1094 (6) (7) (8) (9)
(10) (11) (12) (13) (14) (15) (16,
HAROLD A. .4BRAMSON A N D MANUEL H . GORIN
MILLIGAN AND WEISER: J. Am. Chem. SOC. 69, 1670 (1937). SXITH:J. Am. Chem. SOC. 68, 173 (1936). WARREN:Chem. Rev. 26, 237 (1940). WEISER: Inorganic Colloid Chemistry, Vol. I I . The Hydrous Oxides and Hydroxides. John Wiley and Sons, Inc., New York (1935). WEISER AND MILLIGAX: J. Phys. Chem. 98, 513 61934). WEISER .AKD MILLIGAK: J . Phys. Chem. 39, 25 (1935). WEISER A N D MILLIGAN: J. Am. Chem. SOC. 67, 238 (1935). WEIBERAND MILLIGAN: J. Am. Chem. SOC.69, 1670 (1937). WEISER AND MILLIGAN: Chem. Rev. 26, 1 (1939). WEISER, MILLIGAK, AND PURCELL: Ind. Eng. Chem. 32, 1487 (1940). ZACHARIASEX: J. .im. Chem. S O C . 64, 3841 (1932).
SKIK
REACTIOKS.
IX
THEELECTROPHORETIC DEMONSTRATION O F T H E P A T E N T P O R E S OF THE LIVINGHUMAN SKIN;ITS RELATION TO THE CHARGE OF THE SKIN’ HAROLD A. ABRAMSON
AND
MANUEL H. GORIX
Bzological Laboratory, Cold Spring Harbor, Long Island, New York, and the Medical Service of D r . George Baehr and the Laboratories of the Mount S i n a i Hospital, N e w York, New York Heceioed July 9, 19$0 INTRODUCTIOIi
The active agent in dialyzed extracts of giant ragweed, of short ragweed, and of timothy pollen p a y be trmsported by electrophoresis into the human skin (1). Marked whealing is readily produced in the skin of individuals hypersensitive to these comparatively large molecule.;. It becomes of interest to ascertain by what means these substances of comparatively high molecular weight pass through thc skin. There are three main channels of transport to be considered: ( 1 ) the pores or coils of the sweat glands; (2) thc hair follicles and sebaceous glands; (3) the skin itself, that is, the keratin matrix linking the structures mentioned in (1) and (2). The authors have investigated this problem by the electrophoresis of both basic and acid dyes as well as of copper ions. By means of “development” of thc patterns produced by the electrophoretic technic, it is possible not only to trace more accurately the channels traversed during the electrical transport but also to describe more explicitly than hitherto the relationship between the electrical charge of the dyc ion and that of the pores of the skin. 1 Presented at the Seventeenth Colloid Symposium, held a t Ann Arbor, Michigan, June 6-8, 1940.
SKIN REACTIONS.
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HISTORICAL
ilmong those who in the latter part of the nineteenth century were especially interested in the electrical transport of drugs through the skin, was Morton. Morton (15) stated, “The skin and other membranes and tissues of the human body may be regarded as septa, and, this allowed, it follows that electric osmosis may be made to take place through such septa . . . .I prefer the term Electric Diffusion, which may be defined as the capacity possessed by an electric current for diffusing or transporting liquids and whatever they may contain in solution from one polarity toward the other, preferably through porous septa-thus being applicable t o the tissues of the living body.’’ Morton (15), however, rejected the notion that the cataphoresis of ions into the tissues is dependent upon the charge of the particles. He wrote, “The theory of Alrrheniusas to the free and electrically charged ion advances our views :LS to the naturc of electrolysis and electrolytic conduction, but I cannot see that we arc yet entitled to aqsume that the electrolytic double progrrlssion of the ions which occurs in only a small percent of the molecules which constitute the solution is, in reality, that movement known as cataphoresis or electric diffusion. . . .This view would involve the relation and behavior of electrostatically charged tubu1e.i of small calibre, namely, tht. porous septa.” Before Morton wrote his book, J’ergn6s ( l T ) , an electroplater n h o worked in Cuba, found that he had developed on his hand an incurable ulcer that had been produced by contact with solutions of metallic salts. H e accidentally placed his hand in a bath already prepared for plating and found that the negative pole received a metallic coating. In other words, he observed reversed rlectrophoresis of the metal out of the skin and plating of the metal onto the electrode employed. H e followcd this with a similar number of electrophoretic treatments, using the negative pole. This resulted in a iapid (sure of his iilccr. On the basis of this incident the Vergn6s electrochemical bath wa5 dcl-eloped. Morton 115), to iupport his contention that the pores of the skin ncre important, did another interesting experiment. .‘I, also, there mentioned an experiment IT hich I had tried upon myself and which showed the ease with which even solid particles might be driven into tissuc by the action of an electric current. Some finely powdered lampblack or graphite was incorporated with some salicylate of soda and placed on my arm under the positive electrode. The current was turned on and the particles of graphite were carried into the sweat follicles, making small black spots, like bird shot, w hich were so deeply embedded that they did not disappear for several weeks.” This was apparently the first demonstration that particles may go into the living human skin by electrophoresis. Three decades later Rein (16) also noted that dyestuff seemed to be de-
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HAROLD A. ABRAMSON A N D MANUEL H. GORIN
posited in the pores of the skin. He maintained, however, that positively charged dyes were more readily introduced than negatively charged dyes. He explained this selectivity by assuming that negatively charged dyes were restricted in their passage by the negative charge of the keratin. Our findings are contrary to those of Rein. Although not directly connected with the subject matter of this communication, the recent papers of Hopkins, Kesten, and Hazel (9), Harpuder (8), Kovacs (lo), Kovacs, Saylor, and Wright ( l l ) , and Lowenfish (12) may be consulted for related phenomena. METHODS
Commercially available dyes were employed without purification. These were methylene blue (u.s.P. and “vital”), eosin Y, prontosil sodium, sodium indigotetrasulfonate, pontamine blue, patent blue V, sodium phenolsulfonephthalein, neutral red, and basic and acid fuchsin. Concentrations from 0.1 per cent to 2 per cent were used, but, in general, a solution containing 0.5 per cent of the dye gave excellent results. The dye was applied to the skin through a piece of absorbent cotton or canton flannel. The electrophoresis apparatus consisted of the usual galvanic circuit. Current densities from 0.3 to 1.0 milliampere per square centimeter were suitable, the higher currents being comfortably borne with the positive pole. Observations were made ( 1 ) without magnification, (2) with a hand lens, or ( 3 ) with a Bausch and Lomb wide-angle dissecting microscope. The patterns obtained were photographed by means of a specially designed attachment to an Argus csmera. It allowed the camera to be held against the area to be photographed and a t a fixed distance from it. The distance was chosen to make the ratio of image to object 1:l. Pictures obtained in this way were enlarged for detailed study. EXPERIMENTAL
If a pledget of cotton wet with a 1 per cent solution of methylene blue is held against the skin of the forearm or of the palm of the hand, the skin stains fairly uniformly. With vigorous rubbing and washing the stained cornified layer of the skin may usually be removed, with no evidence of marked selective adsorption of the dyestuff by any particular skin structure. If, however, the pledget of cotton is made the positive pole of an electrophoretic circuit for 1 to 5 min., selective adsorption by the skin can be demonstrated. On superficial examination of the blue-stained area, differential staining of the skin surface may not be observed. If, however, the stained area of the skin is vigorously rubbed and washed free of the blue dye, a remarkable pattern of the channels traversed by the dye is
SKIK REACTIONS.
IS
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"developed." h n example of this pattcrii is illustrated in figure 1 (magnified 3 x). Close examination of the bkin in experiments of thi. type disclosed that the blue dots were the iites of the pow\ of the skin,-thc orifices of the coils of the sweat glands. Little or no methylene blue n a s usually observed a t the bases of the hair follicles, although exceptions occurred. The electrical transport of dye through the skin of the palm of the hand showed that the hair follicles need not be involved at all. Development of the pattern in the palm is more difficult. In certain adult cases the final pattern developed w w a l days after electrophoresis of the dye, as the superficial epithelium was lost. With the uncalloused hand of a three-year-old child, however, porr patterns can be developed in the usual way. Figure 2 is a photograph of a pore pattern obtained in an adult on the hypothenar eminence. Pore patterns produced with methylene blue, by the technic describcd in the foregoing may remain clearly visible for several weeks. The pattern nearly always remains visible for a week. In the course of time an interesting transformation may occur in the appearance of the individual spot. of blue which comprise the pattern. Thii is observed in figures 3a and 3b, which are photographs of pore patterns obtained after 24 hr. and 1 week. respectively. S o t e that the blue spots which have within them paler white areas have taken on the appearancc of doughnut forms. Examination of the size and structure of these doughnut forms indicates that the methylene blue which persists is due to a selective staining of the walls of the sweat glands. The pale areas observed in the centers of the doughnut forms are the orifices of the sweat glands, which gradually clear themselves of the methylene blue contained within the ducts. It is surprising that the methylene blue is not reduced to the leuco form. Evidently the diffusion of oxygen into the cells of the duct is sufficiently great to keep the dyestuff in the oxidized state. One of us with Taylor (6) has qhown that adsorbed dyes may be reduced, but more slowly in all cases studied. In certain instances it has been observed that the cells of the ducts containing methylene blue grow outward from the skin. Tiny papillae may then be seen under the microscope at the sites of the bluestained pores. S o t all of the pores may be stained by methylene blue. This can be readily demonstrated by following the electrophoresis of methylene blue with a second electrophoresis of eosin Y . By this means, red staining pores not previously stained by the first application of methylene blue may in certain instances be observed to appear. According to Rein (16), a negative charge on the skin surface facilitates the passage of positively charged basic dyes, whereas it retards the passage of negatively charged acid dyes. This is in decided contrast with our experience. Not only h a l e we obtained definite patterns Kith positive dyes like patent blue V and basic fuchsin, but we have obtained similar
1098
HhROLD 1. ABlUhlSON AND MANUEL H. GOliIN
patterns using the negative pole with neutral red, eosin Y, sodium prontosil, sodium indigotrt.rasulEonate,sodium phenolsulfont.phthalein,and acid
Fm. 1. (uppor left) Magnification 3.2 X. Pore pattern developed following electrophoresis of methylene blue into tlre skin of the nnteiior aspect of forearm of mnle adult. Not,e the arrangement of the date in rows. Fro. 2. (upper right) Eleotrophomais of methylene blue into the pores of the palm of tFc hand. S o t e the more orderly arrangement of the pores in this region (hypothenar ominenoe). Millimeter soale. Fro. 3. B (lower left): doughnut forms appearing 24 hr. after electrophoresis of !methylene blue into tho pores of the anterior sspeet ai the forearm. b (lower right): note the persistence of the pattern 1 reek niter the electrophoresis of the methylene blue. Millimeter 8 c ~ l e .
S K I N IlEbCTlONS.
IX
1099
fuchsin. Figures 4 and 5 are illustrations of pattwns ohtsined hy sodium phenolsulfonephthalein and by sodium indigotrtrasulfonate. There are differcnces in t,he skin react,ions of crrt,ain individuals. Thus, in subject E. A:A. introduction of iodiom phenolsulfonepl~thnlr'inrciiiltcd in a red pattern which rhangrd to yelk~win time, t h r yrllom patt.t'rn remaining for about a \>-e& In an cxpcrimmt on d. (?., \vl.iio trnd a
Frc. 4. (upper loft) Negatively charged dyes like pltenol indiRot,etmsulfonatc form definite pore patterns in the skin. Note the more diffusr ncture of the pettern when compared with positively charged methylene blrio crnployed to obtain the pattern in figure 1. Magnification 3.2 x . FIG.5. (upper right) Another illustration of x pore pattern obtainod with phenol red, a negatively charged dye. The pattern appears to he more diffuse in this cme also. Magnification 3.2 x. Fic. 6. (lower left) Pore pattern in a war over 30 gcars old; natural siao FIG.7. (lower right) Pore psttcrn in psoriasis; natural size
different type of pore pattern, the red stage w&$ not obsrwed. Evidently in the cells of the pores the dye is in a diffment statr than in the ducts of the pores. This is not unexpected. In experiments with sodium indigotetradfonate-doughnut forms appeared much more readily arid rapidly than in the case of methylene blue. The rapid appearance of the doughnut forms is probably caused by reduction of some of the dye in speeial regions, following its introduction
1100
HAROLD -1. ABRAMSON AND MANUYL H. GORIN
into thr skin. In spit0 of this rapid initial reduction the pore pattern may remain visible for a week with large doughnut forms. Apparently the dyc which has been absorbed by the cells lining the coils of the pores is not readily reduced. I n view of the development of recent technics (7) designed to introduce copper by rlectrical transport in the treatment of fungus diseases of the skin, the path taken by the copper ion was also studied. When copper is introduced into the skin by electrophoresis with the technic described in the foregoing, tiny red dots due to irritation appear and form a pattern of their own without the introduction of any dyestuff. It is not difficult to demonstrate that copper has been introduced through the pores, apparently in the same manner. Chemical tests in the pores for copper were positive. APPLICATION TO DISEaSES OF THE SKIN
Up to this point the experiments which have been described deal with the reaction of the normal skin. There is, however. a wide field of application to dermatological problems. Thus, scars and skin diseases like psoriasis, etc., may be investigated in this way. In figures 6 and 7 are illustrations of the type of application possible. Figure 6 is a pore pattern of a scar 30 years old, and figure 7 is a pore pattern in a lesion of psoriasis. It is hoped that the application of these technics to the study of skin diseases will lead to a more explicit structural and functional comprehension of the rale that the pores of the skin play in these diseases. DISCUSSION
The adherents of the pore or sieve theory of skin permeability (e.g., Rein (12)) maintain that the charge on the pores determines the restrictive forces which operate on the permeability of the membrane. Thus, negatively charged rollodion membranes are well known to restrict the passage of negative ions but to permit positive ions to pass freely (13). In support of the point of view that the charge on the membrane determines the restrictive forces, Mond and Hoffmann (14) have shown that reversal of the sign of charge of the membrane by a dyestuff, rhodamine R, changes the nature of the permeability. This dye, by reversing the sign of the ehargc on collodion membranes, presumably permitted the passage of negative ions but restricted the passage of positive ions. It is evident that the pores of the skin readily permit the passage of both positively charged dye ions and negative ions like the phenolsulfonephthalein ion, in spite of the skin’s negativc charge,. Indeed, one may easily imagine, according to Rein’s scheme, that as positive ions are introduced into the skin they would reverse the sign of the surface charge, making the walls of the pores of the skin positive and in that way would progressively tend
SKIN REACTIONS.
IS
1101
to restrict their own passage by reversal both of the charge and of the electroosmotic flow. I t can be readily observed that if a dye is introduced electrophoretically for 0.5, 2, and 5 min., respectively, on separate sites, much more of the dye goes in as the experimental time increases. This illustrates that the adsorbed positive methylene blue ion apparently does not restrict its own passage as it accumulates on the cell surfaces. Similar observations have been made with other dyes. We are unable, therefore, to correlate the charge of the skin with the passage of the dyes under these circumstances. However, it would be of interest to make skin potential measurements of the type performed by Rein to determine whether these dyes do change the sign of the charge of the membrane which is assumed to give rise to the potentials observed. It is useful t o have the physical picture of the doughnut form in figures 3a and 3b. This enables us t o visualize the pores of the skin discussed in detail (3) in a recent communication. It had been previously found that, contrary to the behavior of ordinary ions, the biologically active substance of giant and dwarf ragweed pollens migrated into the skin, not only from one pole but from both poles (1). In the case of giant ragweed, with the solutions employed, the positive pole was more efficacious than the ncgative pole; in the case of dwarf ragweed, the negative pole was more efficient. This type of electrophoretic behavior was unprecedented, for, as stated previously, usually only one pole is able to introduce an ion of the proper sign. The data were also not in keeping with preliminary measurements made on the electrical charge of the biologically active constituent of the ragweed pollen (5). More recently a single constituent which is negatively charged a t pH 7.0 has been isolated (4) in the Tiselius apparatus. This negatively charged constituent can be introduced into the skin with the positive pole. The fact that the skin acts as a reservoir for histamine and epinephrine, following electrophoresis of these substances (3, 2), may be correlated to a certain point with the persistence of dyes in the cells of the pores over long periods. Since histamine may be demonstrated in the skin for as long a period as a week, there must be some mechanism which prevents diffusion into the dermis. Although this mechanism is probably in some respects unlike that operating in the case of dyestuffs, the persistent pore patterns found for the dyes are presumptive evidence that a mechanism restricting diffusion does exist and that a similar general process may retain the histamine depots. Obstruction of the pores need not prevent the formation of electrophoretic patterns. To investigate the effect of. plugging the pores, an area was treated with albolene before electrophoresis of methylene blue. Although the initial resistance of the skin so treated was very much higher than that of a normal area, with sufficient voltage the resistance soon
1102
HAROLD .I. ABRAMSON A N D MANUEL H . GORIN
dropped to normal. With a current density of 0.5 milliampere for 5 min., a typical pore pattern was obtained. It is possible that electroosmotic forces drive the albolene out of the pores. SUMMARY
When an electric current is applied across the living human skin, the skin may be considered to act like a system of pores through which transfer of substances like ragweed pollen extract may bc achieved both by electrophoretic and by diffusion phenomena. The sites of these pores have been localized by forcing dyestuffs electrically into the skin, thus localizing the channels traversed. Both basic and acid dyes, as well as copper ions, localize primarily in the sweat glands, and pore patterns of the skin may be “developed” following electrophoretic transfer of basic dyes, acid dyes, and metallic ions. The pore patterns may persist for several weeks. During this time some of the small colored spots may decolorize in the center, leaving colored doughnut forms which correspond to selective staining of the cells comprising the orifices of the glands. Electrically patent pores may be plotted over the entire body. The pore patterns in scar tissue and in skin diseases like scleroderma and psoriasis may also be mapped, and in this way characteristics of these diseases be defined. REFERENCES (1) ABRANSON, H . A.: Science 87, 299 (1938);K. Y. State J. Med. 39, 611 (1939). H . A.: Arch. Phys. Therapy, X-ray, Radium 21, 261 (1940). (2) ABRAYSON, (3) ABRAMSON, H. A,, AND GORIN,M. E . : J. Phys. Chem. 49, 3% (1939);Chem. Products (London), February, 1940. (4) ABRAMSON, H. A., MOORE,D. H . , GETTNER, H . , GAGARIX, J., AND JENNINGS, L.: J. Am. Chem. SOC. 62, 1627 (1940);Proc. SOC.Exptl. Biol. Med. 44, 311 (1940). (5) ABRAMSON, H . A,, SOOKNE, A. M., AND MOYER,L. S.: J. Allergy 10, 317 (1939). (6) ABRAMSON, H.A., AND TAYLOR, I. : J. Phys. Chem. 40,519 (1936). H. W.,STRAUES, M. J., AND GREENBERG: J. Am. Med. Assoc. 112, (7) HAGGARD, 1229 (1939). (8) HARPUDER, K . : N. Y. State J. Med. 38, 176 (1938). J. G . , KESTEN,B. M., AND HAZEL,0. G . : Arch. Dermatol. Syphilol. (9) HOPKINS, 88, 679 (1939). (10) KOYACS,J.: Am. J. Med. Sci. 188, 32 (1934). (11) KOYACS,J., SAYLOR, L. L., AND WRIGHT,I. S.: Am. Heart J. 11, 53 (1936). F. P.: Arch. Dermatol. Syphilol. 37, 797 (1938). (12) LOWENFISH, (13) MICHAELIS, L.:Bull. Natl. Research Council, No. 69 (1929). F.: Arch. ges. Physiol. 220, 194 (1928). (14) MOND,R., AND HOFFMAN, (15) MORTON,W. J.: Cataphoresis or Electric Medzcamental Surgery. American Technical Book Company, New York (1898). (16) REIN, H . : Z.Biol. 85, 195 (1926). (17) V E R G N ~ SIn: Morton’s Cataphoresis or Electric Medicamental Surgery, p. 21. American Technical Book Company, New York (1898).
