1 I 1
I
metal cotton and skin body skin cotton and metal saline elecelec- substance to trode trode be introduced ~
336
HAROLD A. ABBRAMBON AND MANUEL E. QORIN
nificant trauma and no discomfort. It is evident that this method, commonly called electrophoresis or iontophoresis (erroneously termed “ionization” (3)) embodies certain advantages in both experimental and therapeutic procedures. (1) The surface of the skin ia unbroken. (2) The current density may be varied over a wide range. (3) Areas differing in size from that of a hair follicle to that of very large surfaces may be studied. (4) The total amount of material introduced and the rate of introduction can be varied by varying both the current density and the concentration. (6)There is excellent uniformity in the distribution of the drug through the akin compared, for example, with that obtained by injection. (6) The skin may act as a reservoir. NATURE O F THE SHIN
The outer layer of living human skin may be considered to be a membrane, consisting of very h e pores which lead from the surface toward the vascular regions below. All the experimental evidence to be presented can be qualitatively explained on the assumption of a fairly rigid inanimate system. Actually, of course, the pores may change their entire nature under the influences of the electrophoretic procedures employed, as well as with changes in the physiological state of the individual during the course of an experiment. A quantitative treatment of electrophoresis through the skin will not be possible until these physiological factors are better understood, but these factors appear to be of secondary importance for the qualitative considerations to be examined. It is evident that if the skin is considered to be a pore-like membrane it would differ fundamentally from a wet collodion membrane when the skin pores are incompletely filled with liquid. Either a gel-like or a pore-like membrane would allow highly diffusible substances to pass readily through it when the inside and outside surface are in contact with fluid. On the other hand, under the same circumstances, a pore-like membrane which contains air spaces of small diameter and is otherwise permeable with difEculty will not readily allow diffusion. Actually, of course, the outer skin layer may be a combination of the two types. If it is, the pore-like part must constitute the outermost layer. ‘When an electrical potential is applied across a membrane of this nature, the inner and outer surfaces being in contact with fluid, electroosmotic forces would tend to displace the air in the pores with fluid. The pores would then contain an unbroken column of conducting fluid in which would be displayed all the phenomena of electrokinetics. Ions would be forced through by the potential drop (iontophoresis or ionic migration), and the columns of liquid as a whole would move either toward the inside or toward the outside depending upon conditions (electro6smosis).
SKIN REACTIONS
337
Besides these two phenomena, diffusion must be considered, for, when a current is passing, diffusion can take place through the, continuous columns of liquid established. If electrophoresis and electroosmosis are in the same direction or one is much larger than the other, the diffusion term may be neglected in qualitative considerations. However, under certain circumstances the electrophoretic and electroiismotic velocities may be of the same order of magnitude and opposite in sign. For these cases the diffusion term must be considered. Shortly, diffusion velocities will be calculated and an example given in which the diffusion term may be of importance. SIbfPLE CASE OF IONTOPHOREBIB: HIBTAMINE HYDROCHLORIDE
The drug histamine is of great importance in skin reactions. When it is introduced into the vascular regions of the skin, the whealing reaction occurs; the skin becomes elevated and resembles a hive or mosquito bite in the zone of pharmocological action. This reaction is very similar to the one obtained in certain allergic individuals when substances to which they are sensitive are introduced into the skin. Histamine (imidazoleethylamine) is a readily Musible diacidic base (8),with a h t dissociation constant about the same as that of ammonia and a second very much weaker. Below pH 4.0 the doubly charged ion, histamine++, predominates in aqueous solution. From pH 4.0 to pH 10 histamine(OH)+ predominates, while above pH 10 solutions of the drug contain mostly the uncharged form, histamine(0H)r. Abramson and Alley (3) have recently made a careful study of the electrophoretic introduction of histamine into living human skin. They found that in the absence of other electrolytes water solutions of histamine salts in dilutions as high as 1:5,000,0002 gave a whealing response when the positive pole was applied to the cotton containing the histamine. They used a constant current density and time interval,-O.5 milliampere per square centimeter for 5 min. In no case was a reaction ever obtained with the negative pole. The method is so sensitive that Abramson and Ochs were able to work out a semi-quantitative procedure for determining minute amounts of histamine in complicated media, e.g., whole blood (5). The effect of salts a t constant current density was carefully studied (see table 2 of reference 8). They found that, as the concentration of potassium chloride was increased, larger concentrations of histamine were necessary to produce a reaction. In 2.4 M potaasium chloride practically no reaction was obtained with the most concentrated histamine solution employed (1:5OOO). The authors explained their findings by pointing With subsequent development of technique histamine in higher dilutions haa been detected.
338
HAROLD A. ABRAhfSON AND MANUEL H. W R I N
out that as the inert salt concentration increases the fraction of the current carried by the histamine decreases, and therefore that the amount of histamine electrically transported into the skin decreases. Further evidence that the galvanic introduction of histamine may be considered to be a simple case of iontophoresis waa furnished by Abramson, Engel, Lubkin, and Ochs (4). These authors observed that histamine which had previously been electrically introduced into the skin with the positive pole could be partially removed by reversing the current. @antities of histamine large enough to cause strong reactions in a new area of skin were successfdly recovered with the negative pole (within 40 min.) from the treated area into a piece of cotton wet with distilled water. This type of wheal produced in a new area of skin after recovery of histamine we shall call a transferred wheal. Unpublished results of the writers on the iontophoresis of histamine demonstrate further the pore-like nature of the outer skin membrane. It was discovered that when the positive pole was applied to a piece of cotton, wet with distilled water, over an area to which histamine had previously been applied by electrophoresis, the whealing reaction reappeared. This type of wheal produced at the primary site of histamine iontophoresis we shall call a secondary wheal. Thus wheals, the severest of which last only a few hours, were caused to reappear, in some instances as long as five days after the electrical introduction of histamine. Furthermore, if the negative pole was applied to an area which had previously received histamine electrically, in some instances as long as four days, and invariably 24 hr. after the original application, histamine could be demonstrated in the cotton by the production of transferred wheals. As yet our attempts to remove histamine with the negative pole from an area to which it had been administered by ordinary intradermal injection have failed. Nor has it been possible to cause, with the positive pole, the reappearance of a histamine wheal (secondary) that had been produced by ordinary intradermal injection. In a few experiments in which the histamine had been pricked into the skin, the histamine being applied only to the most superficial layers of the skin by this method, it was possible to demonstrate the presence of histamine in the skin 24 hr. later, and occasionally after a longer period, by both secondary and transferred wheals. It appears, therefore, that the intradermal injection method distributes histamine primarily to the deeper layers of the skin, where fewer barriers to difIusion exiat. There must be, then, regions in the skin through which histamine can be readily transported electrically, but with difficulty by diffusion. T4e pore-like membrane postulated above has such properties, for, when the current is started, the pores will fill by the outward electroosmotic flow of skin fluid or by the inward movement of cotton fluid. The liquid that
SKIN REACTIONS
339
fills the pores while the current is flowing will be partially trapped and therefore remain in the skin for long periods. The pores, under these conditions, act as reservoirs from which histamine is released only very slowly. The capacity of the skin to act as a reservoir for biologically active materials may be connected with the origin of some skin diseases. On the other hand, galvanic therapy might prove useful in removing depots of toxic materials formed in the skin pores, or valuable for establishing depots of materials of therapeutic import in the cases in which slow absorption is desirable, as in all types of vaccination. ELECTROOSMOSIS
Electroosmosis through animal tissues was first observed by Porret in the early part of the nineteenth century. In 1860 Kuhne (7) apparently observed electroosmosis in muscle. Roever, in 1896, investigated the relationships between the field strength and the volume of liquid transported through the skin of the cow. He found that the volume of liquid transported was proportional to the field strength. Morton (9) found that electroosmosis was responsible for the transfer of many substances into the skin and into the pulp of the tooth. In fact, he noted explicitly that solutions of cocaine were more effective in producing anesthesia when applied by electrophoresis if an alcoholic rather than an aqueous medium was employed. Rein (10) emphasized and clarified to some extent the importance of electroosmosis, but some of his conclusions regarding the sign of the charge and the electrophoresis of ions through the pores do not seem to be completely borne out by more detailed experimentation. The direction of electroosmotic flow in a pore-like membrane depends upon the sign of the average charge on the walls of the pores (2). If the walls are positively charged the solution will move toward the positive pole, and if negatively charged toward the negative pole. In the case of the human skin it is assumed that the walls of the pores are lined with proteins. It is well established that for protein surfaces the charge density at the protein-water interface depends primarily upon the pH of the solution and closely parallels the charge density calculated from the excess of hydroxide or hydrogen ions as determined by titration curves for individual molecules of the same species (1). Therefore the direction of electroosmotic flow in the skin will reverse at the isoelectric point of the pores. According to Rein, the pores of the skin behave as if they are lined with keratin, with an isoelectric point between pH 3.0 and 4.0. At pH values above 4.0, then, the positive pole at the cotton should cause electroosmotic flow into the skin. The introduction of histamine a t pH 12 and of alkaline alcoholic solu-
340
HAROLD A. ABRAM80N AND MANUEL E. QORIN
tions of local anesthetics furnishes examples in which electroosmosis probably predominates. In both of these cases the material successfully introduced is essentially uncharged. There are no examples available for electroosmosis into the skin with the negative pole on the acid side of the assumed isoelectric point of the pores. For optimum electroosmotic velocity several conflicting factors must be taken into consideration. Thus, the charge depends upon the distance from the isoelectric point. However, increasing the pH generally necessitates increasing the total strength of the solution. High ionic strength slows up electroosmosis in two ways: (1) The potential gradient in the pores, a t constant current density, is low because of the high conductivity of the solution in the pores. (9)The t-potential of the pores at constant charge density may be reduced. Abramson and Alley have demonstrated the effect of salts in the case of the electrophoresis of histamine a t pH 12. Under these conditions less than 0.1 per cent of the histamine molecules are charged. Yet solutions as dilute as those in the case of histamine hydrochloride mentioned above caused a positive whealing response. The evidence is strong, therefore, that the electrical introduction of histamine at pH 12 is due primarily to electroosmotic forces. Here, as well as with the iontophoresis of histamine salts, it was found that potassium chloride greatly diminished the transport of histamine into the skin. T E E DIFFUSION TERM
The electrical migration of a charged body and the electroosmotic movement of the solution aa a whole are strictly additive. The velocity of electrical transport of any charged molecule or particle in a potential gradient will be, then, the algebraic sum of the iontophoretic (electrophoretic) velocity of the particle and the electroosmotic velocity of the solution as a whole,
Ve = Vi
+ Ve
(1)
where V. is the resultant velocity due to the electric field, Vi is the velocity the particle would have if the solution as a whole were not moving, and V, is the electroosmotic velocity of the solution. In addition, of course, d8usion of' the material will occur wherever concentration gradients exist. The diffusion term must be added to equation 1, the complete equation becoming
v
= ve
+
v d
=
vi + v e +
v d
(2)
where V is the actual velocity of the molecule or particle and Va its diffusion velocity. In general, Vi and V, are proportional to the potential gradient and
341
SKIN REACTIONS
may be complicated functions of the pH and other variable conditions discussed in the foregoing. Vd is proportional to the concentration gradient of the material and may also change considerably with total salt concentration and pH, as well as with the nature of the medium (cell
c
90
70
-
a F
0
8
a
f 50-
2
c
30-
0.2
Distance in mm.
0.4
FIG.1. The time needed for the establishment of a concentration of 1/1000th of the original concentration as a function of the distance from the original boundary.
membranes), although, a t least as far as proteins are concerned, V d changes much less than Vc and V . as the pH and ionic strength are vaned. Since in general vi and v,are unrelated, V d usually operates in the nature of a correction, the magnitude of which cannot be estimated in the general case.
342
HAROLD A. ABRAWON AND MANUEL E. GORIN
There is, however, one case for which a quantitative treatment is warranted. If the substance to be introduced ie a protein and if it is adsorbed by the walls of the pores, it turns out that Vi and V , can be 0.2
0.4
0. os
0
$ .005
v
-I
0.0005
0 .o0005
Distance i n mm.
FIQ.2. The concentration ratio C/Co as a function of the distances from the original boundary established in 5 min. for various values of the diffusion coefficient.
nearly equal and opposite at all points in the pores, assuming, of course, open pores. If Vi = -V,, equation 2 becomes V = Va, and therefore the problem of calculating V becomes that of calculating Vd. Assuming
343
SKIN REACTION6
cylindrical pores and insensible depletion of the material on the outside of the skin, the equation (6,11,12),
may be used for calculating diffusion; y is given by the expression y = x/2fit
C is the concentration, Co is the original concentration, x is the distance from the original boundary, in this case the outside of the pores, t is the TABLE 1 Fraction, C/C,, of the original concentration of various substances that would be established at the ends of pores of several lengths in 6 min. SUBBTANCE
DX
101
em. per saeond
Egg albumin. . . . . . . . . . . . . 9.58 Serum globulin. . . . . . . . . . . . 104,000 5.40 Helix hemocyanin. . . . . . . . . 1.70 L,900,000 Histamine acid phosphate.. 100.00* 210
* Unpublished
i
0.392 0.315 0.473
j
0.082 , 0.0026 O.M)8 j O.OooO1 0.449 10.260 10.099
value, determined at room temperature, is (100 i 20) X 10-7. TABLE 2
“Average” difusion velocities, SUBBtANCE
9, for C
1
= CO/lOOO i n microns per second
MOLECULAR WEBET
Egg albumin.. . . . . . . . . . . . . . . . . . . . . . . 34,500 Serum globulin. .................... 104,000 Helix hemocyanin.. . . . . . . . . . . . . . . . .4,CW,ooO 210 Histamine acid phosphate.. . . . . . . . . .
I
-
V I N MICRONS PER SECOND
O.Wmm. 10.1mm. 10.2mm.
36.5
1 ’:::1
?:: ,
382.0
18.2
191.O
9.2 5.2 1.7 85.5
, 0.5mm. j
i
3.7 2.1 0.7 38.2
time, and D is the diffusion coefficient. (See figures 1 and 2 for graphic representation of the theory.) In table 1 is given C/Co at the ends of the pores after 5 min., assuming various values for the length of the pores (length 0.05 to 1.0 mm.), for a series of proteins (molecular weight = 34,000 to 4,900,000) and for an easily diffusible substance, histamine hydrochloride. T t iQs e n frnm t a h b 1 thnt. rnmnnmtivdv Inrue rnnrmtratinnn of
protein (from an immunological point of view) may be established by diirusion in 5 min. a t the ends of pores 0.25 mm. or less in length. ALSO,
344
W O L D A. ABRAMBON AND llMNUEL H. QORIN
that concentrations of importance physiologically may be established at the ends of pores as long as 0.5 mm., but that if the pores are much longer than 0.5 mm., no important amounts of proteins will reach the ends by diffusion in 5 min. Histamine (tables 1 and 2) diffuses so rapidly over distances of the order of magnitude of the length of the skin pores (0.1 to 0.2 mm.) that it seems likely that a considerable portion if not most of the histamine, in the case of electrophoresis at pH 12 and even that of iontophoresis of histamine salts, might be carried by diffusion. If so, diffusion explains the great similarity in the action of histamine at pH 12 and of histamine salts. From a physiological point of view it is interesting to calculate diffusion in another way. The time necessary to establish a concentration, say, 1/1000th of that on the outside of the skin, at the ends of pores of various lengths will be calculated for several proteins by means of equation 3. If C equals Co/lOOO, equation 3 becomes
or
From tablbles for the probability integral, the value of y for which the integral equals 0.998 is 2.184. Therefore
or x = 4.368Gt
In table 2 is given x / t , an "average" velocity in microns per second, for several proteins and histamine acid phosphate at various distapces, x , from the original boundary. THE DIFFUSION TERM AND RAQWEED ELECTROPHORESIS
The fact that the active constituent of ragweed extract can be introduced into living human skin by electrophoresis is of paramount importance in considerations of the nature of the outer skin membrane. While the active principle of ragweed pollen has not been isolated, many of its properties have been fairly definitely established. First, the active constituent cannot be removed by dialysis and is therefore of high molecular weight. Secondly, it can be completely removed frOm aqueous solution by adsorption on quartz surfaces. Thirdly, its biological activity follows
SKIN REACTIONS
345
closely that of the protein nitrogen in the solutions, and the active principle can be precipitated by the usual protein precipitants, such as ammonium sulfate. And lastly,a the active principle is apparently an amphoteric substance negatively charged a t pH 7.4. All indications, therefore, point to a protein-like body. Whether or not the active constituent of ragweed is a protein, the fact that it is a large molecule suggests that the effective diameters of the skin pores are greater than those of Cellophane or collodion membranes and that it might be poasible to introduce other proteins into the skin by electrophoresis. When ragweed extract is introduced into the skin of individuals who are skin-sensitive to ragweed, a reaction somewhat similar to that produced by histamine in normal individuals occurs. It was discovered by one of the authors that dialyzed extracts, pH about 7.6, could be introduced into the skin with current densities of -from 0.3 to 0.5 milliampere per square centimeter. These current densities for 5 to 10 min. caused large wheals in individuals hypersensitive to ragweed extract. No wheals were obtained in normal subjects with ragweed extract, nor did the subjects who were sensitive to ragweed give wheals with current alone. The positive pole produced reactions a t least as great as the negative pole, even though the material is probably negatively charged under the conditions employed. An explanation of this phenomenon may be that in the case of ragweed the musion term of equation 2 predominates. This term, of course, operates independently of the polarity. Further analysis of the mechanism involved is being conducted through the study of the electrical charge of ragweed extracts by the moving boundary method. SUbfMARY
In connection with a study of the absorption of drugs through the living human skin, the outer skin is considered to be a membrane made up of very fine pores. Under normal conditions these pores are held to be incompletely filled with liquid. It is postulated that, when an electrical potential is applied across the skin, the electroosmotic forces would tend to displace the air in the pores with liquid. The pores would then contain an unbroken column of fluid in contact with liquid on the outside and with the dermal tissues beneath. The phenomena of electrokinetics would be displayed in the pores. For the case of the movement of charged molecules through these pores therefore, three factors, which may be summed up algebraically, operate. The first factor is the simple migration of the ions themselves in an electric field. The second factor is the elec-
* Unpublished work of Abramson, Sookne, and Moyer on the electrical mobility of microscopic quartz particles covered with a film of adsorbed substances from ragweed extracts.
346
HAROLD A. ABRAMBON AND MANUEL E. OORPl
trobsmotic flow of liquid as a whole. The third factor, considered for the first time, is diffusion over small distances. In the special case where the electrophoretic and the electroosmotic movements balance one another the diffusion factor may predominate. The diffusion term is treated semi-quantitatively by a special integrated form of Fick's law which, assuming cylindrical symmetry, permits the calculation of the quantity of materials at small distances from the depot on the outer skin surface and for small fractions of the original amount in the depot itself. Diffusion velocities of even large molecules may exceed velocities due to electrical mobility with the usual field strengths acrom the skin. The retardation of diffusion in the skin in the absence of a potential gradient is demonstrated by a newly discovered phenomenon associated with the electrical introduction of histamine, a substance of relatively low molecular weight. It was found that histamine which has been electrically transported into the skin remains in the pores for periods as long as a week. Furthermore, by reversing the current histamine can be readily recovered. The foregoing theory and experiments are applied to the interpretation and the electrophoresis of protein extracts of ragweed pollen into the skin of indiyiduals hypersensitive to these substances. In conclusion we wish to express our appreciation for the aid given by Mrs. Margery Engel and Mrs. Henrietta Gettner during the course of these investigations. REFERENCES
(1) ABRAMSON, H. A.: Electrokinetic Phenomena and their Application to Biology and Medicine, Chap. V. The Chemical Catalog Co., Inc., New York
(1934). (2) ABRAMSON,H.A.: Urolog. Cutan. Rev. 82, No. 4 (1938). (3) ABRAMSON, H.A., AND ALLEY,A.: Arch. Phys. Therapy, X-ray, Radium 18, 327 (1937). (4) ABRAMSON, H.A , , ENOEL,M., LUBKIN,V., AND OCHS,I . : Proc. SOC.Exptl. Biol. Med. 38, 65 (1938). ( 5 ) ABRAMBON, H.A,, AND OCHS,I.: J. Lab. Clin. Med., in press. (6) FURTH, O.,BAUER,H., AND PIECH, H.: Biochem. Z. 100,52 (1919). (7) KUHNE,W.: Arch. Anat. Physiol. 2, 673 (1860). (8) LEVY,M. J.: J. Biol. Chem. 109, 361 (1935). (9) MORTON,W. J.: Cataphoresis. American Technical Book Co., New York (1898). (10) REIN, H.:2. Biol. 84, 41 (1926). (11) STEFAN:Sitzber.' Akad. Wiss. Wien, Math.-naturw. Klasse 79, 11, 176 (1879). (12) SVEDBERO, T.:In Alexander's Colloid Chemistry, Vol. I, p. 838. The Chemical Catalog Co., Inc., New York (1926).
