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Effect of CaEDTA administration on urinary hydroxyproline excretion and skin wound healing in the rat
JOURNAL
OF SURGICAL
RESEARCH
346-351 (1974)
17,
Effect of CaEDTA Hydroxyproline GORDON
Administration
Excretion TOBIN,
and Skin Wound
M.D.,
MILOS
ARTHUR
ARONSON,
CHVAPIL,
IN OUR PREVIOIJS WORK, we have been testing several drugs which were expected to interfere with collagen accumulation in various models of fibrotic lesions in a specific fashion. Our approach to the pharmacology of fibrosis was recently reviewed [3] and consists of studying the effects of drugs on collagen synthesis, polymerization, and degradation. While most of the studies dealt with control of collagen synthesis and polymerization, not enough attention has been paid to collagen breakdown as a method to decrease the amount of deposited collagen in fibroproliferative inflammation. Mechanisms regulat’ing mammalian collagenase activity in viva arc still poorly understood. For this reason, we became alerted by the finding of Aronson and Rogerson [l] showing a striking increase in the excretion of urinary hydroxyproline in animals treated with ethylendiamine tetraacetat’e calcium (CaNa,EDTA) . It was assumed that this chelating agent significantly increased collagen breakdown [lo]. These authors presented evidence that both soluble and insoluble collagen are Division of Surgical Biology. Department of Surgery, Arizona Medical Center, Univemit,y of Arizona, Tucson, Arizona 85721. Supported in part by U.S. Public Health Grants ES 00790, AM 16489, and AM 14047. * Dr. Aronson was on sabbatical leave from Cornell University. Present address : Cornell University, N.Y.S. Veterinary College, Ithaca, N.Y. 14850. Submitted for publication October 19, 1973.
M.D.,
@ 1974 by Academic of reproduction in any
Press, form
Inc.
reserved.
Healing PH.D.,*
in the Rat
AND
PH.D.
randomly degraded, since the specific activity of urinary radiohydroxyproline in rats prelabeled with C14-Pro one month before the experiment remained the same during the 4%hr infusion. A close correlation was found between the magnitude of urinary Hyp excretion and the degree of lysosomal labilization in the liver and kidney. With simultaneous administration of CaEDTA and high doses of vitamin A, the enhancement of hydroxyproline excretion was enormous (10 times) [lo]. It occurred to us that administration of CaEDTA could be used in the control of abnormal collagen accumulation in developing fibrotic lesions. Newly formed, incompletely crosslinked collagen structures are more susceptible to collagenase digestion [ 81. Granulation tissue in a healing skin wound represents a structure with “young” collagen. We postulated that this collagen would be preferentially degraded ; thus, the mechanical strength of wounds would be decreased when the animal is treated with CaEDTA. The main goal of this study was to test this Other aspects of chronic hypothesis. CaEDTA administration were also studied in order to better understand the interference of this drug with collagen metabolism. MATERIALS
AND METHODS
This study reports three experiments, one with continuous acute intravenous administration of CaEDTA for 48 hr, and the other two with chronic intraperitoneal administration of CaEDTA for a period of 14 days. 346
Copyright All rights
on Urinary
TOBIN,
Animrrls and S’wger!/
AKONSON
SND
CHVAPIL:
C&EDTA
Ah-11
\VOUNiD
HEALIKG
34i
cept that vit.amin A palmitatc 250,000 IUjkg (Mann Research Laboratory) was Male and female Sprague-Dawley rats given subcutaneously every other day. weighing between 180 and 280 g were used In nnothcr chronic experiment (Table 2) , in this study. Rats were individually skin and colon wounds were made and the housed in stainless steel cages. A 5-cm midrats fed a fluid diet per gavugc iu an atline skin incision was made over the ccrtempt to avoid the weight loss observctl vicothoracic spine and was closed with 6-O after high tloscs of CaEDTA. These aninylon sutures. mals were given CaEDT,4 (4.8 mm/kg/day) In one group of rats treated chronically ip in t,wo divided doses for 14 days:. Conwith CaEDTA (Table 2‘1, a coloton1y was trols were given 0.99 NaCl. performed in addition to the skin incision. A 2-cm longitudinal incision was made ix1 Determincltio?z of Mechnnicd Properties nj the ascending colon and closed with 6-O silk the &in sutures. Breaking strength was measured by an Instron Tester, Model T;\I, using the C cell Acute Administration of CnEDTA with pneumatic clamps. From each skin Twclvc days after making the skin inciwound, three 0.5-cm wide strips were dission, the rats were infused wit’11 CaEDTA sccted by a double-bladed knife. The reprousing the method of Aronson and Rogerson ducibility of individual determinations was [ 11. The rats were lightly anesthetized with from 10 to 15%. ether and a lateral tail vein was cannulated Urine Metnl Analyses with PE 10 tubing by threading the cathcter through a venipuncture made with a 19The urinary content of zinc was dctergauge thin~all needle (Sherwood Med. mined without digestion of the sample with Ind., Delano, Fla.) The rats were immobila Perkin-Elmer Atomic Absorption Specized in plastic tubes and urine was quantitrophotometcr, Model 305. Recoveries of tatively collected. Six rats were infused this metal added to rat urine and, analyzed simultaneously with a Harvard Model 600- by this m&hod, ranged between 93 and 1200 pump, three with 0.9% NaCl for con- 103%. trols, and three with CaEDTA at the rate of 1.08 ml/hr and 6 mmolc/kg/24 hr. In Liver Xetnl Analyses all experiments, the rats were infused conDry substance of the liver was dig&cd tinuously for 48 hr, during which time they with concentrated nitric acid followed by were fasted. The CaEDTA was supplied by a combination of equal parts nitric acid Ciba-Geigy as Sequestrene Nn,Ca. and 30% hydrogen peroxide. The digest was cvaporatcd to dryness, diluted with Chronic Administration of CaEDTA deionized distilled water, and appropriate Male rats initially weighing 280 F 20 g aliquots taken for the analysis of zinc with Atomic Absorption Spccwere divided into groups of 10 each. a Perkin-Elmer CaEDTA was administered ip in two di- tropl1otomctcr, Rlodel 305. vided doses daily at 6 mmolc/kg/day for a period of 14 days, starting wit11 the day Determination, of Collagenow of wounding. Control rats were given 0.9% Hydroryprotine Hydroxyproline content of skin, gut, and NaCl. Urine was quantitatively collected bone was done with dry samples hydroon the fifth and tenth days of treatment. All rats were fed standard lab chow. The lyzed in 6 1V NC1 at 105OC for 16 11r. Hydrolysatcs were decolorized with activated group receiving vitamin A and CaEDTA was treated ident’ically to the controls cx- charcoal, evaporated, and diluted with dis-
348
JOURNAL
OF
SURGICAL
RESEARCH,
VOL.
17,
NO.
5,
NOVEMBER
1976%
RESULTS 0.9 % wNaCl (9)
Eflect of CnEDTA
--EEDTANaz
i-i
12 24 36 Tme (hrs.,
cl
46
Fig. 1. Effect of continuous iv CaEDTA on the urinary excretion proline in rats. iVine rats in each tested. Variability is given by SE. droxyproline are presented as total proline in pmoles in 12-hr samples
infusion of of hydroxygroup were Dat,a on hyof hydroxyof urine.
tilled water to obtain a concentration of 0.5-3 rg hydroxyproline/ml. An Automated Technicon Autoanalyzer procedure was used.
Statistical Analyses The Student’s t-test was used to determine the statistical significance between test and control groups. The data are presented as Mean _+ SE.
Continuous
iv
Infusion
of
Rats infused for 2 days with CaEDTA 12 days after wounding excreted approximately 4 times more Hyp in the urine than controls infused with 0.9% NaCl (Fig. 1 and Table 1). The difference, 6.3 mg of Hyp, represents almost 50 mg of collagen breakdown. In spite of this enhanced breakdown of collagen, no changes in the collagenous Hyp content of skin, bone, or gut were found (Table 1). This was true whether the data were related to weight, area (skin), or an anatomic region (small intestine). Measurements of the breaking strengths of the skin and gut wounds revealed no significant differences between control and CaEDTA-treated rats (Table 1).
Chronic Administration of CaEDTA to Rats with u Skin Wound and a Colotomy The simultaneous wounding of the skin and colon proved to be severely traumatic since more than 50% of the animals, including controls and CaEDTA-treated, died during the experimental period. By the
of CaEDTA on the Breaking Strength and Collagenous Hydroxyproline of Nonwounded and Wounded Tissues in the Rata
Table 1. Effect of iv Infusion
Parameter
studied
Breaking strength Nonwounded abdominal skin (g/O.5 cm) Nonwounded gut (g/sample) Skin wound (g/O.5 cm) Collagenous hydroxyproline Skin: mg/g dry wt mg/cm2 Gut: mg/g wet wt mg/g dry wt Bone (femur): mg/g dry wt mg/femur Urine: mg/48 hr rmoles/48 hr
Control, saline (6)
CaEDTA
1050 * 95 223 ?i 12.5 598 iz 73 90.1 2.62 2.26 10.2 22.9 11.98 2382 18.2
+ 2.5 i 0.14 * 0.02 i 0.38 k 0.61 k 1.02 + 164 +_ 1.25
(6)
1630 + 345 231 k 8.5 678 31 59
** *
*** ***
a Number of animals is given in parenthesis. The data are presented as g between groups, calculated by Student’s l-test, is given by number of asterisks,
85.4 2.51 3.13 14.1 22.37 11.17 8682 66.3
IL + f rk + + f k
1.6 0.11 0.21 0.90 0.52 0.84 638 4.87
?c SE. The significance referring to p < 0.0.5*,
TOBIN,
ARONSON
AND
CHVAPIL:
C&EDTA
AND
WOUND
349
HEALING
Table 2. E$ect of Chronic Administration of CaEDTA on Sonwounded and Wounded Skin and Colon of the Rat as Measured by Breaking Strength” Breaking
strength
(g/O.5 cm strip)
Nonwounded skin Wounded skin Wounded (in y0 of nonwounded Nonwounded colon Wounded colon @Number “Methods.”
of animals
in each group
Controls 7994 638 8.02 89 60
skin)
is given
Chronic Administration of CaEDTA Rats with a Xkin Wound
to
The previous experiment was repeated on a larger group of rats with a skin incision only. The dose of CaEDTA was increased to 6 mmole/kg/day. Because of the reported synergism between vitamin A and CaEDTA on collagen degradation [lo], an additional group of rats was treated with CaEDTA and vitamin A palmitate (250,000 IU/kg) given every second day. In spite of a nearly twofold increase in urinary Hyp excretion, the breaking strength of neither the wounded nor the unwounded skin was affected by CaEDTA treatment either alone or in combination with vitamin A palmitate (Table 3). The data on breaking strength were very homogenous, not only within groups, but also among all three groups of animals ; no significant differences were measured. In previous papers we expressed the hy-
**
+ 611
* 105 xk 0.72 XII 10 + 12
in parentheses.
end of the experimental period, the rats treated with 4.8 mmole CaEDTA/kg/day appeared unclean, depressed, and had diarrhea. Nevertheless, there was no change in the breaking strength of e:ther wound after CaEDTA administration (Table 2). The significantly lower breaking strength of the unwounded skin of CaEDTA-treated rats is possibly related to the greater loss of body weight in this group. When the breaking strength of the skin wound is expressed as % breaking strength of nonwounded adjacent skin, there is no difference between control and CaEDTA-treated rats (Table 2).
C&I>TA
(3)
For details
(3)
5300
-t 506
463 8.73 83 37
+ 89 + 1.12 + 7.7 57.9
see legend
to Table
1 and
potheses that enhanced collagen breakdown is related to labilization of such biostructurcs as lysosomes and that the stability of biomembranes depends on the presence of zinc in the structures [4, 5, lo]. Furthermore, it has been shown that zinc deficiency delays wound healing [ 71. CaEDTA administration is known to deplete the animal in various metals, including zinc [Z]. For these reasons, we measured zinc content in urine, serum, and liver tissue t)o ascertain the magnitude of zinc depletion. CaEDTA administration and the combination of CaEDTA and vitamin A significantly lowered the content of zinc in all t#issues analyzed (Table 3). DISCUSSION The vast literature on the significance of urinary hydroxyproline excretion in health and disease, reviewed by Kivirikko [ 91, indicates that this amino acid is derived exclusively from collagen breakdown. Doubling the amount of urinary Hyp due to a pathological process is considered to be clear evidence of increased collagen breakdown in an affected tissue. Significant to our further discussion is the finding that “only about 10% of the Hyp released by the breakdown of collagen in the tissues is recovered as urinary pcptidc-bound Hyp, which represents more than 95% of the total urinary Hyp” [9]. Although it is impossible to estimate the relative contribution of individual tissue collagens to urinary Hyp, we assume that tissues with high Hyp content and high turnover rates of collagen are the principle source of urinary
350
JOURNAL
OF
SURGICAL
RESEARCH,
Table 3. Effect of Chronic Administration Urinary Hydroxyproline,
Parameter studied
17,
VOL.
NO.
5,
NOVEMBER
1974
of CaEDTA on the Breaking Strength of a Skin Wound, and Zinc Concentrations in the Rata
Control
CaEDTA
EDTA $ vitamin A
608 + 36 7437 + 403
619 f 25 6772 j, 117
1112.5 + 44.4 829.0 t 67.3
849.5 f. 53.3 827.3 i 39.8
170.2 3121.3 54.6 + 3.4
144.2 + 11.5 49.3 * 3.0
Skin wound Breaking strength (g/O.5 cm strip)
skin wound (g) normal skin (g) skin wound in y0 of control
600 3~27 7384 + 280
Urine Hyp
(,ug/24-hr sample) Day 5 Day 10 Zinc Day 5 Day 10 Serum Zinc bg %I Liver Zinc (@g/g wet wt)
597.7 k 20.3 611.8 + 37.2
***
9.2 -t 0.9 8.9 rk 0.54 187 + 4.1
***
40.1 f 2.10
**
161 k 4.3
**
26.9 i 3.28
140 + 6.8
**
***
-
GThere were 10 rats in each group. Data are presented as g + SE. Asterisks refer to p values, p < 0.05*,
Hyp. Thus, bone (containing almost 50% of the body’s collagen), skin (containing 30% of the body’s collagen), and gut could be considered a likely source of urinary Hyp. In light of these considerations, the fourfold increase in urinary Hyp in rats continuously infused for 48 hr with CaEDTA is highly significant. This fourfold increase over control excretion represents almost 50 mg of collagen and corresponds to approximately 500 mg of tissue collagen being degraded. Assuming that a 300 g rat contains 20 g of collagen, it is evident that the collagenous Hyp loss due to CaEDTA administration represents only approximately 2.5% of the total body collagen. If all of the collagen were lost from a single tissue, it would seem that a chemical analysis for Hyp would reveal the change. However, analysis of the skin, gut, and bone (Table 1) did not reveal a significant loss of collagenous Hyp. It seems, therefore, that the enhanced collagen breakdown associated with CaEDTA occurs in several tissues and, as such, the changes are too small to detect by biochemical analysis.
Similar reasoning applies to the urinary Hyp excreted following chronic administration of CaEDTA. Under these conditions, the rate of urinary Hyp excretion was twice that of controls; the total amount of Hyp excreted during the total of the 2-wk CaEDTA treatment was approximately the same as in the acute CaEDTA-treated group. These considerations may explain the administered inability of chronically CaEDTA to affect the breaking strength of skin or colon wounds. The results certainly support the view that collagen newly formed in a skin-incision wound is not preferentially degraded. For all these reasons, there seems to be little hope that CaEDTA could be useful in the control of abnormal collagen accumulation. REFEREYXES 1. Aronson, A. L., and Rogerson, K. M. Effect of calcium and chromium chelates of Ethylenediaminetetraacetate on intestinal permeability and collagen metabolism of the rat. Toxicol. Appl. Pharmacol. 21:440, 1972.
TOBIN,
ARONSON
Ah-11
CHVAPIL:
2. Braide, V. B. Aspects of calcium EDTA toxicity in rats. Ph.D. Thesis, Cornell University, 1973. 3. Chvapil, M. Pharmacology of fibrosis. Presentcd at International Symposium on Wound 11euling, Rotter&m, April 1974, in print. 4. Chvapil, M. Ryan, J. S., and Zukoski, C. F. The effect. of zinc and other metals on the stability of lyaosomcs. PI,OC. SOC. Exp. Biol. Med. 140:642. 1972a. 5. Chvapil, M., Ryan, J. N., and Zukoski, C. F. Effect of zinc on lipid pcroxidation in liver microsomcs and mitochondrin. Proc. Sot. Exp. Biol. Med. 141:150, 1972b. 6. Chvapil, M., Ryan, J. S., Elias, S. L., and Pcng, Y. M. Protective effect of zinc on cnrbon tetrachloride induced liver injury in rats. Ezp. Molec. Pnlhol. 19:156. 1973. 7. Chvapil, M., Elins, S. L., Ryan, J. N., and
CBEDTA
8.
9.
10.
11.
AND
WOUND
HEALISG
%l
Zukoski, C. F. Pathophysiology of zinc. In C. Pfeiffcr (Ed.), Irtternntionnl Review of Neurobiology, Supplement 1, p, 105, New York: Academic Press, 1972. Harris, E. D. Jr., and Farrell, M. D. Resistance to collagenase: A characteristic of collagen fibrils cross-linked by formaldehyde. Biochim. Biophys. Actn 278:133, 1972. Kivirikko, K. I. Urinary excretion of hydroxyproline in health and disensc In D. Hall and D. Jackson (E~s.)~ Internntionel Review of Coxtlectiue Tissue Research. XCW York: Acndcmic Press, 1970. Rogerson, K. M. and Aronson, A. L. Effect of CaEDTA on rat liver and kidney Iysosomrs. Fed. Proc. 32:366, 1973. Webrr, K. M. Die Schadigung des Darmes ADTA und DTPA bei dcr Ratte. Z. ges. Exp. Med. 150:354, 1969.
OF SURGICAL
RESEARCH
346-351 (1974)
17,
Effect of CaEDTA Hydroxyproline GORDON
Administration
Excretion TOBIN,
and Skin Wound
M.D.,
MILOS
ARTHUR
ARONSON,
CHVAPIL,
IN OUR PREVIOIJS WORK, we have been testing several drugs which were expected to interfere with collagen accumulation in various models of fibrotic lesions in a specific fashion. Our approach to the pharmacology of fibrosis was recently reviewed [3] and consists of studying the effects of drugs on collagen synthesis, polymerization, and degradation. While most of the studies dealt with control of collagen synthesis and polymerization, not enough attention has been paid to collagen breakdown as a method to decrease the amount of deposited collagen in fibroproliferative inflammation. Mechanisms regulat’ing mammalian collagenase activity in viva arc still poorly understood. For this reason, we became alerted by the finding of Aronson and Rogerson [l] showing a striking increase in the excretion of urinary hydroxyproline in animals treated with ethylendiamine tetraacetat’e calcium (CaNa,EDTA) . It was assumed that this chelating agent significantly increased collagen breakdown [lo]. These authors presented evidence that both soluble and insoluble collagen are Division of Surgical Biology. Department of Surgery, Arizona Medical Center, Univemit,y of Arizona, Tucson, Arizona 85721. Supported in part by U.S. Public Health Grants ES 00790, AM 16489, and AM 14047. * Dr. Aronson was on sabbatical leave from Cornell University. Present address : Cornell University, N.Y.S. Veterinary College, Ithaca, N.Y. 14850. Submitted for publication October 19, 1973.
M.D.,
@ 1974 by Academic of reproduction in any
Press, form
Inc.
reserved.
Healing PH.D.,*
in the Rat
AND
PH.D.
randomly degraded, since the specific activity of urinary radiohydroxyproline in rats prelabeled with C14-Pro one month before the experiment remained the same during the 4%hr infusion. A close correlation was found between the magnitude of urinary Hyp excretion and the degree of lysosomal labilization in the liver and kidney. With simultaneous administration of CaEDTA and high doses of vitamin A, the enhancement of hydroxyproline excretion was enormous (10 times) [lo]. It occurred to us that administration of CaEDTA could be used in the control of abnormal collagen accumulation in developing fibrotic lesions. Newly formed, incompletely crosslinked collagen structures are more susceptible to collagenase digestion [ 81. Granulation tissue in a healing skin wound represents a structure with “young” collagen. We postulated that this collagen would be preferentially degraded ; thus, the mechanical strength of wounds would be decreased when the animal is treated with CaEDTA. The main goal of this study was to test this Other aspects of chronic hypothesis. CaEDTA administration were also studied in order to better understand the interference of this drug with collagen metabolism. MATERIALS
AND METHODS
This study reports three experiments, one with continuous acute intravenous administration of CaEDTA for 48 hr, and the other two with chronic intraperitoneal administration of CaEDTA for a period of 14 days. 346
Copyright All rights
on Urinary
TOBIN,
Animrrls and S’wger!/
AKONSON
SND
CHVAPIL:
C&EDTA
Ah-11
\VOUNiD
HEALIKG
34i
cept that vit.amin A palmitatc 250,000 IUjkg (Mann Research Laboratory) was Male and female Sprague-Dawley rats given subcutaneously every other day. weighing between 180 and 280 g were used In nnothcr chronic experiment (Table 2) , in this study. Rats were individually skin and colon wounds were made and the housed in stainless steel cages. A 5-cm midrats fed a fluid diet per gavugc iu an atline skin incision was made over the ccrtempt to avoid the weight loss observctl vicothoracic spine and was closed with 6-O after high tloscs of CaEDTA. These aninylon sutures. mals were given CaEDT,4 (4.8 mm/kg/day) In one group of rats treated chronically ip in t,wo divided doses for 14 days:. Conwith CaEDTA (Table 2‘1, a coloton1y was trols were given 0.99 NaCl. performed in addition to the skin incision. A 2-cm longitudinal incision was made ix1 Determincltio?z of Mechnnicd Properties nj the ascending colon and closed with 6-O silk the &in sutures. Breaking strength was measured by an Instron Tester, Model T;\I, using the C cell Acute Administration of CnEDTA with pneumatic clamps. From each skin Twclvc days after making the skin inciwound, three 0.5-cm wide strips were dission, the rats were infused wit’11 CaEDTA sccted by a double-bladed knife. The reprousing the method of Aronson and Rogerson ducibility of individual determinations was [ 11. The rats were lightly anesthetized with from 10 to 15%. ether and a lateral tail vein was cannulated Urine Metnl Analyses with PE 10 tubing by threading the cathcter through a venipuncture made with a 19The urinary content of zinc was dctergauge thin~all needle (Sherwood Med. mined without digestion of the sample with Ind., Delano, Fla.) The rats were immobila Perkin-Elmer Atomic Absorption Specized in plastic tubes and urine was quantitrophotometcr, Model 305. Recoveries of tatively collected. Six rats were infused this metal added to rat urine and, analyzed simultaneously with a Harvard Model 600- by this m&hod, ranged between 93 and 1200 pump, three with 0.9% NaCl for con- 103%. trols, and three with CaEDTA at the rate of 1.08 ml/hr and 6 mmolc/kg/24 hr. In Liver Xetnl Analyses all experiments, the rats were infused conDry substance of the liver was dig&cd tinuously for 48 hr, during which time they with concentrated nitric acid followed by were fasted. The CaEDTA was supplied by a combination of equal parts nitric acid Ciba-Geigy as Sequestrene Nn,Ca. and 30% hydrogen peroxide. The digest was cvaporatcd to dryness, diluted with Chronic Administration of CaEDTA deionized distilled water, and appropriate Male rats initially weighing 280 F 20 g aliquots taken for the analysis of zinc with Atomic Absorption Spccwere divided into groups of 10 each. a Perkin-Elmer CaEDTA was administered ip in two di- tropl1otomctcr, Rlodel 305. vided doses daily at 6 mmolc/kg/day for a period of 14 days, starting wit11 the day Determination, of Collagenow of wounding. Control rats were given 0.9% Hydroryprotine Hydroxyproline content of skin, gut, and NaCl. Urine was quantitatively collected bone was done with dry samples hydroon the fifth and tenth days of treatment. All rats were fed standard lab chow. The lyzed in 6 1V NC1 at 105OC for 16 11r. Hydrolysatcs were decolorized with activated group receiving vitamin A and CaEDTA was treated ident’ically to the controls cx- charcoal, evaporated, and diluted with dis-
348
JOURNAL
OF
SURGICAL
RESEARCH,
VOL.
17,
NO.
5,
NOVEMBER
1976%
RESULTS 0.9 % wNaCl (9)
Eflect of CnEDTA
--EEDTANaz
i-i
12 24 36 Tme (hrs.,
cl
46
Fig. 1. Effect of continuous iv CaEDTA on the urinary excretion proline in rats. iVine rats in each tested. Variability is given by SE. droxyproline are presented as total proline in pmoles in 12-hr samples
infusion of of hydroxygroup were Dat,a on hyof hydroxyof urine.
tilled water to obtain a concentration of 0.5-3 rg hydroxyproline/ml. An Automated Technicon Autoanalyzer procedure was used.
Statistical Analyses The Student’s t-test was used to determine the statistical significance between test and control groups. The data are presented as Mean _+ SE.
Continuous
iv
Infusion
of
Rats infused for 2 days with CaEDTA 12 days after wounding excreted approximately 4 times more Hyp in the urine than controls infused with 0.9% NaCl (Fig. 1 and Table 1). The difference, 6.3 mg of Hyp, represents almost 50 mg of collagen breakdown. In spite of this enhanced breakdown of collagen, no changes in the collagenous Hyp content of skin, bone, or gut were found (Table 1). This was true whether the data were related to weight, area (skin), or an anatomic region (small intestine). Measurements of the breaking strengths of the skin and gut wounds revealed no significant differences between control and CaEDTA-treated rats (Table 1).
Chronic Administration of CaEDTA to Rats with u Skin Wound and a Colotomy The simultaneous wounding of the skin and colon proved to be severely traumatic since more than 50% of the animals, including controls and CaEDTA-treated, died during the experimental period. By the
of CaEDTA on the Breaking Strength and Collagenous Hydroxyproline of Nonwounded and Wounded Tissues in the Rata
Table 1. Effect of iv Infusion
Parameter
studied
Breaking strength Nonwounded abdominal skin (g/O.5 cm) Nonwounded gut (g/sample) Skin wound (g/O.5 cm) Collagenous hydroxyproline Skin: mg/g dry wt mg/cm2 Gut: mg/g wet wt mg/g dry wt Bone (femur): mg/g dry wt mg/femur Urine: mg/48 hr rmoles/48 hr
Control, saline (6)
CaEDTA
1050 * 95 223 ?i 12.5 598 iz 73 90.1 2.62 2.26 10.2 22.9 11.98 2382 18.2
+ 2.5 i 0.14 * 0.02 i 0.38 k 0.61 k 1.02 + 164 +_ 1.25
(6)
1630 + 345 231 k 8.5 678 31 59
** *
*** ***
a Number of animals is given in parenthesis. The data are presented as g between groups, calculated by Student’s l-test, is given by number of asterisks,
85.4 2.51 3.13 14.1 22.37 11.17 8682 66.3
IL + f rk + + f k
1.6 0.11 0.21 0.90 0.52 0.84 638 4.87
?c SE. The significance referring to p < 0.0.5*,
TOBIN,
ARONSON
AND
CHVAPIL:
C&EDTA
AND
WOUND
349
HEALING
Table 2. E$ect of Chronic Administration of CaEDTA on Sonwounded and Wounded Skin and Colon of the Rat as Measured by Breaking Strength” Breaking
strength
(g/O.5 cm strip)
Nonwounded skin Wounded skin Wounded (in y0 of nonwounded Nonwounded colon Wounded colon @Number “Methods.”
of animals
in each group
Controls 7994 638 8.02 89 60
skin)
is given
Chronic Administration of CaEDTA Rats with a Xkin Wound
to
The previous experiment was repeated on a larger group of rats with a skin incision only. The dose of CaEDTA was increased to 6 mmole/kg/day. Because of the reported synergism between vitamin A and CaEDTA on collagen degradation [lo], an additional group of rats was treated with CaEDTA and vitamin A palmitate (250,000 IU/kg) given every second day. In spite of a nearly twofold increase in urinary Hyp excretion, the breaking strength of neither the wounded nor the unwounded skin was affected by CaEDTA treatment either alone or in combination with vitamin A palmitate (Table 3). The data on breaking strength were very homogenous, not only within groups, but also among all three groups of animals ; no significant differences were measured. In previous papers we expressed the hy-
**
+ 611
* 105 xk 0.72 XII 10 + 12
in parentheses.
end of the experimental period, the rats treated with 4.8 mmole CaEDTA/kg/day appeared unclean, depressed, and had diarrhea. Nevertheless, there was no change in the breaking strength of e:ther wound after CaEDTA administration (Table 2). The significantly lower breaking strength of the unwounded skin of CaEDTA-treated rats is possibly related to the greater loss of body weight in this group. When the breaking strength of the skin wound is expressed as % breaking strength of nonwounded adjacent skin, there is no difference between control and CaEDTA-treated rats (Table 2).
C&I>TA
(3)
For details
(3)
5300
-t 506
463 8.73 83 37
+ 89 + 1.12 + 7.7 57.9
see legend
to Table
1 and
potheses that enhanced collagen breakdown is related to labilization of such biostructurcs as lysosomes and that the stability of biomembranes depends on the presence of zinc in the structures [4, 5, lo]. Furthermore, it has been shown that zinc deficiency delays wound healing [ 71. CaEDTA administration is known to deplete the animal in various metals, including zinc [Z]. For these reasons, we measured zinc content in urine, serum, and liver tissue t)o ascertain the magnitude of zinc depletion. CaEDTA administration and the combination of CaEDTA and vitamin A significantly lowered the content of zinc in all t#issues analyzed (Table 3). DISCUSSION The vast literature on the significance of urinary hydroxyproline excretion in health and disease, reviewed by Kivirikko [ 91, indicates that this amino acid is derived exclusively from collagen breakdown. Doubling the amount of urinary Hyp due to a pathological process is considered to be clear evidence of increased collagen breakdown in an affected tissue. Significant to our further discussion is the finding that “only about 10% of the Hyp released by the breakdown of collagen in the tissues is recovered as urinary pcptidc-bound Hyp, which represents more than 95% of the total urinary Hyp” [9]. Although it is impossible to estimate the relative contribution of individual tissue collagens to urinary Hyp, we assume that tissues with high Hyp content and high turnover rates of collagen are the principle source of urinary
350
JOURNAL
OF
SURGICAL
RESEARCH,
Table 3. Effect of Chronic Administration Urinary Hydroxyproline,
Parameter studied
17,
VOL.
NO.
5,
NOVEMBER
1974
of CaEDTA on the Breaking Strength of a Skin Wound, and Zinc Concentrations in the Rata
Control
CaEDTA
EDTA $ vitamin A
608 + 36 7437 + 403
619 f 25 6772 j, 117
1112.5 + 44.4 829.0 t 67.3
849.5 f. 53.3 827.3 i 39.8
170.2 3121.3 54.6 + 3.4
144.2 + 11.5 49.3 * 3.0
Skin wound Breaking strength (g/O.5 cm strip)
skin wound (g) normal skin (g) skin wound in y0 of control
600 3~27 7384 + 280
Urine Hyp
(,ug/24-hr sample) Day 5 Day 10 Zinc Day 5 Day 10 Serum Zinc bg %I Liver Zinc (@g/g wet wt)
597.7 k 20.3 611.8 + 37.2
***
9.2 -t 0.9 8.9 rk 0.54 187 + 4.1
***
40.1 f 2.10
**
161 k 4.3
**
26.9 i 3.28
140 + 6.8
**
***
-
GThere were 10 rats in each group. Data are presented as g + SE. Asterisks refer to p values, p < 0.05*,
Hyp. Thus, bone (containing almost 50% of the body’s collagen), skin (containing 30% of the body’s collagen), and gut could be considered a likely source of urinary Hyp. In light of these considerations, the fourfold increase in urinary Hyp in rats continuously infused for 48 hr with CaEDTA is highly significant. This fourfold increase over control excretion represents almost 50 mg of collagen and corresponds to approximately 500 mg of tissue collagen being degraded. Assuming that a 300 g rat contains 20 g of collagen, it is evident that the collagenous Hyp loss due to CaEDTA administration represents only approximately 2.5% of the total body collagen. If all of the collagen were lost from a single tissue, it would seem that a chemical analysis for Hyp would reveal the change. However, analysis of the skin, gut, and bone (Table 1) did not reveal a significant loss of collagenous Hyp. It seems, therefore, that the enhanced collagen breakdown associated with CaEDTA occurs in several tissues and, as such, the changes are too small to detect by biochemical analysis.
Similar reasoning applies to the urinary Hyp excreted following chronic administration of CaEDTA. Under these conditions, the rate of urinary Hyp excretion was twice that of controls; the total amount of Hyp excreted during the total of the 2-wk CaEDTA treatment was approximately the same as in the acute CaEDTA-treated group. These considerations may explain the administered inability of chronically CaEDTA to affect the breaking strength of skin or colon wounds. The results certainly support the view that collagen newly formed in a skin-incision wound is not preferentially degraded. For all these reasons, there seems to be little hope that CaEDTA could be useful in the control of abnormal collagen accumulation. REFEREYXES 1. Aronson, A. L., and Rogerson, K. M. Effect of calcium and chromium chelates of Ethylenediaminetetraacetate on intestinal permeability and collagen metabolism of the rat. Toxicol. Appl. Pharmacol. 21:440, 1972.
TOBIN,
ARONSON
Ah-11
CHVAPIL:
2. Braide, V. B. Aspects of calcium EDTA toxicity in rats. Ph.D. Thesis, Cornell University, 1973. 3. Chvapil, M. Pharmacology of fibrosis. Presentcd at International Symposium on Wound 11euling, Rotter&m, April 1974, in print. 4. Chvapil, M. Ryan, J. S., and Zukoski, C. F. The effect. of zinc and other metals on the stability of lyaosomcs. PI,OC. SOC. Exp. Biol. Med. 140:642. 1972a. 5. Chvapil, M., Ryan, J. N., and Zukoski, C. F. Effect of zinc on lipid pcroxidation in liver microsomcs and mitochondrin. Proc. Sot. Exp. Biol. Med. 141:150, 1972b. 6. Chvapil, M., Ryan, J. S., Elias, S. L., and Pcng, Y. M. Protective effect of zinc on cnrbon tetrachloride induced liver injury in rats. Ezp. Molec. Pnlhol. 19:156. 1973. 7. Chvapil, M., Elins, S. L., Ryan, J. N., and
CBEDTA
8.
9.
10.
11.
AND
WOUND
HEALISG
%l
Zukoski, C. F. Pathophysiology of zinc. In C. Pfeiffcr (Ed.), Irtternntionnl Review of Neurobiology, Supplement 1, p, 105, New York: Academic Press, 1972. Harris, E. D. Jr., and Farrell, M. D. Resistance to collagenase: A characteristic of collagen fibrils cross-linked by formaldehyde. Biochim. Biophys. Actn 278:133, 1972. Kivirikko, K. I. Urinary excretion of hydroxyproline in health and disensc In D. Hall and D. Jackson (E~s.)~ Internntionel Review of Coxtlectiue Tissue Research. XCW York: Acndcmic Press, 1970. Rogerson, K. M. and Aronson, A. L. Effect of CaEDTA on rat liver and kidney Iysosomrs. Fed. Proc. 32:366, 1973. Webrr, K. M. Die Schadigung des Darmes ADTA und DTPA bei dcr Ratte. Z. ges. Exp. Med. 150:354, 1969.