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Polarization and permeability (quantitative measurements)
POLARIZATION AND PERMEABILITY (QUANTITATIVE MEASUREMENTS) I M. Spiegel-Adolf and E. A. Spiegel From the Departments of Colloid Chemistry and Exp. Neurology, Temple University School of Medicine, Philadelphia, Pa. Received November 7, I9~5 INTRODUCTION
The measurement of the permeability of membranes is of interest to colloid chemists as well as biologists. If one wishes to ascertain the permeability of cellular membranes within the organs of animals, the usual chemical methods as a rule can hardly be applied, particularly when rapid changes of permeability are to be recorded. Due to their semipermeability to electrolytes, the cell membranes are the seat of electrical double layers. They show polarization phenomena, i.e., an electromotive force in opposite direction develops, when a direct current flows through the organ. If an alternating current passes through the organ this counterforce, and consequently the impedance measured, is smaller the higher the frequency of the A. C. Gildemeister (1) has pointed out that determination of the capacitance may serve as an indicator of the polarizability and thus indirectly of the permeability. The measurement of the polarization capacity requires complete balancing of the Wheatstone bridge, so that its zero arm is practically free of current. This is sometimes rather inconvenient, particularly when one deals with rapid changes due to experimental procedures. Therefore, we developed a method based upon another manifestation of polarization; viz., the fact that the difference between the conductivity at a certain high frequency (CA) and that at a certain low frequency (C~) is larger the more polarizable the membrane under study (2). From the 100(Ch -- Cz) . measurements of Ch and C~ the polarization index A -C~ is calculated (2). Applying such measurements to the brain, it was found that some epileptogenous agents diminish the density of the cell membranes, while hypnotics and anesthetics act in the opposite direction (3, 4). To analyze the basic chemical and colloid chemical conditions, experiments on artificial membranes have been performed (2, 5). In 1Aided b y a grant from the J o h n and Mary R. Mark]e Foundation. 21
22
M. SPIEGEL-ADOLF AND E. SPIEGEL
artificial membranes containing body constituents it could be shown that proteins may be substituted by other colloids, while the presence of lipids seems to be necessary for achieving polarizability. By perforating polarizable collodion or lecithin-collodion membranes repeatedly, a progressive reduction of A was produced (5). These findings pointed to a relationship between permeability and polarizability. However it seemed desirable to present a more direct evidence of such a relationship by a quantitative study. In the experiments to be reported here, frog's skins were used in order to obtain data from a natural membrane. Method: The experiments were performed on the skin of the trunk of winter frogs. On such isolated preparations, a series of alternating measurements of A and of the permeability for an electrolyte (NaC1) or for a non-electrolyte (urea) were made during the first stages of post mortem deterioration of the specimen. The skin was inserted flat between the ground flanges of the central and one external compartment of a Pauli electrodialysis apparatus, the original wire mesh P t electrodes of which had been coated eleetrolytically with platinum black. The surface inserted measured 452 mm.~. Between the central and the other external compartment no membrane was inserted, so that the apparatus was changed into a system consisting of 2 cells only. For the determination of A, the apparatus was filled with 0.1 N NaCI solution on either side of the skin. The bridge arrangement was the same as previously described (3, 4). Frequencies of 547 and 5120 cycles respectively were used. The measurements were made at room temperature. For the dialysis experiments the 0.1 N NaC1 solution in the apparatus was replaced in the compartment facing the inner side of the skin by a 5.4% dextrose solution, and in the compartment facing the external surface of the skin, by a N NaC1 solution. Each dialysis lasted 50 minutes and was performed at room temperature. This time interval was chosen because it permitted one to make from 5-6 dialysis experiments on one specimen within 1-2 days. After each dialysis experiment both the NaC1 and the dextrose solution were replaced by fresh solutions. Since the maximum amount dialyzed did not exceed 1.2% of the initial concentration, the gradient was practically constant during a single experiment. NaCI entering the dextrose solution was determined, following each dialysis experiment, by measuring the conductivity of samples of the dialysate (dextrose-NaC1 mixture) at 30°C. To convert the conductivity values to NaC1 concentration, a standardization graph which had previously been obtained from NaCl-dextrose mixtures of known concentrations was used. In a second series of experiments, the permeability of the skin preparations for urea was tested, a solution containing 16.2% urea and 5.4% de~xtrose solution being on the outer surface and a 5.4% dextrose solution on the inner side of the skin. With this group also each dialysis lasted 50 minutes. The urea content of the dialysate was determined by means of a Zeiss interferometer, a 5.4% dextrose solution serving for comparison. The conversion of the interferometric readings into urea concentrations was made by means of a standardization curve obtained by interferometry of "5.4% dextrose solutions containing urea in various concentrations. Before and after each dialysis experiment the A of the membrane was determined by double measurements. RESULTS
If an isolated preparation of frog's skin is kept in a 0.1 N NaC1 solution, the polarizability of the specimen, as determined by the polar-
POLARIZATION AND PERMEABILITY
23
ization index A, increases in the first 1-4 hours, and then gradually declines reaching values close to zero about 5-50 hours post mortem (Fig. 1). The dialysis experiments were, as a rule, performed during the decrease of the A values. In order to compare polarizability and permeability, the average of A for each dialysis period was computed from the values before and after dialysis, and the mean A was plotted against the amount of NaC1 or urea which had passed through the membrane in the correspon.ding interval of 50 minutes. A 18,
16-
14-
12-
I0-
8
S
4
2 0
FIG. 1
Post Mortem Changesof A (Frog Skin) Abscissa: Time after death Fig. 2 shows the result of such a typical experiment, in which NaC1 permeated into the dextrose solution. A linear relationship between the changes of A and of the permeability for NaC1 is demonstrable. It is evident that the intersection of the graph and the x-axis indicates the value of A for which the membrane is completely impermeable to NaCl. The intersection of the graph and the y-axis shows the amount of NaC1 that penetrates the membrane after a complete loss of A. This value is finite, and it may be inferred that the membrane has some semi-permeability left after the loss of polarizability. The first derivative of the above graph can be used as characteristic of the membrane. Changes of A are a function of time. If time is substituted for ~ in the above graph, the resultant first derivative at a specified time indicates the rate at which salt is dialyzed at this time, and this corresponds to the permeability.
24
M. S P I E G E L - A D O L F
A N D E. SPIEGEL
b
nNaGI 13
xtO*: 12 II I0 9 8 7, 6' 5" 4
Fro. 2 Relationship Between A and Permeability for NaCl (Frog Skin) Ordinate of curve a is conductivity of dialysate. Ordinate of curve b is NaC1 concentration of dialysate.
The relationship between polarizability and permeability for a nonelectrolyte, such as urea, is illustrated in Fig. 3. The amount of urea passing through the frog's skin in 50 minute intervals is plotted against the average value of the A measurements performed before and after each dialysis. Contrary to the results obtained with NaC1, the resulting graph is non-linear. It shows, however, that with decreasing A the amount of urea passing through the membrane increases. Thus it seems warrantable to infer that changes of A indicate changes of permeability for an electrolyte, such as NaC1, as well as for non-electrolytes, such as urea.
180 '
|40 120 -
I00 80 60 40 20
Fro. 3 ]{elationship Between ~ and Permeability for Urea (Frog Skin) Ordinate: Urea concentration of dialysate
POLARIZATION AND PERMEABILITY
25
Another characteristic of the urea experiments requiring comment is the fact that the permeability for urea rises very steeply with decreasing A values. If an extrapolation of the graph in Fig. 3 is permissible, it seems to indicate that after a complete disappearance of A the membrane probably would hold back only little, if any, urea while only a rather limited amount of NaCI can permeate the membrane at a zero value of A. The differences are obvious on comparison of the NaC1-A and the urea-A graphs. Differences in the behavior of semipermeable membranes toward ionized and non-dissociated substances have been observed by Weatherby (6), who refers them to the electrical status of both the membrane and the permeating substance. One of us (Spiegel-Adolf (7)) has been able to show that neutral NaC1 solutions give a positive charge to globulins within the concentration range used in these experiments. No such changes can be produced by urea. This may account, at least partly, for the differences in the shape of the NaC1 and of the urea curves. Changes in the degree of hydration of the membrane proteins produced by the urea (Heim (8), Spiegel-Adolf (9)) may also play a role. SUMMARY
The relationship between polarizability and permeability was studied on isolated frog skins during the initial stages of post mortem deterioration. The polarizability was determined by measuring the conductivity at a high (Ch) and. at a low frequency (Cz) of A. C. and calculating the difference of these measurements expressed in percentage of Cz (polarization index A). In one series of experiments the permeability for NaC1 was determined, and in a second series that for urea. Both groups of experiments indicated that decrease of polarizability is associated with increase of permeability. ~:~EFERENCES 1. GILDEMEISTER,1VL,Handbuch der normalen und pathologischen Physiologie. Eclited by A. Bethe et al. 8, II, 657 (1928). 2. SPIEGEL-ADOLF,M., AND SPIEGEL, E. A., Proc. Soe. Exptl. Biol. Med. 32, 139 (1934). 3. SPIEGEL, E. A., AND SPIEGEL-ADOLF,M., Am. J. Psychiatry 92, 1145 (1936). 4. SPIEGEL, E. A., AND SPIEGEL-ADOLF,M., or. Nervous Mental Disease 90, 188 (1939). 5. SPIEGEL-ADOLF,M., J. Gen. Physiol. 20, 695 (1937). 6. WEATHERBY,J. H., J. Cellular Comp. Physiol. 21, 1 (1943). 7. SPIEGEL-ADOLF,M., Globuline. Th. Steinkopff. Dresden-Leipzig, 1930. 8. HEIM, F., Biochem. Z. 291, 88 (1937). 9. SPIEGEL-ADOLF,M., Federation Proc. 3, 63 (1944).
The measurement of the permeability of membranes is of interest to colloid chemists as well as biologists. If one wishes to ascertain the permeability of cellular membranes within the organs of animals, the usual chemical methods as a rule can hardly be applied, particularly when rapid changes of permeability are to be recorded. Due to their semipermeability to electrolytes, the cell membranes are the seat of electrical double layers. They show polarization phenomena, i.e., an electromotive force in opposite direction develops, when a direct current flows through the organ. If an alternating current passes through the organ this counterforce, and consequently the impedance measured, is smaller the higher the frequency of the A. C. Gildemeister (1) has pointed out that determination of the capacitance may serve as an indicator of the polarizability and thus indirectly of the permeability. The measurement of the polarization capacity requires complete balancing of the Wheatstone bridge, so that its zero arm is practically free of current. This is sometimes rather inconvenient, particularly when one deals with rapid changes due to experimental procedures. Therefore, we developed a method based upon another manifestation of polarization; viz., the fact that the difference between the conductivity at a certain high frequency (CA) and that at a certain low frequency (C~) is larger the more polarizable the membrane under study (2). From the 100(Ch -- Cz) . measurements of Ch and C~ the polarization index A -C~ is calculated (2). Applying such measurements to the brain, it was found that some epileptogenous agents diminish the density of the cell membranes, while hypnotics and anesthetics act in the opposite direction (3, 4). To analyze the basic chemical and colloid chemical conditions, experiments on artificial membranes have been performed (2, 5). In 1Aided b y a grant from the J o h n and Mary R. Mark]e Foundation. 21
22
M. SPIEGEL-ADOLF AND E. SPIEGEL
artificial membranes containing body constituents it could be shown that proteins may be substituted by other colloids, while the presence of lipids seems to be necessary for achieving polarizability. By perforating polarizable collodion or lecithin-collodion membranes repeatedly, a progressive reduction of A was produced (5). These findings pointed to a relationship between permeability and polarizability. However it seemed desirable to present a more direct evidence of such a relationship by a quantitative study. In the experiments to be reported here, frog's skins were used in order to obtain data from a natural membrane. Method: The experiments were performed on the skin of the trunk of winter frogs. On such isolated preparations, a series of alternating measurements of A and of the permeability for an electrolyte (NaC1) or for a non-electrolyte (urea) were made during the first stages of post mortem deterioration of the specimen. The skin was inserted flat between the ground flanges of the central and one external compartment of a Pauli electrodialysis apparatus, the original wire mesh P t electrodes of which had been coated eleetrolytically with platinum black. The surface inserted measured 452 mm.~. Between the central and the other external compartment no membrane was inserted, so that the apparatus was changed into a system consisting of 2 cells only. For the determination of A, the apparatus was filled with 0.1 N NaCI solution on either side of the skin. The bridge arrangement was the same as previously described (3, 4). Frequencies of 547 and 5120 cycles respectively were used. The measurements were made at room temperature. For the dialysis experiments the 0.1 N NaC1 solution in the apparatus was replaced in the compartment facing the inner side of the skin by a 5.4% dextrose solution, and in the compartment facing the external surface of the skin, by a N NaC1 solution. Each dialysis lasted 50 minutes and was performed at room temperature. This time interval was chosen because it permitted one to make from 5-6 dialysis experiments on one specimen within 1-2 days. After each dialysis experiment both the NaC1 and the dextrose solution were replaced by fresh solutions. Since the maximum amount dialyzed did not exceed 1.2% of the initial concentration, the gradient was practically constant during a single experiment. NaCI entering the dextrose solution was determined, following each dialysis experiment, by measuring the conductivity of samples of the dialysate (dextrose-NaC1 mixture) at 30°C. To convert the conductivity values to NaC1 concentration, a standardization graph which had previously been obtained from NaCl-dextrose mixtures of known concentrations was used. In a second series of experiments, the permeability of the skin preparations for urea was tested, a solution containing 16.2% urea and 5.4% de~xtrose solution being on the outer surface and a 5.4% dextrose solution on the inner side of the skin. With this group also each dialysis lasted 50 minutes. The urea content of the dialysate was determined by means of a Zeiss interferometer, a 5.4% dextrose solution serving for comparison. The conversion of the interferometric readings into urea concentrations was made by means of a standardization curve obtained by interferometry of "5.4% dextrose solutions containing urea in various concentrations. Before and after each dialysis experiment the A of the membrane was determined by double measurements. RESULTS
If an isolated preparation of frog's skin is kept in a 0.1 N NaC1 solution, the polarizability of the specimen, as determined by the polar-
POLARIZATION AND PERMEABILITY
23
ization index A, increases in the first 1-4 hours, and then gradually declines reaching values close to zero about 5-50 hours post mortem (Fig. 1). The dialysis experiments were, as a rule, performed during the decrease of the A values. In order to compare polarizability and permeability, the average of A for each dialysis period was computed from the values before and after dialysis, and the mean A was plotted against the amount of NaC1 or urea which had passed through the membrane in the correspon.ding interval of 50 minutes. A 18,
16-
14-
12-
I0-
8
S
4
2 0
FIG. 1
Post Mortem Changesof A (Frog Skin) Abscissa: Time after death Fig. 2 shows the result of such a typical experiment, in which NaC1 permeated into the dextrose solution. A linear relationship between the changes of A and of the permeability for NaC1 is demonstrable. It is evident that the intersection of the graph and the x-axis indicates the value of A for which the membrane is completely impermeable to NaCl. The intersection of the graph and the y-axis shows the amount of NaC1 that penetrates the membrane after a complete loss of A. This value is finite, and it may be inferred that the membrane has some semi-permeability left after the loss of polarizability. The first derivative of the above graph can be used as characteristic of the membrane. Changes of A are a function of time. If time is substituted for ~ in the above graph, the resultant first derivative at a specified time indicates the rate at which salt is dialyzed at this time, and this corresponds to the permeability.
24
M. S P I E G E L - A D O L F
A N D E. SPIEGEL
b
nNaGI 13
xtO*: 12 II I0 9 8 7, 6' 5" 4
Fro. 2 Relationship Between A and Permeability for NaCl (Frog Skin) Ordinate of curve a is conductivity of dialysate. Ordinate of curve b is NaC1 concentration of dialysate.
The relationship between polarizability and permeability for a nonelectrolyte, such as urea, is illustrated in Fig. 3. The amount of urea passing through the frog's skin in 50 minute intervals is plotted against the average value of the A measurements performed before and after each dialysis. Contrary to the results obtained with NaC1, the resulting graph is non-linear. It shows, however, that with decreasing A the amount of urea passing through the membrane increases. Thus it seems warrantable to infer that changes of A indicate changes of permeability for an electrolyte, such as NaC1, as well as for non-electrolytes, such as urea.
180 '
|40 120 -
I00 80 60 40 20
Fro. 3 ]{elationship Between ~ and Permeability for Urea (Frog Skin) Ordinate: Urea concentration of dialysate
POLARIZATION AND PERMEABILITY
25
Another characteristic of the urea experiments requiring comment is the fact that the permeability for urea rises very steeply with decreasing A values. If an extrapolation of the graph in Fig. 3 is permissible, it seems to indicate that after a complete disappearance of A the membrane probably would hold back only little, if any, urea while only a rather limited amount of NaCI can permeate the membrane at a zero value of A. The differences are obvious on comparison of the NaC1-A and the urea-A graphs. Differences in the behavior of semipermeable membranes toward ionized and non-dissociated substances have been observed by Weatherby (6), who refers them to the electrical status of both the membrane and the permeating substance. One of us (Spiegel-Adolf (7)) has been able to show that neutral NaC1 solutions give a positive charge to globulins within the concentration range used in these experiments. No such changes can be produced by urea. This may account, at least partly, for the differences in the shape of the NaC1 and of the urea curves. Changes in the degree of hydration of the membrane proteins produced by the urea (Heim (8), Spiegel-Adolf (9)) may also play a role. SUMMARY
The relationship between polarizability and permeability was studied on isolated frog skins during the initial stages of post mortem deterioration. The polarizability was determined by measuring the conductivity at a high (Ch) and. at a low frequency (Cz) of A. C. and calculating the difference of these measurements expressed in percentage of Cz (polarization index A). In one series of experiments the permeability for NaC1 was determined, and in a second series that for urea. Both groups of experiments indicated that decrease of polarizability is associated with increase of permeability. ~:~EFERENCES 1. GILDEMEISTER,1VL,Handbuch der normalen und pathologischen Physiologie. Eclited by A. Bethe et al. 8, II, 657 (1928). 2. SPIEGEL-ADOLF,M., AND SPIEGEL, E. A., Proc. Soe. Exptl. Biol. Med. 32, 139 (1934). 3. SPIEGEL, E. A., AND SPIEGEL-ADOLF,M., Am. J. Psychiatry 92, 1145 (1936). 4. SPIEGEL, E. A., AND SPIEGEL-ADOLF,M., or. Nervous Mental Disease 90, 188 (1939). 5. SPIEGEL-ADOLF,M., J. Gen. Physiol. 20, 695 (1937). 6. WEATHERBY,J. H., J. Cellular Comp. Physiol. 21, 1 (1943). 7. SPIEGEL-ADOLF,M., Globuline. Th. Steinkopff. Dresden-Leipzig, 1930. 8. HEIM, F., Biochem. Z. 291, 88 (1937). 9. SPIEGEL-ADOLF,M., Federation Proc. 3, 63 (1944).