From the Universi ty Medical Clinic B , Rigshospilalet, Copenhagen, Denmark, (Chief: Professor Erik Warburg, M.D.)

iind f r o m the Department of Medicine, Duke Hospital, Duke Universi ty Medical Schooll), Durham, N . C., U . S . A . (Chairman: E . A . Stead Jr., M . D . ) .

A N E W DEVICE FOR MEASURING COLLOID OSMOTIC PRESSURE BY

Anders T y b j m g Hansen.')

Measurements of colloid osmotic pressure in biological fluids by incans of osmotic cells have formed the basis of fundamental investigations on circulatory problems (Starling, 1909) and on proteins (S. P. L. Sorensen, 1917). Nevertheless, the available methods suffer from certain drawbacks which have prevented their general use in clinico-physiologic studies. The main objections are that a measurement requires (1) too much time and ( 2 ) too large a sample. Furthermore, the maintenance of a rigid tempera- ture control and the preparation of uniform and suitable membranes give rise to much trouble. This is true more or less for both the direct method (as used by Siar l ing) and the compensation method (introduced by S. P. L. Senensen) although the latter methods has proved more profitable than the former.

EVALUATION OF FACTORS OF CONSEQUENCE IN THE USE OF COMPENSATION METHODS

The time required to accomplish a measucement of the colloid osmotic pressure by means of a compensation method is dependent on ( a ) the time to establish an equilibrium between the crystalloids on either side of the membrane and ( b ) the duration of the measuring procedure itself which cannot be carried out until the osmotic forces of the crystalloids have been eliminated.

The equlibrium between the crystalloids is obtained more rapidly the thinner the membrane. It is further enhanced if a small amount of reference liquid can be used because a uniform concentration is more quickly obtained in a narrow space of liquid. However, the same time-saving effect may

1) Fellow of The Rockefeller Foundation, 1950 to 1951. 2) Address : A. Tybjserg Hansen, M. D., Higshospitalet, Copenhagen, Denmark. Submitted for publication August 8, 1951.

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be secured even if a larger amount of reference liquid is used provided nieasures are taken to prevent motion of the layers of solution next to the membrane. If such requirements are fulfilled a state of equilibrium is poss- ible in spite of considerable differences of concentration throughout the larger part of the liquid compartment.

By reducing the thickness of the semipermeable membrane (without changing its permeability quantitatively) the time required for balancing the extrinsic pressure against the colloid osmotic pressure is reduced to the same degree as is the diffusion time mentioned above.

As the device that detects the movements of the solvent across the membrane actually registers volume changes, the area of the membrane is of importance too. However, mechanical objections and especially the antagonistic effect on the efforts to reduce the quantity of the sample renders i t inadvisable to cut down the adjustment time by increasing the area of the membrane.

From the above considerations it is readily seen that besides a reduction of the thickness of the membrane, the basis for any improvement of the compensation method is an increase in the sensitivity with which the movement of the liquid across the membrane is detected.

OWN APPLICATION OF THE COMPENSATION METHOD

In the methods hitherto used the motion of liquid across the membrane has been visualized by means of optical magnification.

With reference to the very substantial improvements that have been obtained switching from optical to electrical methods in other fields of pressure measurements, it seems appropriate to apply these favourable experiences also to colloid osmotic pressure measurements.

This has been attempted independently by Meehan e t al. (1950) and by the writer (1950). Meehan et al. use a strain gage system. Their method is not intended as a compensation method (but may possibly be used for such a type of measurement). The sample required amounts to several cc. (personal communication (Human) ). The osmometer comes to equilibrium in about 15 minutes. Its modulus of volume elasticity is rather low, approx- imately 5 X 106 dynes X cm-5.

The manometer applied by the writer is an electric condenser manometer described in detail elsewhere ( 1943,1949)- The modulus of volume elasticity is 4 x 109 dynes x cm-5. That means that a change of pressure of 1 cm water in the manometer causes a change of volume of about 0.00025 cubic mm. The other dynamic quantities : effective mass and damping forces, which are so important when rapid pressure variations are to be recorded, are of minor significance in connection with the much slower displacement of liquid that takes place through a semipermeable membrane, as in measure- ments of colloid osmotic pressure.

In the compensation type of osmometers the aim is to exert a pressure

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in the compartment containing the osmotic active phase so as to bring to a standstill the motion of liquid across the semipermeable membrane caused by the osmotic forces. When this equilibrium is established the counter- balancing pressure equals the osmotic pressure.

In the method here to be presented the function of the manometer is to indicate whether or not this state of equilibrium is obtained, i. e. it functions as the zero indicator so widely used in different types of electric measure- men ts.

In case of equilibrium the output of the electric aggregate does not change when the pressure chamber of the manometer is alternatingly closed off from, and connected with, the ambient air. When no equalization of osmotic and counterbalancing pressure has yet been reached the pressure in the chamber will rise or fall during the period of time in which the chamber is closed, indicating a displacement of liquid out of, or into, the cell con- taining the sample. It is thus indicated whether the extrinsic pressure should be lowered or raised in order to attain the state of equilibrium.

This manner of using the manometer eliminates the serious consequences of leakages which are never completely avoidable. A leakage will lower the sensitivity leading to a prolonged adjustment time and possibly to a wider range of standard deviation. However, the determinations approximate the correct value. This is not true if the manometer is used to measure osmotic pressure directly. In that case the pressure is determined as the (negative) pressure prevailing in the manometer after a course of time which is sufficient to secure a steady state when no extrinsic pressure is applied. However, this steady state results from an equilibrium between a flow out of the pressure chamber into the compartment containing the colloid phase and a flow into the chamber by way of existing leakages. The pressure in the chamber will, therefore, not equal the osmotic pressure, but always be lower than this pressure. Furthermore, as the leakage undoubtedly will vary from one measurement to another there are no ways to estimate the part played by the leakage in every single case.

DESCRIPTION OF THE ASSEMBLY The construction of the device by means of which the preliminary

experiments were undertaken appears diagrammatically in Figure 1. The water manometer on which the balancing pressure is read is not shown. A hollow cylinder (11) is supplied with an inner thread in one end so as to be tightly connected to the condenser manometer. A brass rod ( 10) supports a perforated nickel plate (9) on which the semipermeable membrane rests ( 8 ) . The membrane is kept in place by means of a disc ( 4 ) . Tightness is secured by means of an interposed rubber gasket ( 7 ) . The disc is supplied with a concentric hole in which the semipermeable membrane is exposed to the sample. The diameter of the hole is 5 mm. as is the bore of the cylinder. At ( 5 ) . is shown one of the two screws by which the disc is tightened against the membrane. The glass plate ( 6 ) , showing a little open-

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ing, forms a cover for the space where the sainple is to be kept. A Lucite plate ( 2 ) together with the disc ( 4 ) forms a dhamber. This chamber is connected to a water manometer by means of a channel drilled into the Lucite plate. ( 1 ) is one of the two hand screws by means of which the Lucite cover is held tightly against the disc (41, with a rubber gasket ( 3 ) heing

Fig. I . Sketch of the gadget which is t o be connected to the electric condenser mano- meter when used to measure colloid osmotic pressure. See explanation in text. - A diminished longitudinal .section of the manometer is inserted in the lower l e f t corner (relative size approxi~mately 118). A ) thread f o r connection w i th ( 1 1 ) . B ) pressure chamber. C ) flexible plate and front electrode. I)) rear electrode. E ) constructional details. F) three w a y stopcock to be operated when measuring colloid osmotic pressure.

interposed. The three way stopcock (12) makes it possible to change the reference liquid in the cylinder without dismounting the membrane.

The membrane is made by submerging a slide in a solution of collodion ( 5 % Mallinckrodt P. C.) diluted by equal quantities of ether and alcohol. The slide is taken out of the solution, held in a vertical position (one short end down), dried 1 to 2 minutes, submerged in distilled water and left there for at least 20 minutes. From then on it is never allowed to dry. The thickness of the membrane is 5 to 10 microns, measured prior to the submersion in distilled water. The membrane is impermeable for proteins, easy to reproduce and amazingly rugged when well supported. It may be used for a large number of measurements.

Figure 2 shows the assembly including electric aggregate (left), labora- tory stand with pressure flask, pumping balloon and the condenser mano- meter firmly clamped at one of the branches of the side cock. A t the right is the voltmeter on which the pressure variations are indicated. (Full deflection for 3 volts, resistance 20000 Ohms). As will be noted no thermo- static temperature control is needed.

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Fig. 2. Thc equipment assembled for use. (1) electric condenser manometer. (2) electric aggregate. ( 3 ) water manometer. ( 4 ) voltmeter.

USE OF THE APPARATUS

‘The determination of the colloid osmotic pressure takes place as follows: The manometer is prepared as usual, i. e. it is filled with distilled water by boiling. The cylinder (11) is filled with physiologic saline solution and connected to the manometer. The collodion membrane is mounted. The sample (less than 20 cubic mni. is required) is placed in the hole in the plate, and covered by the glass plate. The Lucite cover is then fastened. The manometer which has until now been kept in a vertical position is turned to a horizontal position so that hydrostatic corrections are made superfluous. By means of the balloon the pressure is raised to a level which is higher than the colloid osmotic pressure. The stopcock (F) is closed so that only a very slight movement of liquid (ultrafiltrate) will take place across the membrane into the manometer. After one minute’s time the cock is opened to the ambient air and the pressure lowered, and after closing the stopcock the deflection of the index of the voltmeter is observed. By repeating this procedure the pressure that just balances the colloid osmotic pressure is determined.

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The entire measurement may be accomplished within 3 minutes. Before a new sample is introduced the external compartment is rinsed with saline.

TESTING OF THE APPARATUS

The device has been tested in different ways. When distilled water is used as the reference liquid a sample of physio-

logic saline solution will not show any osmotic effect, indicating that the diffusion equilibrium of the crystalloids is obtained very rapidly. If a concentrated saline solution is used an osmotic difference is observed but it is eliminated within a short time.

Calculations from the determination of colloid osmotic pressure in samples of known concentrations of pure ox serum albumine ( 5 per cent and 2.5 per cent) give a molecular weight of about 67000. The results of colloid osmotic pressure determinalions on samples of human plasma are in accordance with data from the literature. The accuracy with which the determinations have been accomplished by means of the experimental outfit here described is about rt 0.5 cm of water.

Some of the results of the preliminary measurements are listed in Table 1.

Table 1. The three groups of determinations are from three subsequent days. The temperature

of the room was 25"-26" C. The samples consisted of solutions of pure ox serum albumine (Armour) 5 per cent or 2.5 per cent. The solvent was either distilled water or physiologic saline (denoted D. W. and P. S. respectively). Each figure represents the mean value of several observations using the same sample. When not specifically noted the subsequent samples are taken from the same protein solution and the same collodion membrane has been used.

5/12 50 17.5 - 18.5 - 17.6 - 18.6 - 18.3 - 17.8 - 17.9 (5 70; D. W.; 1 hour)

18.1 - 17.8 (5 %; P. S.; new membrane) 18.6 - 18.5 - 18.6 (5 %; P. S.)

5/13 50 18.1 - 18.2 - 18.4 ( ( 5 %; D. W.; new membrane)

18.5 (5 %; D. W.; 2 hours later)

18.6 - 19.0 (5 70; D. W.; 6 hours later)

9.3 - 9.8 (2.5 %; D. W.; made by diluting the 5 % solution)

9.6 (2.5 70; D. W.)

5/14 50 18.8 (5 %; D. W.; new membrane) 18.6 (5 %; D. W.)

REFERENCES Buchthal, F. st E. Warbug: Acta physiol. Scand. 5: 55, 1943. Hansen, Anders Tybjrerg : Pressure Measurement in the Human Organism. Dissertation,

Copenhagen, Denmark, Teknisk Forlag 1949. Hansen, Anders Tybjeerg: A New Method for Measuring Colloid Osmotic Pressure. Oral

Communication at the Autumn Meeting of The American Physiological Society. Columbus, Ohio, Sept. 1950. Summary in Am. J. Physiol. 163: No. 3, 1950.

Meehan, J. P., C. C. Hyman and M. Sosnow: Measurement of Colloid Osmotic Pressures with an Unbonded Strain Gage Transducer. Statham Laboratories Instrument Notes, No. 15, 1950.

Starling, E. H.: The Fluids of the Body. Chicago 1909, Keener and Co. pp. 177. Ssrensen, S. P. L.: Comptes-Rendus des Travaux du Laboratoire Carlsberg, 12: p. 262-

369, 1917.

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