capillary tube is placed in a fluid tank containing standard fluid having known surface tension, the capillary is fixed at an angle of 0 < a ≤ π/2 related to the plane of the standard fluid in the tank, a scale on the basis of the known surface tension of the standard fluid is made in such a manner that at the height corresponding to the fluid level in the capillary the value of the surface tension, while at the plane of the fluid level in the fluid tank the 0 point of the scale are indicated; thereafter the test fluid is poured from the fluid tank into a capillary of the same volume, and the capillary is set at the previously given angle α in relation to the fluid level in the fluid tank; in the capillary the fluid is pulsated and both before and after pulsation the surface tension values are read off the scale.

In a preferred embodiment according to the invention that the capillaries applied are disposable glass capillaries for single use.

In a further preferred embodiment according to the invention distilled water is used as standard fluid.

According to another favourable solution of the invention the longitudinal axis of the capillary is set at such an angle in relation to the plane of the fluid in the fluid tank that the length of the standard fluid column in the capillary will be identical, possibly in mm, with the measuring number of its fluid's surface tension.

It can also be a favourable solution that pulsation is carried out for a standard period of 5 minutes, using a frequency of 1/sec. It can be another feature of the method according to the invention that the fluid is pulsated by means of a pump.

It is also a preferred embodiment according to the invention that pulsation of the fluid is generated by periodically pressing the air cushion above the fluid in the capillary.

Description of the Preferred Embodiments

In the following our invention will be illustrated by examples of application.

Example 1.

The test liquid is an amniotic fluid and the purpose of the test is to determine the surfactant activity of the amniotic fluid; to be able to this the surface tension of the amniotic fluid must be measured. 50 μl of distilled water is measured into a fluid tank, e.i. a cuvette, as a standard fluid that can easily be obtained in everyday medical practice. A 100 mm long glass capillary having an inside diameter of 0.95 mm is placed, in the cuvette. The capillary is placed in the cuvette perpendicularly to the plane of the water surface, i.e. at an angle of or = 90° in relation to it. At the final level of the water creeping up in the capillary the known surface tension of distilled water is indicated; at 20 °C this value is 72 dyn/cm. In the test set-up given in this example the fluid level forming in the capillary can also be determined by calculation. Thus: h=l =30 , 9mm.

To the fluid surface in the cuvette point 0 of the scale is assigned, to the upper point of the fluid level the value

water =72 dyn cm

is attached, while the section between the above two points is linearly divided. Thereafter, 50 μl of test fluid is measured into another cuvette in which a glass capillary having the same inside diameter as that of the previous one is placed in such a manner that the capillary should have an angle of = 90° in relation to the surface of the test fluid. With the help of the scale division made in the case of the standard fluid - distilled water - the value of the surface tension is read, which also in this case is obtained in the measuring unit of the scale, i.e. in dyn/cm. This reading is the value of the maximum surface tension of the test fluid. Thereafter, the fluid in the capillary is periodically pulsated for 5 minutes with a frequency of 1/sec. The capillary is again placed next to the scale and now the value of the minimum surface tension of the test fluid can be read off.

Example 2.

The same procedure is used as in the previous example, but in this case the capillary containing the distilled water is held inclined at an angle of or in relation to the plane of the fluid being in the cuvette. Thus, according to the above,

and α =28 . 24 Holding the capillary at this or angle in relation to the plane of the water surface in the cuvette, the actual surface tension can be read off the mm scale. For the sake of simplicity slightly lower than normal density values at room temperature were disregarded in the above calculations and thus we calculated with the value

₉=1, 000^=1-2- m^J cm

which represents a negligible error in diagnostics.

Example 3.

Everything was done as in examples 1 and 2 in such a manner that the pulsation of the fluid in the capillary was generated in the following way. To the upper end of the capillary one end of a rubber tubing having a ball-like buffer tank was connected and the other end of this rubber tubing could be separated from the external atmosphere. With the help of this set-up the fluid can be pulsated in such a way that the air cushion trapped above the fluid surface in the capillary and the rubber tubing is periodically brought into movement. This is done by plugging the rubber tubingto eliminate the effect of the external atmosphere, then by pressing on the ball¬ like buffer tank, then releasing it, simultaneously opening the rubber tubing towards the atmosphere. This "pumping" of the air cushion above the fluid surface makes the fluid in the capillary to move along the longitudinal axis of the capillary. During the tests it is very important to maintain the inside radius of the capillaries at a constant value, because the or angle depends on the radius of the capillary and thus the error percentage of the capillaries used will result in a double percentage error of the scale. At the same time, care should be taken that the capillaries be clean and free of any grease. The advantage of the method according to the invention is that it makes the measurement of biological fluids possible in everyday diagnostic medical practice with the usage of simple means and in a directly readable manner. In this way the process meets the criteria of the "methods that can be used at the bed side" and does not require a special knowledge.

Claims

CLAEMS

1. A method for the measurement of the surface tension of biological fluids, especially for the determination of the maximum and minimum surface tension of biological fluids having aqueous phase, during which the maximum surface tension of the test fluid is measured, then the fluid is periodically moved, after which the minimum surface tension is measured, characterized in that a capillary tube is inserted in the fluid tank containing the standard fluid having a known surface tension, the capillary is fixed at an angle of 0 < or < π/2 in relation to the plane of the surface of the standard fluid in the fluid tank, a scale is made on the basis of the known surface tension of the standard fluid in such a manner that at the height identical with the fluid level formed in the capillary the value of the surface tension, while at the plane of the fluid surface in the fluid tank the 0 point of the scale are marked, after which the test fluid is poured over from the fluid tank into a capillary of the same volume, the capillary is set to the fluid level in the fluid tank at the or angle given above; the fluid is pulsated in the capillary and both before and after pulsation the values of the surface tension are read off the scale.

2. The method according to Claim 1, characterized in that the capillaries used are disposable glass capillaries for single use.

3. The method according to Claim 1 or 2, characterized in that distilled water is used as standard fluid.

4. The method according to any of Claims 1 - 3 characterized in that the longitudinal axis of the capillary is set at such an α angle in relation to the plane of the fluid in the fluid tank that the length of the standard fluid column in the capillary be identical, possibly in mm, with the measuring number of its fluid's surface tension.

5. The method according to any of Claims 1 - 4 characterized in that pulsation is performed for a standard period of 5 minutes, with a frequency of 1/sec.

6. The method according to any of Claims 1 - 5 characterized in that pulsation of the fluid is generated by means of a pump.

7. The method according to any of Claims 1 - 6 characterized in that the pulsation of the fluid is generated in such a manner that the air cushion above the fluid in the capillary is periodically pressed.