Measurement of Surface Tension in Urines of 495 Out-Patients of a Private Office

Acknowledgement

I am deeply grateful to Professor Dr. med. Hans-Joachim Merker, Director of the Anatomical Institute of the Free University Berlin for his initiation of this work. It was his question why human urine was sometimes foamy that started this investigation.

1. Introduction

Several proposals for methods to investigate the changes in the surface tension of biological or body fluids have been made already, since it has been suspected that such changes might reflect a pathophysiological status of the respective organism. Data on systematic measurements of the surface tension of various physiological fluids have been published, but not yet for urine during various urological diseases.

Measurements of the surface tension of amniotic fluid were carried out clinically in conjunction with the respiratéory distress syndrome (6, 7, 9). other measurements of the surface tension were performed on bile, blood, cerebrospinal fluid, serum, lymph, saliva and tears.

ABSOLOM et al. (1) investigated in 1983, whether substrates with different surface tensions would induce a different degree of conformational change in adsorbed protein molecules, and whether these differences in the degree of change would be reflected by differences in the surface tension of the adsorbed layers. Their results were in good agreement with the relative hydrophobicity of the investigated proteins, as determined by other, independent methods.

MYSELS (10) carried out surface tension studies of bile salts dissolved in water with the purpose to show that, on the base of certain assumptions, the results of measurements of the surface tension of the solution may be translatable directly into the monomer activity and thus yield an indication for correlation.

It is well known, that at any border surface between air and a liquid intermolecular forces of attraction become effective with the tendency to minimize the surface of the liquid. From measurements of the surface tension in alveoli İt İS e. g. known that the layer of liquid on the alveolar wall has to contain substances reducing the surface tension. Subotances showing this property consist of molecules with strong mutual forces of attraction, but with low forces of attraction against the other molecules of the liquid. For this reason, such molecules accumulate at the surface of the liquid, reducing the surface tension. They are therefore also called "surface active substances" or "surfactants".

Some surface active substances have been successfully indentified chemically. The alveolar film of liquid e.g. contains a mixture of proteins and lipoids, with derivates of lecithin most likely determining the specific surface activity.

At the urothelium also, e.g. at least by means of a scanning electron microscope, a high membrane turnover can be demonstrated also (8). Accordingly the question arises, whether in various disorders, such as hyperuricemia, diabetes mellitus, chronical pyelonephritis, diathesis for calcium oxalate lithiasis, carcinomas of the urothelium etc. a change in the surface tension of the urine can be observed or not, as a differential test with respect to patients with e.g. Lumbar symptoms.

Several methods exist for measuring the interfacial or the surface tension, respectively. Physically the surface of a substance constitutes a special f©Um of the border surface, i.e. the surface forms the border surface between gaseous and liquid phases of substances, while a border surface represents the area between two condensed phases of substances.

The surface tension is a physically measurable tensorial force, the molecules in the border region of the condensed phase of a liquid are exposed to. While the actual tensorial force cannot be measured directly, the resulting surface tension can be determined.

Methods based on measurement of a force are e.g. the ring method, by which the force required to pull a ring immersed into the liquid with a wetted circumference of defined length through the border surface is measured, or the plate method, using principally a similar approach, but not requiring hydrostatic corrections (12).

In France, the stirrup method, employing a length of wire stretched horizontally in a frame, has found some use.

Pressure measuring methods observe the rise of a liquid in capillaries or determine the maximum pressure in gas bubbles.

Optical methods generally are based on optical measuring of a distance or of an angle on a drop of the liquid. Common procedures are the so-called "Pending drop method" or the "Sessile drop method" and similar approaches.

2. Materials and methods

A tensiometer Model к 10 (Kruess Corp., 2000 Hamburg, Fed. Rep. Germany) based on the "Ring-method" was used for measuring the surface tension of the urine.

2.1. The Ring Method

A platinum ring suspended horizontally is immersed into the liquid and subsequently lifted out of the liquid again. By means of this method, the force Keax required to pull the ring with a wetted circumference of length Lb through the border surface is measured.

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Fig.: The force acting on the ring as a function of the height of the film of liquid

The ring method was known in the past century already. Following the description of a "Ring-Tensiometer" by LECOMPTE DU NOÜY in 1919, the procedure became worldwide the preferred method. The principal advantages of the method are ะ

- simplicity of execution
- short measuring intervals
- high resolution of the measured values
- neglibility of the wetting angle
- availability of additional information from the shape of the "Film rupture characteristic".

All these facts are most likely the reason for the undiminished popularity and preferred use of the ring method.

A ring with geometrically precisely defined dimensions, made of Ptlr 20 alloy and suspended horizontally, is used as the measuring probe.

The selection of the platinum-iridium alloy mentioned is based on the following properties:

- excellent wettability
- chemical inertness and stability
- high melting point
- considerably mechanical strength

The wetted length Lb ist determined by the geometrical configuration of the ring, characterized by the mean diameter 2 R of the ring and by the diameter 2 r of the wire the ring is made of.

The following dimensions of the ring have generally been accepted ะ

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These dimensions represent compromises between the demands for:

- high measuring accuracy (R as large as possible, r as small as possible)
- convenient size for easy handling
- good mechanical stability of the ring
- minimal quantity of liquid sample required

(R as small as possible, г as large as possible)

The standard configuration of the ring as described requires the volume of the liquid sample to be at least 10 ml. Special rings offered by KRÜSS Corporation, Hamburg, Fed. Rep. of Germany, allow evaluation of samples with a volume as low as 0.2 ml with reduced accuracy, stretching the method that far is particularly valuable in medical applications, where frequently samples of body fluids are obtainable in very limited quantities only.

2.2. Details of the Measuring procedure

2.2.1. Border surface between a liquid and a gaseous phase

Preceding immersion, the force measuring system, including the horizontally suspended ring, is carefully tared.

The ring is subsequently immersed into the liquid to be investigated until completely wetted and subsequently lifted again until the force required to lift the ring has reached the maximum, respectively until rupture of the film of liquid occurs.

During the lifting procedure the surface tension acts along the wetted line on the ring, with the point of action migrating along the circumference of the wire forming the ring.

As illustrated in the figure, the resultant of the forces acting on the ring reaches a maximum for the tangents of the film of liquid on the circumference of the ring being perpendicular to the plane of the ring. This maximum is measured.

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Contrary to the widespread opinion, it is therefore not necessary to lift the ring until rupture of the film of liquid occurs, to determine the surface tension of the liquid. Except for the hydrostatic correction required, the wetting angle is also primarily without an influence of the result of the measuring procedure.

2.2.2. Border surface between two liquid phases

Except for somewhat more elaborate preliminaries, the procedure for determination of the tension of the border surface between two liquid phases is similar to the measurement of the surface tension of a liquid.

In the course of the first step, the force measuring system is tared carefully with the ring completely immersed into the phase with lower specific gravity.

Subsequently a new beaker is approximately half filled with the phase with the higher specific gravity, the ring cleaned and immersed into that phase. A sufficient volume of the phase with lower specific gravity is then very carefully placed on top of the first phase. Measuring of the tension of the border surface between the two phases is then performed as described before by measuring the maximum of the force observed during lifting the ring out of the phase with the higher specific gravity.

Taring of the force measuring system with the ring immersed into the phase with the lower specific gravity serves for compensation of the buoyancy of the ring and of the surface tension acting on the vertical supports for the ring.

2.2.3. Evaluation of the results of the Measurements

The results of the measurements are evaluated using the expression

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In addition to the resultant from the surface tension, the hydrostatic weight of the volume of liquid Vh below the ring lifted together with the ring is included in the result of the measurement. This additional force has to be eliminated by means of a correction factor.

To achieve the necessary stability, the ring also cannot comply with the theoretical requirement of R/r » ®

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Consequently, during each measuring procedure, not only one, but two maxima of the force resulting from the surface tension are actually occuring, with the tangent of the surface of the outer film of liquid to the circumference being first perpendicular to the plane of the ring and then the tangent of the inner film of liquid. This detail is important for measurements calling for utmost precision only however.

In a paper publihed in 1930, HARKINS and JORDAN presented an extensive table of values for F determined empirically. The two scientists had noted similar geometrical shapes of the films of liquid to be associated with similar correction factors. It proved to be possible to describe the geometrical shapes by the dimensionless expressions

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with V representing the volume of liquid lifted by capillary action.

The table covers the ranges for

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Parallel to the work of HARKINS and JORDAN, theoretical determination of the shapes of the inner and outer meniscus (FREUD/FREUD) confirmed the correctness of the table and the error limit of 0.25 % only. The table of correction factors remains actual to the present day.

Modern tensiometers feature linear compensation, being adjusted to have a correction factor of unity for water at a temperature of 20 degrees centigrade.

Correction of values for the border surface tension measured is advantageously performed using the equation by ZIDEMA and WALTERS (1941) standardized by the American Society for Testing and Materials (ASTM)ะ

where

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2.2.4« Re»arks concerning the measuring accuracy

The degree of accuracy ebtainable using the ring method is primarily limited by the correction factor F, since physical determination of the geometrical configuration of the ring and measurement of the force incurred during lifting of the ring can be carried out with a five digit accuracy at least. Absolute precision down to less than 0.01 nM/m cannot be achieved in practice, while the results are quite reproducible down to 0.01 nM/m.

2.2.5. Meaaureient on solutions containing tensides

Measurements on solutions containing surface active molecules, such as tensides, pose special problems. As the name implies, surface active molecules aggregate at the border surfaces. Due to their bipolar characteristics they practically form an interface between the borders of the phases, resulting in lowering of the tension of the border surface, resp. of the surface.

Very small amounts of tensides are already capably of causing considerable effects. When measuring surface tension of such solutions, the time required for the process of aggregation of the tenside molecules constitutes the main problem. While for high concentrations of tensides equilibrium is reached within fractions of a second, depending on the chemical structure of the tenside molecules hours may be required for low concentrations. Accordingsly the surface tension may be dependent on the "age" of a surface itself.

If now new surfaces are formed during the measurement of the surface tension, as this is the case during lifting of the ring, the age of the surface composed of "old" peripheral areas and the "young" areas of the collar directly below the ring is no longer precisely defined.

In the practice of measurement, such conditions may cause controversial results - each new measurement at the same sample yielding a different value, which is mostly lower than the preceding result, quasi indicating some sort of "drift". Numerous investigators then questioned the correct operation of their measuring set-up. In such cases it is important not to disturb the order of the molecules at the border surface by avoiding rupture of the film of liquid after transgressing the maximum of the force encountered during lifting of the ring.

To bypass the problems described, an arrangement featured by the digital tensiometer made by KRÜSS Corp. in Hamburg, is particularly helpful: Following an automatic stop of the lift exactly in the point the maximum force and a subsequent minute lowering of the ring, the maximum can be tested again within a few seconds without significant formation of new surface. This provision permits reliable determination of an eventual drift in the results.

Concludingly it may be mentioned, that surface films exposed to repeated expansion and compression, such as this is the case during lifting and lowering of the ring, may influence the measured value in a fashion which frequently cannot be reproduced reliably.

2.3. Procedure

The fresh middle portion of the discharge of urine, or urine obtained by means of catheterization of a total of 495 patients of a urological practice with different disorders of the urinary tract. All patients (190 female, 305 male) were examined by means of infusion-urography, sonography and/or cystoscopy. In addition to the usual cytological investigations, the following serum parameters were determined: Level of uric acid, Level of urea, Level of creatinine, Blood count, Levels of Ca, Na and к in the serum, Level of parathormone in the serum (for patients suffering from calcium-oxalate-lithiasis).

The values for the surface tension of the urine obtained were classified according to the following criteria:

- Age of the patient
- Sex of the patient
- pH of the urine
- Sediments observed in the urine (according to BROSIG: Zentrifugation 5' with 1500 rounds/minute. Magnification 10 high-powered fields (400x) were counted.
- quantity of bacteria in urine (culture on Urotube, Fa. Roche, Basel, Switzerland. Infection: 105 - IO7 counts)
- Urological disorders present
- Metabolic disorders present
- Missing urological disorders (Lumbar symptoms)
- Erythrocyte count in the urine
- Leucocyte count in the urine

The levels of uric acid, creatinine, Ca, Na and к and the others were determined in the urine.

Preceding each measurement, the measuring vessel was rinsed repeatedly with acetone and subsequently pulled through a flame.

Evaluation of the results was performed on a personal computer compatible with the AT series of computers made by IBM Corporation.

The results were entered into data acquisition forms designed for electronic data processing and initially acquired with the data bank system "d Base II +". Actual statistical evaluation was carried out using the program parcel "SPSS-PC +”. Variance analysis and single and multi factorial regression procedures were employed. Group differences were declared to be significant for p < 0.05.

Results

Simple computation of the mean value for all 495 cases yielded a mean surface tension of 44.27 nM/m (SD 4.50). For women (ท = 190) a mean value of 44.35 nM/m ( SD 5.04) and for men (ท ะ 305 ) a mean value of 44.22 nM/m ( SD 4.15) was determined. No difference specific to sex exists therefore.

When classified according to the age of the patients, the values measured showed no remarkable differences either. Fig. 1 offers a review of the mean surface tension measured with respect to the age and the sex of the patients.

Statistica ะ

Table 1:

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