COLLOIDAL PROPERTIES OF THE SURFACE OF THE LIVING CELL.

I. CONDUCTIVITY OF BLOOD TO DIRECT ELECTRIC CURRENTS.

BY J. F. McCLENDON.

(From the Laboratory of Physiological Chemistry, University of Minnesota, Minneapolis.)

(Received for publication, April 6, 1926.)

Stewart showed that blood cells act as insulators, but his and most later measurements were made with alternating currents, and since the mathematics is more complicated than with direct currents, the results with alternating currents are reserved for the second paper of this series.

Clerk Maxwell’ devised a formula for conductivity of a uniform suspension of spheres of equal radii in a medium

R = Rz X 2 RI + Rz + PO& - &J

2Rl+Ra- 2 PC& - &I

in which R is the specific resistance (resistance of a cube of 1 cm. edge) of the suspension, RI of the spheres, Rz of the medium, and p the ratio of the total volume of the spheres to that of the sus- pension (volume of spheres per cc.). Clerk Maxwell pointed out that the suspension must be dilute (radii of spheres small com- pared to their distances from one another) in order for the formula to hold. McClendor? attempted to test, experimentally, a formula derived from a paper by Breit.3 MacDougal14 used the same formula as Maxwell as indicated by McClendonP It now appeared that Maxwell’s, MacDougall’s, and Breit’s forms of

1 Maxwell, J. C., Electricity and magnetism, Oxford, 3rd edition, 1892, i, 440, equation 17.

f McClendon, J. F., J. Biol. Chem., 1924, lix, p. lvi. 3 Breit, G., Konin. Akad. Wetensch., Amsterdam, 1922, xxv, 293. 4 MacDougall, F. H., Science, 1924, lix, 403. 6 McClendon, J. F., Science, 1924, Ix, 523.

653

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

654 Colloidal Properties of Living Cell. I

mathematics were essentially the same. The fact that they did not check with my measurements on blood might be explained by the fact that the corpuscles are not spheres, but since Clerk Maxwell states that the formula holds only for dilute suspensions it was thought best to use a graphical method for comparing the results rather than making an analytical study of them. Fricke6 published a formula and I sent a copy to Dr. Breit who kindly examined it for me and replied: ‘(Fricke’s formula does not check with MacDougall’s or mine. His error is made essentially in equation (4). This equation presupposes that the effective conductivity is the average conductivity (a volume average) which has no logical foundation. MacDougall’s formula and mine agree.” Fricke’ developed his formula further to take into consideration the shape of the blood cells and considers them as ablate spheroids with the ratio of arcs = 4.25. GramS deter- mined the conductivity of blood using direct current and com- pared these results with that of Maxwell’s formula, considering the cells as insulating spheres, and found the resistance greater than that calculated from t.he formula. His measurements were made instantaneously.

EXPERIMENTAL.

Since Ohe alternating current method of Kohlrausch has been used almost exclusively for the determination of the conductivity of electrolyte solutions for many years, the direct current methods have been neglected and it was necessary to spend considerable time in finding a method that would work. In several physica laboratories it has been the custom to determine the potential between two points in a solution by determining the potential difference between two platinum wires inserted at these two points. This could be checked by reversing the wires to see if the same result was obtained. A similar method was first tried with blood and some other tissues. The conductivity vessel shown in Fig. 1 was first used. It consisted of two amalgamated zinc elec- trodes in concentrated solution of zinc sulfate connected by three- way stop-cocks with a horizontal tube containing the blood. The

6 Fricke, H., J. Gen. Physiol., 1924, vi, 375. 7 Fricke, H., Physic. Rev., 1925, xxiii, 575. 8 Gram, H. C., J. Biol. Chem., 1924, lix, 33.

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. F. McClendon 655

FIG. 1. Conductivity vessel for direct current, consisting of two amal- gamated zinc electrodes in zinc sulfate solution connected by a-way stop- cocks to a horizontal tube containing blood. The current passing through the apparatus was measured as well as the potential difference between two platinum vx-ires inserted in the openings of the horizontal tube.

FIG. 2. Conductivity vessel similar to Fig. 1 except that calomel clec- trodes are used and the platinum wires are sealed in.

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

656 Colloidal Properties of Living Cell. I

apparatus was immersed in a thermostat filled with oil. In the case of solid tissues, a disc of the tissue was inserted in the mid- point of the tube. The total current passed through the appara- tus was measured by means of a Rawson multimeter. This instrument is a sensitive milliammeter whose range can be varied by simply turning a switch. The potential difference between two points in the blood in the tube was determined by inserting two platinum wires in the two openings at the top of the horizontal . tube, t.he platinum wires being fixed a certain distance apart in a rigid frame.. The wires could at any time be withdrawn and put in in the reverse order. The potential difference between the wires was measured by means of a Dolezalek electrometer with a platinum sputtered quartz suspension giving one division of the

FIG. 3. Motor-driven commutator for reversing the current every 30 seconds.

scale per millivolt. This instrument was in a grounded shield and the key was well shielded. It was found impossible to make very accurate determinations with this apparatus due to the fact that the electrometer had a large capacity so the apparatus shown in Fig. 2 was made. In this case the zinc electrodes were replaced by large calomel electrodes and the platinum wires were sealed in. In order to reduce polarization at the calomel elec- trodes and to correct for any differences in the platinum wires, an electric motor was used t,o drive a commutator which re- versed the current every 30 seconds (Fig. 3). After a rough determination was made a final adjustment could be sharpened during a 30 second interval and a final reading could be verified during a 30 second interval. With this apparatus some difficulty

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. F. McClendon 657

was experienced by slow mixing of the blood with the Ringer’s solution saturated with calomel which filled the calomel electrode vessel, so the electrode shown in Fig. 4 was made. In this apparatus the platinum wires were made further apart to increase the accuracy, and the calomel electrodes were above the level of the blood so that no blood corpuscles could fall by gravity into the calomel electrodes. The results were a little better but not wholly satisfactory. On the assumption that some of the trouble might be due to diffusion of the blood into the Ringer’s solution in the calomel electrode, the hole in the stop-cock was filled with an agar gel made up in Ringer’s solution. Since the resistance of the solution in the calomel electrode was very small compared with that of the blood in the narrow tube it was thought advisable

- - - -

FIG. 4. Conductivity vessel similar to Fig. 2 except that the calomel elec- trodes are above the level of the horizontal tube.

to do away with the platinum wires and measure the difference in potential between the mercury in the two calomel electrodes and assume that it was the same as the difference of potential in the blood at the points where the Ringer’s solution in the agar gel came in contact with it. For this purpose the apparatus shown in Fig. 5 was constructed. At the top of each calomel electrode vessel there was inserted a hollow glass stopper with a horizontal fused-in glass tube. At right angles to the insertion of the tube a hole was bored in the hollow stopper. The remaining hollow space in the stopper was filled with agar gel made up in Ringer’s solution. There was another hole in the neck of the calomel electrode vessel to allow escape of Ringer’s solution when inserting the stopper. This was closed after the stopper wasinserted. The

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

658 Colloidal Properties of Living Cell. I

two stoppers were placed so as to connect the fused-in tube with the horizontal tube to be filled with blood, also with a side neck

ending in a funnel on one side and an overflow tube on the other.

FIG. 5. Conductivity vessel similar to Fig. 2 except that the stoppers of. the stop-cocks are filled with agar gel to prevent mixing of the blood and; KC1 solutions.

FIG. 6. Conductivity vessel similar to Fig. 5 except that a horizonta1 tube is in the shape of a U and can be placed in the centrifuge.

Blood was placed in the funnel and allowed to run through the horizontal tube, displacing all the air bubbles or liquid from pre- vious determinations. Then the stoppers were turned through a

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. F. McClendon 659

right angle, connecting the agar gel with the blood in the hori- zontal tube. The difference in potential between the mercury in the calomel electrodes was determined by means of a Leeds and Northrup potentiometer and the total current flowing was determined by the multimeter. The resistance of the blood was

determined by Ohm’s law, I = i in which I is the current in

amperes, E is the electromotive force in volts, and R is the resist- ance in ohms. In this apparatus, after determining the resistance of the blood to direct current, an alternating current could be used by inserting the conductivity vessel in a Wheatstone bridge arrangement. In order to make determinations on very high concentrations of corpuscles the conductivity vessel was modified to the form shown in Fig. 6. In this case the tube containing the blood was U-shaped and could be placed in a centrifuge to pack the corpuscles further.

Since NewberyO had claimed that he could make more accurate determinations with direct current than by the Rohlrausch method, using alternating currents, a modified Newbery appara- tus was made as shown in Fig. 7. The apparatus consisted of two large calomel electrodes and above them were two connecting vessels filled with agar gel made up in Ringer’s solution. The blood was contained in a horizontal tube, the two ends of which were inserted in the two connecting vessels, fitting into ground glass joints. When the blood tube was fully inserted, that is when the joint was tight, a capillary tube not filled with agar gel reached almost to the open end of the blood tube. This capillary tube was filled with Ringer’s solution and connected to a small calomel electrode. Around the open end of this capillary tube was lyound a platinum wire which served as an electrode for the alternating current for comparative measurements. When the apparatus was used for direct current, a current was passed through the blood from the large calomel electrodes and deter- mined by the multimeter. At the same time a potential differ- ence at the two ends of the tube containing blood as picked off by means of the capillary tubes and small calomel electrodes was

Q Newbery, E., J. Chem. Xoc., 1919, cxiii, 701. Eastman, E. D., (J. Am. Chem. Sot., 1920, xiii, 1648) concludes that Newbery was in error nearly 0.7 per cent and Kohlrausch’s measurements correct.

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

660 Colloidal Properties of Living Ceil. I

determined by means of the potentiometer. The determinations with alternating current required a large inductance inserted in series with the circuit which passed through the blood by means of the platinum wires in order to produce resonance. This in- ductance had to be so large because the series capacity at the surface of the platinum wires was so small, but this part of the technique will be considered in the second paper which concerns itself with alternating currents.

In these determinations there was always found to be a change in resistance with time. Since the apparatus was in all cases immersed in a constant temperature oil bath and the tube contain-

FIG. 7. Modified Newbery conductivity vessel for blood. The current is run through two large calomel electrodes below, and the voltage is picked off by two small calomel electrodes at the twa ends. Two platinum loops are used to pick off the current when using the Kohlrausch method. The segments in which the platinum loops and capillary tubes from the small calomel electrodes are inserted, are filled with agar gel.

ing the blood was very narrow, and with a large surface to the oil which was circulated at a rapid rate, it was concluded that tem- perature could not account for this difference. In all my work for the past 15 years on the conductivity of cell suspensions, I have taken into consideration the effect of settling of the cells, contrary to the objections raised by Brooks.lO Since these deter- minations took some time, and although by reversing the stop- cocks the blood could be stirred up in some of these electrodes, there was some opportunity for settling. Beef corpuscles, how-

10 Brooks, S. C., J. Gen. Physiol., 1925-26, viii, 349.

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. F. McClendon 661

ever, were used exclusively as they do not settle as rapidly as human corpuscles; in fact, they settle very slowly under most conditions. Another change which may affect the conductivity is change in shape of the corpuscles. Crenation may occur in corpuscles suspended in salt solution without any change in salt concentration as shown by Brinkman and Van Dam.ll Crenation may occur fairly rapidly in the corpuscles in contact with a cover- glass under a microscope and if this cover-glass is passed through a flame previous to placing it over the corpuscles, the crenation is inhibited. Therefore, it was concluded that the crenation was due to an electric charge on the cover-glass which was removed by the ionized gas or electron emission in the flame. If an electric charge on the glass may produce crenation, it seems possible that the passage of an electric current under certain conditions may produce crenation, although this would probably occur only with corpuscles in salt solution.

Another change seemed, however, to be the basis of the change in conductivity, and that was the orientation of the corpuscles with a long axis parallel to the current lines. Such an orientation has been observed by other workers and this would be progressive with time so that the resistance would decrease from the instan- taneous resistance and approach a maximum, which would be arrived at when the corpuscle was rotated as much as it had room to rotate in the suspension. It would therefore rotate more in a dilute suspension than in a concentrated one. Since some time was required for accurate determinations, the measurements made were the final values; that is to say, the rotation had ap- proached a maximum so there was little change with time. The results are shown in Fig. 8 in which the resistance of the blood is recorded in units such that the resistance of the serum in the same conductivity vessel is equal to 1, or in other words, the specific resistance of the blood is divided by the specific resistance of the serum and this measured on the abscissa in Fig. 8. The cell volume in percentage of total volume of the suspension is meas- ured on the ordinate. The cell volume in a precipitate of blood cells taken from a large centrifuge was determined by means of a hematocrit rotating at a speed of about 20,000 revolutions in a minute (centrifuged to translucence). This cell precipitate was

11 Brinkman, I. R., and Van Dam, E., Biochem. Z., 1920, cviii, 35.

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

662 Colloidal Properties of Living Cell. I

‘go I II I1 1 I / 1 I /,,,,,I,

go- ---c ________ -___

1

FIG. 8. Graph showing the per cent cell volume on the ordinate and the resistance of blood on the abscissa regarding the resistance of serum as unity. The smooth curve represents the resistance of blood as measured and the dashed curve as that calculated from Clerk Maxwell’s formula assuming the corpuscles to be spheres of infinite resistance.

FIG. 9. Graph of calculated curves similar to Fig. 8. The lower curve marked F 4.25 is from Fricke’s caIcuIations assuming the resistance of the corpuscles to be infinite and corpuscles to be oblate spheroids with a ratio of axes = 4.25. The remaining curves were calculated by F. Fetter using Maxwell’s formula. The ratio of the specific resistance of the corpuscle substance to that of the serum from 6 to infinity is marked at the end of each curve.

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. F. McClendon 663

then kept stirred to keep it uniform and its specific gravity deter- mined as well as the specific gravity of the serum by means of the pycnometer. The various cell volumes were obtained by weighing corpuscle precipitate and serum and mixing, since the weight can be determined much more accurately than any volu- metric measurements.

The continuous curve in Fig. 8 shows the results of measure- ments and the dotted curve shows the calculations made from Maxwell’s formula assuming the corpuscles to be spherical in- sulators. For an equal cell volume, the resistance of the blood is less than calculated by Maxwell’s formula. The determinations made by Gram show the resistance of the blood to be greater than calculated by Maxwell’s formula. The difference between these two sets of determinations probably arises from the fact that Gram made his determination instantaneously, in which case no time for orientation of the corpuscles was given, and present dctcrminations were made after orientation had occurred. In Fig. 9 are given a number of curves using Maxwell’s formula and assuming various specific resistances of the corpuscles from 7 to infinity, and also it shows the curve using Fricke’sformula, assum- ing the diameter of the corpuscle to be 4.25 times its thickness and its resistance infinite. Fricke’s formula probably harmonizes more with Gram’s data, but the present determinations more with Maxwell’s formula, assuming a slight conductivity of the cor- puscles. Since Fricke has shown that the ellipsoid shape of the corpuscles increases the resistance when they are at random arrangement over that which they would have if they were spheres, and has emphasized also that orientation will decrease the resist- ance, the orientation acting in an opposite direction from the effect of flattening of the corpuscles on the resistance, one would expect the present determinations to approach more toward Maxwell’s formula. Since it is well known that chlorine ions may go in and out of the corpuscles, it is safe to assume them to have a slight conductivity. The passage of ions must be very slow, however, or else we would not get the relatively high resistance of corpuscles which has led many investigators to assume that they were insulators. Since the results with alternating current shed further light on this question, a discussion of it is reserved for the second paper of the series.

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from

J. F. McClendonDIRECT ELECTRIC CURRENTSCONDUCTIVITY OF BLOOD TO

SURFACE OF THE LIVING CELL: I. COLLOIDAL PROPERTIES OF THE

1926, 68:653-663.J. Biol. Chem. 

  http://www.jbc.org/content/68/3/653.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  ml#ref-list-1

http://www.jbc.org/content/68/3/653.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be

by guest on March 21, 2020

http://ww

w.jbc.org/

Dow

nloaded from