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Cutaneous Resorption of Heparin and Heparinoids Investigated by Autoradiography*
Vol. 51, No.2 Printed in U.S.A.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
1968 by The Williams & Wilkins Co.
Copyright
CUTANEOUS RESORPTION OF HEPARIN AND HEPARINOIDS INVESTIGATED BY AUTORADIOGRAPHY* K. REBER, M.D.
The question whether heparin and hep- tion was carried out, partly on unstained slides, arinoids are absorbed after cutaneous application still remains controversial. Jorpes in his extensive review (1) on heparin does not even mention this way of application. Heparin ointments are not mentioned at the "Symposium on Heparin" related in the American Journal of Cardiology 1964 (2). Finne denies any effectiveness of a heparinoid ointment (3). So does Konrad in a study with cattle (4). On the other hand there are numerous papers mainly of clinical origin which claim some effectiveness of such ointments. Histochemical attempts to demonstrate absorbed heparin (5, 6) are not convincing in our view for reasons dealed with below.
partly on slides stained with toluidine blue. The
method is open to two objections:
1. There is the risk that the label is enzymati— cally split off from the compound. The aittoradiog-
raphy shows, strictly speaking, only the locali-
zation of S, and not that of the biologically
active compound. Positive findings would only indicate therefore that S35 (or sulfate) had penetrated into the skin but this would not prove the presence of the biologically active substance itself. This possibility exists, though we estimate it to be extremely low, since intracutaneously or subcutaneously injected heparin displays its undiminished biological activity, whereas desuhfated heparin remains inactive. 2. The labeled substance could diffuse into the
tissue during the preparation of the histological sections, for instance during fixation of the tissue or during thawing of the cryostat sections or dur-
Clear cut proofs for a systemic effectiveness ing other phases of preparation, simulating absorp-
in humans of epicutaneously applied hep- tion. It is difficult to exclude these possibilities by arinoids are lacking. The absorption, if any, adequate controls. However, this bias is extremely must be very small. Therefore, we attempted unlikely, considering our results (see below). to show, at least locally, absorbed heparin in RESULTS AND DISCUSSION the tissue by means of autoradiography which
seemed especially suitable due to its high
No difference could be found between chem-
sensitivity.
ically depilated and electrically shaved skin, nor between the various heparinoids used, nor MATERIAL AND METHODS with regard to the time at which the skin was We used S labelled heparin, the heparinoid Ro taken after application of the ointment, nor 1-8307 and several other S labelled heparin de- between cryostat and paraffin sections. Radiorivatives with a radioactivity of 0.5 to 1.0 milhi— activity was limited to the epidermis and to curie per rng in the form of an ointment containing 5% of heparin or heparinoid. The S label was the surface of the hair, penetrating into the
depth of the hair follicles. In a few cases a To test each substance, 6 rats with a body diffuse radioactivity of a limited degree could weight of approximately 200 gm were depilated on be shown in the most superficial layers of the the left side with a depilatory ointment and elecintroduced in the N-sulfo-groups.
trically shaved on their right side. 0.5 gm of the coriurn, especially in the immediate vicinity of radioactive ointment was rubbed in during 10 the hair follicles or adjoining glands (Figs. 1— minutes in an area measuring about 2 cm in 3). The identical findings in Carnoy fixed tisdiameter. After 30 minutes, 2 and S hours, two siie and in cryostat sections as well as the animals were killed. The treated skin was care- sharp localization of the label usually found fully washed with water and soap, excised and the pieces cut in two halves. One half was fixed in (for instance on the surface of hairs) speak Carnoy, embedded in paraffin and cut into 5 against the possibility of the radioactivity being
i
thick sections. The other half was cut on a cryostat without fixation. Cryostat and paraffin sections were covered with
an artefact produced by diffusion during the histological preparation. Control slides from stripping film Kodak AR 10 and exposed for 4 untreated animals never gave rise to silver After development microscopical evaluaT * From the Department of Experimental Medi-
weeks.
cine, F. Hoffrnann-La Roche & Co., Ltd., Basle. 113
grain formation. In conclusion, we estimate it, therefore, as probable that the small amounts of radioactivity found beneath the epidermis
114
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
-
Autoradiographies from unstained sections. Skin inuncted with labelled heparin ointment. All sections cut perpendicular to the skin surface.
Fio. 1. Radioactivity hmited mainly to the epidermis, penetrating only scarcely into
the deeper layers of the cutis. X 120.
Fio. 2. Longitudinal section of a hair. Radioactivity limited strictly to its surface.
>< 240.
FIG. 3.
X 187.
Radioactivity in thc immediate vicinity of hair follicles and adjoining glands.
indicate pcnetration of labeled heparin (oid) through the epidermis in vivo.
Preparations stained with toluidine blue showed that there is no parallelism between radioactivity and metachromasia. On the other
concentration of a label in a tissue but they cannot supply information on the movements of this label. Though we can only find small amounts of label in all preparations, it is possible that significant amounts have been ab-
hand, the numerous and strongly metachro- sorbed. matically colored mast cells as well as other In fact, this assumption seems probable, if metachromatically colored structures showed no radioactivity. The Offhand explanation appears
we consider our knowledge on the effectiveness
to be that toluidine blue staining is much less sensitive compared to the autoradiographic method and that therefore the absorbed quantities of heparin or heparinoids must be very small, as compared to the quantities of lieparin already present in mast cells and other structures. In interpretation of our findings, one must hear in mind that these preparations like all histological preparations are "snapshots". They may provide a picture of the momentary
dermis. Scheuplein (7) states ". . . the rich vascularity of the superficial layers of the
and intensity of microcirculation within the dermis should drastically limit the number of molecules free to diffuse deeper into the dermis . . ." and ". . . most molecules reaching the vascularized region . . . would be swept into
systemic circulation. The stratum corneuin limits the accumulation of substances within the viable epidermis by preventing their entrance except at rates so slow that the relatively rapid diffusion from the aqueous viable
115
CUTANEOUS ABSORPTION OF HEPARIN
cells to the cutaneous microcirculation is adequate to maintain their concentration at very low levels".
SUMMARY
Autoradiography after epicutaneous application of S35 labeled heparin and heparinoid oint-
The assumption that considerable amounts of ments shows small amounts of radioactivity heparin may cross the epidermal barrier is beneath the epidermal barrier. Despite possible corroborated by the observation that even technical errors, this finding is interpreted as
systemic effects may be observed in rats with chemically depilated skin (8).
indicating penetration of the labeled substances
in vivo. The concentration of radioactivity Our fiidings are in good agreement with found in the tissue is very small because the those of MacKee et al. (9) who studied the absorbed substances are immediately transproblem of percutaneous penetration in a ported away. Accumulation in defined strucgeneral manner as early as in 1945. Their con-
tures beneath the epidermal barrier could not clusion: "the most likely route of maximum be demonstrated. penetration of the inuncted materials was as follows: From the outside into the horny layer; follicle openings; down the follicle, out of the follicle and into the cutis ." is corroborated
REFERENCES 1. Jorpes, J. E.: Heparin, its chemistry, pharmacology and clinical use. Amer. I Mcd., 33: 692, 1962.
by our observations, with the limitation that 2. Symposium on Heparin. Amer. J. Cardiol., 14: 19M. only very small quantities of material have 3. Finne, P. H.: Hirudoid-anticoagulant ointment. been found in our experiments.
A failure to demonstrate significant effect. Ada med. scand., 172: 289, 1962. The histological findings of Wegmann and 4. Konrad, F. M.: Klinische Tlntersuchungen an
Garraud (6) and Lindner (5) who claim an Grosstieren die perkutane Wirkung und Anwendbarkeit antikoagulämischer Stoffe in increase of metachromatic substance in the Salbenform. Chr. Vet. 64—115/Diss., Leipzig, tissue after application of heparin ointments 1963. could not be reproduced in our work. The 5. Lindner, J.: Die perkutane resorption von heparin. Med KIm., 58: 1374, 1963. variations in the distribution pattern of the 6. Wegmann, R. and Garraud, R.: Le probléme de rnetachromatic substances from one section to
l'absorption cutanée de l'héparine. Etude his-
tochimique. Presse Med., 69: 898, 1961. the other, even from one part of the same 7. Scheuplein, ii.. J.: Mechanism of percutaneous section to the other, are remarkable. Thereabsorption. II. Transient diffusion and the
relative importance of various routes of skin fore, the increase in content of metachromatic penetration. J. Invest. Derm., 48: 79, 1967. substance would have to be very large indeed 8, Reber, K.: Clearing factor activation by application of heparin ointment to rats. In press. to reach the level of significance. Pictures as G. M., Sulzberger, M. B., Hermann, published in the above mentioned papers can 9. MacKee, F. and Baer, R. L.: Histologic studies on percutaneous penetration with special refereasily be obtained from the same single prepaence to the effect of vehicles. J. Invest. Derm., ration; thus, they carry no conviction. 6: 43, 1945.
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.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
1968 by The Williams & Wilkins Co.
Copyright
CUTANEOUS RESORPTION OF HEPARIN AND HEPARINOIDS INVESTIGATED BY AUTORADIOGRAPHY* K. REBER, M.D.
The question whether heparin and hep- tion was carried out, partly on unstained slides, arinoids are absorbed after cutaneous application still remains controversial. Jorpes in his extensive review (1) on heparin does not even mention this way of application. Heparin ointments are not mentioned at the "Symposium on Heparin" related in the American Journal of Cardiology 1964 (2). Finne denies any effectiveness of a heparinoid ointment (3). So does Konrad in a study with cattle (4). On the other hand there are numerous papers mainly of clinical origin which claim some effectiveness of such ointments. Histochemical attempts to demonstrate absorbed heparin (5, 6) are not convincing in our view for reasons dealed with below.
partly on slides stained with toluidine blue. The
method is open to two objections:
1. There is the risk that the label is enzymati— cally split off from the compound. The aittoradiog-
raphy shows, strictly speaking, only the locali-
zation of S, and not that of the biologically
active compound. Positive findings would only indicate therefore that S35 (or sulfate) had penetrated into the skin but this would not prove the presence of the biologically active substance itself. This possibility exists, though we estimate it to be extremely low, since intracutaneously or subcutaneously injected heparin displays its undiminished biological activity, whereas desuhfated heparin remains inactive. 2. The labeled substance could diffuse into the
tissue during the preparation of the histological sections, for instance during fixation of the tissue or during thawing of the cryostat sections or dur-
Clear cut proofs for a systemic effectiveness ing other phases of preparation, simulating absorp-
in humans of epicutaneously applied hep- tion. It is difficult to exclude these possibilities by arinoids are lacking. The absorption, if any, adequate controls. However, this bias is extremely must be very small. Therefore, we attempted unlikely, considering our results (see below). to show, at least locally, absorbed heparin in RESULTS AND DISCUSSION the tissue by means of autoradiography which
seemed especially suitable due to its high
No difference could be found between chem-
sensitivity.
ically depilated and electrically shaved skin, nor between the various heparinoids used, nor MATERIAL AND METHODS with regard to the time at which the skin was We used S labelled heparin, the heparinoid Ro taken after application of the ointment, nor 1-8307 and several other S labelled heparin de- between cryostat and paraffin sections. Radiorivatives with a radioactivity of 0.5 to 1.0 milhi— activity was limited to the epidermis and to curie per rng in the form of an ointment containing 5% of heparin or heparinoid. The S label was the surface of the hair, penetrating into the
depth of the hair follicles. In a few cases a To test each substance, 6 rats with a body diffuse radioactivity of a limited degree could weight of approximately 200 gm were depilated on be shown in the most superficial layers of the the left side with a depilatory ointment and elecintroduced in the N-sulfo-groups.
trically shaved on their right side. 0.5 gm of the coriurn, especially in the immediate vicinity of radioactive ointment was rubbed in during 10 the hair follicles or adjoining glands (Figs. 1— minutes in an area measuring about 2 cm in 3). The identical findings in Carnoy fixed tisdiameter. After 30 minutes, 2 and S hours, two siie and in cryostat sections as well as the animals were killed. The treated skin was care- sharp localization of the label usually found fully washed with water and soap, excised and the pieces cut in two halves. One half was fixed in (for instance on the surface of hairs) speak Carnoy, embedded in paraffin and cut into 5 against the possibility of the radioactivity being
i
thick sections. The other half was cut on a cryostat without fixation. Cryostat and paraffin sections were covered with
an artefact produced by diffusion during the histological preparation. Control slides from stripping film Kodak AR 10 and exposed for 4 untreated animals never gave rise to silver After development microscopical evaluaT * From the Department of Experimental Medi-
weeks.
cine, F. Hoffrnann-La Roche & Co., Ltd., Basle. 113
grain formation. In conclusion, we estimate it, therefore, as probable that the small amounts of radioactivity found beneath the epidermis
114
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
-
Autoradiographies from unstained sections. Skin inuncted with labelled heparin ointment. All sections cut perpendicular to the skin surface.
Fio. 1. Radioactivity hmited mainly to the epidermis, penetrating only scarcely into
the deeper layers of the cutis. X 120.
Fio. 2. Longitudinal section of a hair. Radioactivity limited strictly to its surface.
>< 240.
FIG. 3.
X 187.
Radioactivity in thc immediate vicinity of hair follicles and adjoining glands.
indicate pcnetration of labeled heparin (oid) through the epidermis in vivo.
Preparations stained with toluidine blue showed that there is no parallelism between radioactivity and metachromasia. On the other
concentration of a label in a tissue but they cannot supply information on the movements of this label. Though we can only find small amounts of label in all preparations, it is possible that significant amounts have been ab-
hand, the numerous and strongly metachro- sorbed. matically colored mast cells as well as other In fact, this assumption seems probable, if metachromatically colored structures showed no radioactivity. The Offhand explanation appears
we consider our knowledge on the effectiveness
to be that toluidine blue staining is much less sensitive compared to the autoradiographic method and that therefore the absorbed quantities of heparin or heparinoids must be very small, as compared to the quantities of lieparin already present in mast cells and other structures. In interpretation of our findings, one must hear in mind that these preparations like all histological preparations are "snapshots". They may provide a picture of the momentary
dermis. Scheuplein (7) states ". . . the rich vascularity of the superficial layers of the
and intensity of microcirculation within the dermis should drastically limit the number of molecules free to diffuse deeper into the dermis . . ." and ". . . most molecules reaching the vascularized region . . . would be swept into
systemic circulation. The stratum corneuin limits the accumulation of substances within the viable epidermis by preventing their entrance except at rates so slow that the relatively rapid diffusion from the aqueous viable
115
CUTANEOUS ABSORPTION OF HEPARIN
cells to the cutaneous microcirculation is adequate to maintain their concentration at very low levels".
SUMMARY
Autoradiography after epicutaneous application of S35 labeled heparin and heparinoid oint-
The assumption that considerable amounts of ments shows small amounts of radioactivity heparin may cross the epidermal barrier is beneath the epidermal barrier. Despite possible corroborated by the observation that even technical errors, this finding is interpreted as
systemic effects may be observed in rats with chemically depilated skin (8).
indicating penetration of the labeled substances
in vivo. The concentration of radioactivity Our fiidings are in good agreement with found in the tissue is very small because the those of MacKee et al. (9) who studied the absorbed substances are immediately transproblem of percutaneous penetration in a ported away. Accumulation in defined strucgeneral manner as early as in 1945. Their con-
tures beneath the epidermal barrier could not clusion: "the most likely route of maximum be demonstrated. penetration of the inuncted materials was as follows: From the outside into the horny layer; follicle openings; down the follicle, out of the follicle and into the cutis ." is corroborated
REFERENCES 1. Jorpes, J. E.: Heparin, its chemistry, pharmacology and clinical use. Amer. I Mcd., 33: 692, 1962.
by our observations, with the limitation that 2. Symposium on Heparin. Amer. J. Cardiol., 14: 19M. only very small quantities of material have 3. Finne, P. H.: Hirudoid-anticoagulant ointment. been found in our experiments.
A failure to demonstrate significant effect. Ada med. scand., 172: 289, 1962. The histological findings of Wegmann and 4. Konrad, F. M.: Klinische Tlntersuchungen an
Garraud (6) and Lindner (5) who claim an Grosstieren die perkutane Wirkung und Anwendbarkeit antikoagulämischer Stoffe in increase of metachromatic substance in the Salbenform. Chr. Vet. 64—115/Diss., Leipzig, tissue after application of heparin ointments 1963. could not be reproduced in our work. The 5. Lindner, J.: Die perkutane resorption von heparin. Med KIm., 58: 1374, 1963. variations in the distribution pattern of the 6. Wegmann, R. and Garraud, R.: Le probléme de rnetachromatic substances from one section to
l'absorption cutanée de l'héparine. Etude his-
tochimique. Presse Med., 69: 898, 1961. the other, even from one part of the same 7. Scheuplein, ii.. J.: Mechanism of percutaneous section to the other, are remarkable. Thereabsorption. II. Transient diffusion and the
relative importance of various routes of skin fore, the increase in content of metachromatic penetration. J. Invest. Derm., 48: 79, 1967. substance would have to be very large indeed 8, Reber, K.: Clearing factor activation by application of heparin ointment to rats. In press. to reach the level of significance. Pictures as G. M., Sulzberger, M. B., Hermann, published in the above mentioned papers can 9. MacKee, F. and Baer, R. L.: Histologic studies on percutaneous penetration with special refereasily be obtained from the same single prepaence to the effect of vehicles. J. Invest. Derm., ration; thus, they carry no conviction. 6: 43, 1945.
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.