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The Effect of Vitamin A and Hydrocortisone on the Normal Alkaline Phosphatase Response to Skin Wounding in Rats*
Vol. 49, No. 3
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1567 by The Williams & Wilkins Co.
THE EFFECT OF VITAMIN A AND HYDROCORTISONE ON THE NORMAL ALKALINE PHOSPHATASE RESPONSE TO SKIN WOUNDING IN RATS* JOHN P. MANNING, Pn.D. AND GENE DIPASQIJALE, MS.
groups as follows: 1) control, 2) vitamin A, 3)
INTRODUCTION
hydrocortisone and 4) vitamin A + hydrocortisone.
The histochemical distribution of alkaline
In addition, fifteen, normal, intact rats were in-
phosphatase activity has been studied in cluded for evaluation of basal enzyme levels. The hair was removed from the dorsal surface normal and wounded skins of rats (1—4),
an electric clippers and the skin wounds guinea pigs (2, 5, 6) and man (7—9) and the with were incised under light ether anesthesia. The activity of this enzyme has been associated midline incision was approximately 1½" long and with fibrous protein synthesis (1, 5, 10).
Normally, mammalian skin contains only trace amounts of alkaline phosphatase located in the stratum granulosum, hair follicles, sebaceous glands and some connective tissue cells (4, 7, 9). During wound healing, however, an increase in the phosphatase reaction is noted in the keratohyalin granules of the stratum granulosum associated with keratinization and in connective tissue at the time
located 2" below the interscapular region. The 50 mg vitamin A acetate and/or 5 mg hydrocortisone
acetate (Merck, Sharp and Dome) were locally applied along the full extent of the incision and the wounds were closed with stainless steel clips. The tensile strength was determined on ten animals from wounded treatment groups on 4, 7, 10 and 14 days according to a previously published
procedure (12). Five other rats were killed by decapitation at 025, 0.50, 0.75, 1, 2, 4, 7, 10, 14, 17, 20 and 26 days. The wounds were rapidly removed
by cutting 3.5 mm on either side of the incision (total width 7 mm). A section of skin from the
of maximum collagen formation. The presence of the enzyme at these sites suggested a rela-
same dorsal area was taken from the normal, untreated animals. After weighing on an tionship between the keratin-collagen syn- intact, analytical balance a piece of the excised tissue thesis and alkaline phosphatase activity (1, (excluding the areas of the stainless steel wound 5, 10). In addition, Danielli, Fell and Kodicek
clips) was quenched between two blocks of dry ice for 1 minute. The frozen skin was placed in a
(5) demonstrated in scorbutic guinea pigs,
rubber finger cot and shattered by hitting it times with a hammer. The maccrated tissue reaction were severely retarded and these several was transferred to a flask containing 10 ml of that both wound healing and the phosphatase
phenomena paralleled the degree of vitamin cold distilled water and homogenized with a Vir C deficiency.
Tis No. 45 homogenizer until a uniform suspension
The purpose of the present study was to was obtained (approximately 5 minutes). The evaluate the tensile strength and alkaline homogenate was then diluted to a specific concentration (1%) and aliquots used in the alkaline
phosphatase activity during the healing proc-
ess in control wounded rats and animals phosphatase procedure according to Manning,
Babson and Butler (15). Ten units of treated with vitamin A and hydrocortisone. Steinetz, alkaline phosphatase activity liberates 1 mg of
Large doses of vitamin A in animals has
phenolphthalein from phenolphthalein diphosphate
been reported to stimulate bone growth while a deficiency retards osteogenesis and causes epithelial metaplasia (11). On the other hand, the local or systemic administration of cortisone or hydrocortisone acetate significantly
in 1 hour at pH 9.6 and 37° C under standard
depresses scar tissue formation and wound
by analysis of variance and the "F test" for
tensile strength (12—14).
significance (16). The mean and standard errors were calculated for tbe tensile strength results and submitted to the "t tests" of Student.
MATERIALS AND METHODS
conditions.
Dry weights were obtained by drying a piece of the wounded skin in a vacuum oven at 60° C for 24 hours.
The biochemical data was evaluated statistically
Albino rats (Carworth Farms), weighing an
RRSULTS AND DIscussioN
average of 200 gm were divided into 4 treatment Received for publication January 11, 1967.
* Department of Physiology, Warner-Lambert Repearch Institute, Morris Plains, New Jersey.
The data in Table 1 indicate that wounding, per se, resulted in enhanced phosphatase
225
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
226
levels at all time periods tested, relative to the to the wounded controls, vitamin A and the combined treatment increased whereas hydronormal intact skins (11.26 units 1.04). During the normal healing process, bio- cortisone decreased significantly the first peak chemically determined alkaline phosphatase in the alkaline phosphatase reaction (Fig. 1 demonstrated two peaks; at 12 hours during and Table 1). In animals receiving vitamin eschar formation and around day 10 corre- A + hydrocortisone the second phase was 2.5x sponding with collagen synthesis (Fig. 1). The greater while animals treated with these comactivity at day 26 was similar to the intact pounds individually, demonstrated an alkaline control and these results are comparable to phosphatase response similar to the controls earlier histochemical reports (1, 4). Relative (Table 1). With hydroeortisone, however, the TABLE 1 The average* (±S.E.) alkaline phosphalase levels of conlrol and treated, wounded rats over the 26 day period Time (days)
——
Treatment 0.25 23.0
Control
6.3 23.6
Hydroeorti.sone
Vitamin A
+ Hy- 21.4
droeortisone
4.4
22.6
98.61
20.1
30.6
54.2)
3.4 41.8
50.6) 9.5
11.5
26
29.2
30.0
24.2
12.0
3.1
0.7 103.2)
5.0
2.6 35.2
2.3 46.2
40.4
46.6
20
4.1
1.2
2.3
17
35.4
45.8
30.2
3.0
14
3.1
9.5
3.4 26.6
25.8
41.4
2.6 50.4
49.4
12.1
7.1
10
30.0
6.9
86.41
34.2
7
1.2
13.7
7.0 82.6)
47.81 4.2
4
3.5
13.6 44.2
5.9
2
20.1
93.61
20.3 43.81
2.0
1
2.7
19.2 121.8*
41.8
0.75
33.0
73.2 1.3
vitamin A
0.50
49.4
44.6
2.3
25.4
6.7
1.7
11.8
2.0
1.8
52.6) 6.1
42.6
26.2
6.8 39.0
2.2
38.0
1.1
3.1 24.4
6.9
2.2
0.51
* Expressed in units/gm dry tissue. Ten Units of alkaline phosphatase activity liberates 1 mg phenolphthalein from phenolphthalem diphosphate in 1 hr at pH 9.6 and 37CC under standard conditions. Significantly greater than control p < 0.05. Significantly less than control p < 0.05.
— Control X Wounded Control
D Vitamin A
0 5
o Hydrocortisone V Vitamin A + Hydrocortisone
S
5. -5
C
a.
S
4 5,
C C -C
0 C
0
4 Time [days]
Fio. 1. The alkaline phosphatase activity during the healing process in wounded control rats and animals treated with vitamin A and hydrocortisone.
STUDIES ON WOUND PHOSPHATASE
227
Wolbach (24), Wolf and Varandani (25) and Wolf (26) demonstrated the necessity of vitamin A for the synthesis of mucopolysachealing is obscure, although its presence at eharides. If the hypothesis presented in this high levels coincident with other biochem- communication is reasonable, one might exical events, certainly implicates this enzyme pect enhanced mueopolysaccharide formation, second peak of phosphatase activity was delayed
to day 20 (Fig.1). The role of alkaline phosphatase in wound
in the epidermal and dermal regeneration
alkaline phosphatase activity and tensile
processes. The first peak in the phosphatase reaction (12 hours) which is associated with the invading leueoeytes of the esehar, may be involved in catabolic mechanisms for the removal of dead cells and other tissue debris. If one considers the hydrolytic activity of alkaline phosphatase on carbohydrate phosphates, anabolie processes are indicated as well, since increased deposition of glycogen and produc-
strength in vitamin A-treated wounded animals. On the other hand, hydrocortisone administration, which decreases scar tissue formation and
tion of nucleic acids and mucopolysaceharides
A reasonable explanation for these phenom-
tensile strength should also depress the phosphatase reaction during the collagen phase. In contrast, however, neither the tensile strength (except at day 4) was increased in the former ease nor the alkaline phosphatase activity depressed in the latter one.
is noted at this time (10, 17—22). It is of ena is that the sequence of events is so
interest that the "productive" and "collagen critically controlled in relation to quantities phases" of wound healing as suggested by and types of substrates present, time, etc. that Dunphy and Tldupa (18) corresponds to our an increase in any one component does not necessarily result in enhanced would healing. peaks in alkaline phosphatase activity.
As demonstrated by Williamson and Guschl- The reverse is not true, however, since a bauer (21, 22) and Guschlbauer and William- deficiency in a critical element will most son (19), the synthesis of IRNA and DNA is probably lead to an abnormal healing process. maximal 8 days after wound initiation. These 5TJMMARY same investigators reported that the phosphate pool was essentially unchanged although the Alkaline phosphatase activity and tensile
turnover rate was significantly increased; thus, the second phase in the alkaline phosphatase response to wound healing may be necessary to maintain the level of the phos-
strength was evaluated duing the healing
phate pool. With enhanced formation of glycogen (energy source), mucopolysaecharide (homogeneous matrix necessary for collagen deposition) and RNA (protein synthesis), the conditions necessary for fibrous protein production (keratinization and collagen formation) are maximal around the eighth day of
phosphatase reaction were noted at 12 hours and ten days after wound initiation in con-
wound healing.
If the enzymatic results are compared with tensile strengths (Fig. 2 and Table 2), one is immediately aware that no direct correlation is possible since the wound strengths were
similar in vitamin A and control animals and lowered in rats receiving hydrocortisone and the combined treatment. Apparently, the events for the production of tensile strength are so complex and interrelated, that the true
correlation of it with alkaline phosphatase is completely obscured. For example, Perumal, Samy, Lakshuanan, Jungalwala and Rao (23),
process in wounded control rats and animals treated with vitamin A, hydrocortisone and
vitamin A + bydrocortisone. Peaks in the
trol animals. Vitamin A alone or in combination with the glucocorticoid increased, while hydrocortisone decreased, significantly the
first phase of the enzyme activity. The second
peak was enhanced 2.5 x by vitamin A + hydrocortisone, while rats receiving these compounds individually, demonstrated an alkaline phosphatase level similar to the controls. With hydrocortisone alone the second phase of phosphatase activity was delayed to day 20. Wound tensile strength, assessed at days 4,
7, 10 and 14 was similar in control and vitamin A animals. In contrast, the glucoeorticoid significantly lowered the tensile strength and this effect was only partially reversed by
vitamin A. No direct correlation could be made between the alkaline phosphatase activity and wound tensile strength.
THE JOURNAL OF' INVESTIGATIVE DERMATOLOGY
228
Wounded Control
o Vitamin A o Hydrocortisone , Vitamin A + Hydrocortisone 800
700 E
0, 0)
60G
C 4)
U,
Soc 'a
C
I-a, C
400
0 300
200
100
0 FIG.
4
i
à ib
Time (days)
2. The tensile strength during the healing process in wounded control rats and ani-
mals treated with vitamin A and hydrocortisone.
The
TABLE 2 average* (±S.E.) tensile strength of control and treated, wounded rat skins Time (days)
Treatment
4
Control Vitamin A Hydrocortisone
Vitamin A + Hydrocorti-
14
10
7
129 165
9.5
453 419
58.5
668
16.7
6.lt
20.8
690
30.8
772 763
68 91
4.3
246
30.01:
300
26.9
594
31
3.01:
330
23.01:
497
29.21:
630
421:
sone
*gm.
Significantly greater than control p < 0.01. Significantly less than control p < 0.01.
56 30
229
STUDIES ON WOUND PHOSPHATASE
ship of food intake to the effect of cortisone
The alkaline phosphatase results are discussed in relation to the formation of fibrous
acetate on skin wound healing. Proc. Soc.
proteins (collagen and keratin) during the
13. DiPasquale, C., Tripp, L. and Steinetz, B. C.: Effect of locally applied anti-inflammatory
healing process.
Exptl. Biol. Med., 117:
118, 1964.
substances on rat skin wounds. Proc. Soc.
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Exptl. Biol. Med., in press.
14. Sandberg, N.: Time relationship between administration of cortisone and wound healing in rats. Acta Chem. Scand., 127: 446, 1964.
15. Manning, J. P., Steinetz, B. C., Babson, A. L.
and Butler, M. C.: A simple and reliable method for estimation of alkaline phospha-
196, 1943.
tase in tissue homogenates. Enzymologia, 31:
2. Raekallio, J.: Histochemieal demonstration of enzymatic response to injury in experimental
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observations on wound healing in denervated
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1965.
baeka, J. and Puittinen, J.: ilistochemical
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53, 1964.
4. Washburn, W. W.: Comparative histoehemical observations of wound healing in adult rats
and cultured adult human epithelium. III. Alkaline and acid phosphatase. J. Invest. Derm., 24:
Ames, Iowa, State University Press, 1956.
847, 1955.
19. Guschlbauer, W. and Williamson, M. B.: Metabolism of nucleic acids during regeneration of wound tissue. III. The rate of formation of DNA. Arch. Bioehem. Biophys.,
537, 1955.
5. Danielh, J. F., Fell, II. B. and Kodicek, E.:
The enzymes of heahng wounds. II. Tbe effect of different degrees of vitamin C-defi-
ciency on the phosphatase activity in experimental wounds in the guinea pig. Brit. J. Exptl. Pathol., 26: 367, 1945.
100: 251, 1963.
20. Trott, J. H. and Peikoff, M. D.: A histochemical study of glycogen in the epithelium of wounds healing by second intention in the abdomen of mice. J. Histochem. Cytochem., 11: 613, 1963.
6. Raekallio, J.: Enzymes histochemically de- 21. Williamson, M. B. and Guschlbauer, W.: Memonstrable in the earliest phase of wound tabolism of nucleic acids during regeneration of wound tissue. J. Biol. Chem., 236: 1463, healing. Nature, 188: 234, 1960. 1961. 7. Fisher, I. and Ghck, D.: Histochemistry XIX. Localization of alkaline phosphatase in normal and pathological human skin. Proc. Soc. Exptl. Biol. Med., 66: 14, 1947.
8. Kurban, A. K. and Afifi, A. K.: Alkaline phos-
phatase activity in skin as shown by staining en bloc. Stain Technol., 37:7,
1962.
9. Pirilk, V. and Eranko, 0.: Distribution of histochemically demonstrable alkaline phosphatase in normal and pathological human skin. Acta Path. Microb. Scand., 27: 650, 1950.
10. Johnson, F. R. and MeMinn, R. M. H.: The
cytology of wound healing of body surfaces in mammals. Biol. Rev. Cambridge Phil. Soc., 35: 364, 1960. 11. Robbins, S. L.: Textbook of Pathology, p. 247.
Philadelphia and London, W. B. Saunders Company, 1957.
12. DiPasquale, C. and Steinetz, B. C.: Relation-
22. Williamson, M. B. and Cuschlbauer, W.: Metabolism of nucleic acids during regeneration of wound tissue. II. The rate of formation of RNA. Arch. Biochem. Biophys., 100: 245, 1963.
23. Perumal, A. S., Samy, T. S. A., Lakshmanan, M. R., Jungalwala, F. B. and Rao, P. B. R.: Biosynthesis of sulfated mucopolysaccharides in relation to vitamins A and C. Biochim. Biophys. Acta, 124:
95, 1966.
24. Wolbach, S. B.: Vitamin A deficiency and excess in relation to skeletal growth. J. Bone Joint Surg., 29: 171, 1947. 25. Wolf, C. and Varandani, P. T.: Studies on the function of vitamin A in mucopolysaccharide biosynthesis. Biochim. Biophys. Acta, 43: 501,
26.
1960.
Wolf, G.: Some thoughts on the metabolic role of vitamin A. Nutr. Rev., 20: 161, 1962.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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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
Copyright
Printed in U.S.A.
1567 by The Williams & Wilkins Co.
THE EFFECT OF VITAMIN A AND HYDROCORTISONE ON THE NORMAL ALKALINE PHOSPHATASE RESPONSE TO SKIN WOUNDING IN RATS* JOHN P. MANNING, Pn.D. AND GENE DIPASQIJALE, MS.
groups as follows: 1) control, 2) vitamin A, 3)
INTRODUCTION
hydrocortisone and 4) vitamin A + hydrocortisone.
The histochemical distribution of alkaline
In addition, fifteen, normal, intact rats were in-
phosphatase activity has been studied in cluded for evaluation of basal enzyme levels. The hair was removed from the dorsal surface normal and wounded skins of rats (1—4),
an electric clippers and the skin wounds guinea pigs (2, 5, 6) and man (7—9) and the with were incised under light ether anesthesia. The activity of this enzyme has been associated midline incision was approximately 1½" long and with fibrous protein synthesis (1, 5, 10).
Normally, mammalian skin contains only trace amounts of alkaline phosphatase located in the stratum granulosum, hair follicles, sebaceous glands and some connective tissue cells (4, 7, 9). During wound healing, however, an increase in the phosphatase reaction is noted in the keratohyalin granules of the stratum granulosum associated with keratinization and in connective tissue at the time
located 2" below the interscapular region. The 50 mg vitamin A acetate and/or 5 mg hydrocortisone
acetate (Merck, Sharp and Dome) were locally applied along the full extent of the incision and the wounds were closed with stainless steel clips. The tensile strength was determined on ten animals from wounded treatment groups on 4, 7, 10 and 14 days according to a previously published
procedure (12). Five other rats were killed by decapitation at 025, 0.50, 0.75, 1, 2, 4, 7, 10, 14, 17, 20 and 26 days. The wounds were rapidly removed
by cutting 3.5 mm on either side of the incision (total width 7 mm). A section of skin from the
of maximum collagen formation. The presence of the enzyme at these sites suggested a rela-
same dorsal area was taken from the normal, untreated animals. After weighing on an tionship between the keratin-collagen syn- intact, analytical balance a piece of the excised tissue thesis and alkaline phosphatase activity (1, (excluding the areas of the stainless steel wound 5, 10). In addition, Danielli, Fell and Kodicek
clips) was quenched between two blocks of dry ice for 1 minute. The frozen skin was placed in a
(5) demonstrated in scorbutic guinea pigs,
rubber finger cot and shattered by hitting it times with a hammer. The maccrated tissue reaction were severely retarded and these several was transferred to a flask containing 10 ml of that both wound healing and the phosphatase
phenomena paralleled the degree of vitamin cold distilled water and homogenized with a Vir C deficiency.
Tis No. 45 homogenizer until a uniform suspension
The purpose of the present study was to was obtained (approximately 5 minutes). The evaluate the tensile strength and alkaline homogenate was then diluted to a specific concentration (1%) and aliquots used in the alkaline
phosphatase activity during the healing proc-
ess in control wounded rats and animals phosphatase procedure according to Manning,
Babson and Butler (15). Ten units of treated with vitamin A and hydrocortisone. Steinetz, alkaline phosphatase activity liberates 1 mg of
Large doses of vitamin A in animals has
phenolphthalein from phenolphthalein diphosphate
been reported to stimulate bone growth while a deficiency retards osteogenesis and causes epithelial metaplasia (11). On the other hand, the local or systemic administration of cortisone or hydrocortisone acetate significantly
in 1 hour at pH 9.6 and 37° C under standard
depresses scar tissue formation and wound
by analysis of variance and the "F test" for
tensile strength (12—14).
significance (16). The mean and standard errors were calculated for tbe tensile strength results and submitted to the "t tests" of Student.
MATERIALS AND METHODS
conditions.
Dry weights were obtained by drying a piece of the wounded skin in a vacuum oven at 60° C for 24 hours.
The biochemical data was evaluated statistically
Albino rats (Carworth Farms), weighing an
RRSULTS AND DIscussioN
average of 200 gm were divided into 4 treatment Received for publication January 11, 1967.
* Department of Physiology, Warner-Lambert Repearch Institute, Morris Plains, New Jersey.
The data in Table 1 indicate that wounding, per se, resulted in enhanced phosphatase
225
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
226
levels at all time periods tested, relative to the to the wounded controls, vitamin A and the combined treatment increased whereas hydronormal intact skins (11.26 units 1.04). During the normal healing process, bio- cortisone decreased significantly the first peak chemically determined alkaline phosphatase in the alkaline phosphatase reaction (Fig. 1 demonstrated two peaks; at 12 hours during and Table 1). In animals receiving vitamin eschar formation and around day 10 corre- A + hydrocortisone the second phase was 2.5x sponding with collagen synthesis (Fig. 1). The greater while animals treated with these comactivity at day 26 was similar to the intact pounds individually, demonstrated an alkaline control and these results are comparable to phosphatase response similar to the controls earlier histochemical reports (1, 4). Relative (Table 1). With hydroeortisone, however, the TABLE 1 The average* (±S.E.) alkaline phosphalase levels of conlrol and treated, wounded rats over the 26 day period Time (days)
——
Treatment 0.25 23.0
Control
6.3 23.6
Hydroeorti.sone
Vitamin A
+ Hy- 21.4
droeortisone
4.4
22.6
98.61
20.1
30.6
54.2)
3.4 41.8
50.6) 9.5
11.5
26
29.2
30.0
24.2
12.0
3.1
0.7 103.2)
5.0
2.6 35.2
2.3 46.2
40.4
46.6
20
4.1
1.2
2.3
17
35.4
45.8
30.2
3.0
14
3.1
9.5
3.4 26.6
25.8
41.4
2.6 50.4
49.4
12.1
7.1
10
30.0
6.9
86.41
34.2
7
1.2
13.7
7.0 82.6)
47.81 4.2
4
3.5
13.6 44.2
5.9
2
20.1
93.61
20.3 43.81
2.0
1
2.7
19.2 121.8*
41.8
0.75
33.0
73.2 1.3
vitamin A
0.50
49.4
44.6
2.3
25.4
6.7
1.7
11.8
2.0
1.8
52.6) 6.1
42.6
26.2
6.8 39.0
2.2
38.0
1.1
3.1 24.4
6.9
2.2
0.51
* Expressed in units/gm dry tissue. Ten Units of alkaline phosphatase activity liberates 1 mg phenolphthalein from phenolphthalem diphosphate in 1 hr at pH 9.6 and 37CC under standard conditions. Significantly greater than control p < 0.05. Significantly less than control p < 0.05.
— Control X Wounded Control
D Vitamin A
0 5
o Hydrocortisone V Vitamin A + Hydrocortisone
S
5. -5
C
a.
S
4 5,
C C -C
0 C
0
4 Time [days]
Fio. 1. The alkaline phosphatase activity during the healing process in wounded control rats and animals treated with vitamin A and hydrocortisone.
STUDIES ON WOUND PHOSPHATASE
227
Wolbach (24), Wolf and Varandani (25) and Wolf (26) demonstrated the necessity of vitamin A for the synthesis of mucopolysachealing is obscure, although its presence at eharides. If the hypothesis presented in this high levels coincident with other biochem- communication is reasonable, one might exical events, certainly implicates this enzyme pect enhanced mueopolysaccharide formation, second peak of phosphatase activity was delayed
to day 20 (Fig.1). The role of alkaline phosphatase in wound
in the epidermal and dermal regeneration
alkaline phosphatase activity and tensile
processes. The first peak in the phosphatase reaction (12 hours) which is associated with the invading leueoeytes of the esehar, may be involved in catabolic mechanisms for the removal of dead cells and other tissue debris. If one considers the hydrolytic activity of alkaline phosphatase on carbohydrate phosphates, anabolie processes are indicated as well, since increased deposition of glycogen and produc-
strength in vitamin A-treated wounded animals. On the other hand, hydrocortisone administration, which decreases scar tissue formation and
tion of nucleic acids and mucopolysaceharides
A reasonable explanation for these phenom-
tensile strength should also depress the phosphatase reaction during the collagen phase. In contrast, however, neither the tensile strength (except at day 4) was increased in the former ease nor the alkaline phosphatase activity depressed in the latter one.
is noted at this time (10, 17—22). It is of ena is that the sequence of events is so
interest that the "productive" and "collagen critically controlled in relation to quantities phases" of wound healing as suggested by and types of substrates present, time, etc. that Dunphy and Tldupa (18) corresponds to our an increase in any one component does not necessarily result in enhanced would healing. peaks in alkaline phosphatase activity.
As demonstrated by Williamson and Guschl- The reverse is not true, however, since a bauer (21, 22) and Guschlbauer and William- deficiency in a critical element will most son (19), the synthesis of IRNA and DNA is probably lead to an abnormal healing process. maximal 8 days after wound initiation. These 5TJMMARY same investigators reported that the phosphate pool was essentially unchanged although the Alkaline phosphatase activity and tensile
turnover rate was significantly increased; thus, the second phase in the alkaline phosphatase response to wound healing may be necessary to maintain the level of the phos-
strength was evaluated duing the healing
phate pool. With enhanced formation of glycogen (energy source), mucopolysaecharide (homogeneous matrix necessary for collagen deposition) and RNA (protein synthesis), the conditions necessary for fibrous protein production (keratinization and collagen formation) are maximal around the eighth day of
phosphatase reaction were noted at 12 hours and ten days after wound initiation in con-
wound healing.
If the enzymatic results are compared with tensile strengths (Fig. 2 and Table 2), one is immediately aware that no direct correlation is possible since the wound strengths were
similar in vitamin A and control animals and lowered in rats receiving hydrocortisone and the combined treatment. Apparently, the events for the production of tensile strength are so complex and interrelated, that the true
correlation of it with alkaline phosphatase is completely obscured. For example, Perumal, Samy, Lakshuanan, Jungalwala and Rao (23),
process in wounded control rats and animals treated with vitamin A, hydrocortisone and
vitamin A + bydrocortisone. Peaks in the
trol animals. Vitamin A alone or in combination with the glucocorticoid increased, while hydrocortisone decreased, significantly the
first phase of the enzyme activity. The second
peak was enhanced 2.5 x by vitamin A + hydrocortisone, while rats receiving these compounds individually, demonstrated an alkaline phosphatase level similar to the controls. With hydrocortisone alone the second phase of phosphatase activity was delayed to day 20. Wound tensile strength, assessed at days 4,
7, 10 and 14 was similar in control and vitamin A animals. In contrast, the glucoeorticoid significantly lowered the tensile strength and this effect was only partially reversed by
vitamin A. No direct correlation could be made between the alkaline phosphatase activity and wound tensile strength.
THE JOURNAL OF' INVESTIGATIVE DERMATOLOGY
228
Wounded Control
o Vitamin A o Hydrocortisone , Vitamin A + Hydrocortisone 800
700 E
0, 0)
60G
C 4)
U,
Soc 'a
C
I-a, C
400
0 300
200
100
0 FIG.
4
i
à ib
Time (days)
2. The tensile strength during the healing process in wounded control rats and ani-
mals treated with vitamin A and hydrocortisone.
The
TABLE 2 average* (±S.E.) tensile strength of control and treated, wounded rat skins Time (days)
Treatment
4
Control Vitamin A Hydrocortisone
Vitamin A + Hydrocorti-
14
10
7
129 165
9.5
453 419
58.5
668
16.7
6.lt
20.8
690
30.8
772 763
68 91
4.3
246
30.01:
300
26.9
594
31
3.01:
330
23.01:
497
29.21:
630
421:
sone
*gm.
Significantly greater than control p < 0.01. Significantly less than control p < 0.01.
56 30
229
STUDIES ON WOUND PHOSPHATASE
ship of food intake to the effect of cortisone
The alkaline phosphatase results are discussed in relation to the formation of fibrous
acetate on skin wound healing. Proc. Soc.
proteins (collagen and keratin) during the
13. DiPasquale, C., Tripp, L. and Steinetz, B. C.: Effect of locally applied anti-inflammatory
healing process.
Exptl. Biol. Med., 117:
118, 1964.
substances on rat skin wounds. Proc. Soc.
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