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On the Diffusion of Tritiated Water Through Skin
Vol. 55, No. 3
TNE JOURNAL OF INYESTIOATIYE DERMATOLOGY
Copyright
Printed in U.S.A.
1566 by The Williams & Wilkins Co.
LETTER TO THE EDITOR ON THE DIFFUSION OF TRITIATED WATER THROUOH SKIN
THOMAS J. FRANZ, CPT, MC
A recent paper in this journal by Downes et Their results demonstrate one of the fundamenal (1) reported on the use of a simple in Vitro tal properties of the tracer technique, that it is method for determining the diffusion of water impossible to assess net movement simply from through skin by measuring the unidirectional flux of tritiated (radioactive water. The method consists in mounting a piece of skin as a barrier be-
the measurement of a unidirectionol flux. In the
tween two glass chambers and measuring the appearance of tritiated water in the solution
some 300—400 mg/cm2/hr. With a small net movement of 5 mg/cm2/hr the unidirectional
bathing one side of the skin after a known
flux was still over 300 mg/cm2/hr. Treatment of the bladder with vasopressin resulted in a forty-
absence of any net movement of water, Rays
and Leaf measured a unidirectional flux of
amount has been added to the solution bathing
the opposite side of the skin. The method is
fold increase in net water movement, but only a twofold increase in the unidirectional flux. Results demonstrating the same phenomenon have also been obtained in studies on frog skin and toad skin (3). Although useful information can be obtained from the measurement of tritiated water diffusion across the skin, the method described by Downes et al will not allow one to obtain information on the true rate of water loss through the skin. It is unfortunate that earlier workers, who actually measured net water loss, also used
indeed a valid one for assaying the diffusion of water through skin, however, the authors offer it as a substitute for earlier, more difficult, methods which also claim to measure the diffusion of water. The basic problem is as follows. Although the method of Downes et al measures the diffusion of water through the skin, it does not measure net movement of water. The value of 0.49 mg/ cm2/hr cited by them as the rate of diffusion of water through mouse skin does not mean that that much water is being lost from one of the chambers, by passage into the other chamber. Indeed, with isotonic saline on both sides of the skin there should be no real movement, no net
the term diffusion in connection with their measurements; for present evidence indicates that water movement across both living and non-living membranes in response to a pressure gradient is largely non-diffusional in nature (2, 3, 4, 5). Thus, until more definitive experiments are done on skin, it would be better to think of water movement through it in response to a
movement of water (barring the presence of active transport mechanisms). The 0.49 mg/ cm2/hr measured to be moving through the skin in one direction is balanced by an equal amount moving through in the opposite direction. This is simply an exchange of water and takes place
pressure difference as being non-diffusional.
across all membranes even when there is no reol movement of water, no net movement, taking place.
In contrast to this work the earlier methods (usually gravimetric and admittedly more difficult) measured the net amount of water travers-
Derm., 49: 230, 1967. 2. Hays, R. M. and Leaf, A.: Studies on the movement of water through the isolated toad blad-
der and its modification by vasopressin. J.
ing the skin, that which is lost from one side and not replaced. That the two methods can
Cen. Physiol., 45: 905, 1962.
give vastly different values is clearly exemplified
by the work of Hays and Leaf (2) who studied the movement of water across the isolated toad bladder using both methods simultaneously. From The Dermatology Research Program, U.S. Army Medical Research Unit, Presidio, San Francisco, California 94129.
REFERENCES 1. Downes, A. M., Sweeney, T. M. and Matolsty, A. G.: Studies of epidermal water barrier, 1. improved in vitro method for determining the diffusion of water through the skin. J. Invest.
3. Koefoed-Johnsen, V. and Ussing, H. H.: The contributions of diffusion and flow to the passage of D2O through living membranes. Acta. Physiol. Scand., 28:
60, 1953.
4. Robbins, E. and Mauro, A.: Experimental study of the independence of diffusion and hydrodynamic permeability coefficients in collodion membranes. J. Gen. Physiol., 43: 523, 1960.
5. Mauro, A.: Nature of solvent transfer in osmosis. Science, 126: 252, 1957.
260
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
TNE JOURNAL OF INYESTIOATIYE DERMATOLOGY
Copyright
Printed in U.S.A.
1566 by The Williams & Wilkins Co.
LETTER TO THE EDITOR ON THE DIFFUSION OF TRITIATED WATER THROUOH SKIN
THOMAS J. FRANZ, CPT, MC
A recent paper in this journal by Downes et Their results demonstrate one of the fundamenal (1) reported on the use of a simple in Vitro tal properties of the tracer technique, that it is method for determining the diffusion of water impossible to assess net movement simply from through skin by measuring the unidirectional flux of tritiated (radioactive water. The method consists in mounting a piece of skin as a barrier be-
the measurement of a unidirectionol flux. In the
tween two glass chambers and measuring the appearance of tritiated water in the solution
some 300—400 mg/cm2/hr. With a small net movement of 5 mg/cm2/hr the unidirectional
bathing one side of the skin after a known
flux was still over 300 mg/cm2/hr. Treatment of the bladder with vasopressin resulted in a forty-
absence of any net movement of water, Rays
and Leaf measured a unidirectional flux of
amount has been added to the solution bathing
the opposite side of the skin. The method is
fold increase in net water movement, but only a twofold increase in the unidirectional flux. Results demonstrating the same phenomenon have also been obtained in studies on frog skin and toad skin (3). Although useful information can be obtained from the measurement of tritiated water diffusion across the skin, the method described by Downes et al will not allow one to obtain information on the true rate of water loss through the skin. It is unfortunate that earlier workers, who actually measured net water loss, also used
indeed a valid one for assaying the diffusion of water through skin, however, the authors offer it as a substitute for earlier, more difficult, methods which also claim to measure the diffusion of water. The basic problem is as follows. Although the method of Downes et al measures the diffusion of water through the skin, it does not measure net movement of water. The value of 0.49 mg/ cm2/hr cited by them as the rate of diffusion of water through mouse skin does not mean that that much water is being lost from one of the chambers, by passage into the other chamber. Indeed, with isotonic saline on both sides of the skin there should be no real movement, no net
the term diffusion in connection with their measurements; for present evidence indicates that water movement across both living and non-living membranes in response to a pressure gradient is largely non-diffusional in nature (2, 3, 4, 5). Thus, until more definitive experiments are done on skin, it would be better to think of water movement through it in response to a
movement of water (barring the presence of active transport mechanisms). The 0.49 mg/ cm2/hr measured to be moving through the skin in one direction is balanced by an equal amount moving through in the opposite direction. This is simply an exchange of water and takes place
pressure difference as being non-diffusional.
across all membranes even when there is no reol movement of water, no net movement, taking place.
In contrast to this work the earlier methods (usually gravimetric and admittedly more difficult) measured the net amount of water travers-
Derm., 49: 230, 1967. 2. Hays, R. M. and Leaf, A.: Studies on the movement of water through the isolated toad blad-
der and its modification by vasopressin. J.
ing the skin, that which is lost from one side and not replaced. That the two methods can
Cen. Physiol., 45: 905, 1962.
give vastly different values is clearly exemplified
by the work of Hays and Leaf (2) who studied the movement of water across the isolated toad bladder using both methods simultaneously. From The Dermatology Research Program, U.S. Army Medical Research Unit, Presidio, San Francisco, California 94129.
REFERENCES 1. Downes, A. M., Sweeney, T. M. and Matolsty, A. G.: Studies of epidermal water barrier, 1. improved in vitro method for determining the diffusion of water through the skin. J. Invest.
3. Koefoed-Johnsen, V. and Ussing, H. H.: The contributions of diffusion and flow to the passage of D2O through living membranes. Acta. Physiol. Scand., 28:
60, 1953.
4. Robbins, E. and Mauro, A.: Experimental study of the independence of diffusion and hydrodynamic permeability coefficients in collodion membranes. J. Gen. Physiol., 43: 523, 1960.
5. Mauro, A.: Nature of solvent transfer in osmosis. Science, 126: 252, 1957.
260
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.