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Penicillamine and wound healing in young guinea pigs
__-
-
PENICILLAMINE IN
E. E.
SEIFTER,
F.
YOUNG
GEEVER, PH.D.,
AND WOUND HEALING GUINEA PIGS
M.D., AND
IN a recent report of experimental wound healing in guinea pigs [6] we described increased breaking strength of wound strips following in vitro treatment of the wound strips with mercuric chloride solution. Since mercuric ions readily react with mercaptans, it seemed possible to us that mercury was acting by cross-linking two mercaptans. The role of sulfur-containing proteins (i.e., noncollagenous) in contributing to the strength of experimental wounds is not clear, and the finding regarding the action of mercury stimulated our interest in that direction. Recently, Nimni and Bavetta [lo] reported impaired wound healing in young rats after ingestion of penicillamine, 6,6-dimethylcysteine, a mercapto amino acid of interest because of its metal-chelating properties. In this report we describe our experience with penicillamine and its effect on wound healing in young guinea pigs. Since Crawhall and Thompson had postulated earlier [4] that penicillamine decreased the availability of cysteine to the animal, we also tested the ability of cysteine fed simultaneously with penicillamine to neutralize or otherwise affect the latter’s action on wound healing. From the Departments of Surgery, Pathology and Biochemistry of The Albert Einstein College of Medicine, New York, N.Y. Submitted for publication June 30, 1966. 160
S.
YOUSSEF,
S.
M.
M.D.,
LEVENSON,
MATERIAL Wounding Sponges
M.D.
AND and
METHODS
Implantation
of
Polyvinyl
In two experiments spaced six weeks apart standard dermal incisions and porous polyvinyl sponge implantations were performed on young male guinea pigs of the Hartley strain* and the wounds excised and sponges removed at 14 and 21 days postoperatively, at which time the animals were killed. The average body weight at operation was 298 gm. and the range from 227 to 362 gm. All animals were maintained on a special guinea pig test diett fed ad libitum and to which vitamin C was added separately in an amount of 0.1% for 10 days prior to operation to permit the animals to become accustomed to the diet. One day prior to operation they were separated into three groups according to body weight: Group A controls continued on the same dietary regimen to which 0.01% pyridoxine HCl (vitamin Be)$ was added; Group B with 0.25y0 nr-penicillamine$ and 0.01% pyridoxine HCl added daily to the basic diet; and Group C with 0.25% m-penicillamine, 0.375$ L-cysteine and 0.01% pyridoxine HCl * Tumblebrook Farm, Brant Lake, N.Y. ) General Biochemicals, Chagrin Falls, Ohio; ReidBriggs vitamin C-deficient guinea pig diet. Ohio. t Nutritional Biochemicals, Cleveland,
GEEVER
ET
AL.:
added daily to the basic diet. Extra pyridoxine HCI was added to all groups because of the belief that penicillamine might induce a pyridoxine deficiency. Food consumption was measured throughout the experiment. In the first 2 days the animals in Group C ate somewhat less than the animals in Groups A and B. Therefore, the intake of the latter groups was controlled by modified pair-feeding: the average intake per animal in Group C for 2 days was given for the succeeding 2 days to each animal in Groups A and B. Each guinea pig was housed in a separate cage. Under general anesthesia with 6 to 8 mg. of intraperitoneal pentobarbital all animals were subjected to paravertebral skin incision and subcutaneous implantation of three porous polyvinyl sponges. The operations were performed by the same individual under strictly aseptic conditions. The operative site parallel to and lateral to the vertebral column was clipped, depilated with Nair, washed off with gauze and warm water, prepared with Wescodyne solution (approx. 1.6% available iodine) and covered with a sterile cloth drape with a centrally located 8.0 X 1.5 cm. opening. A dermal wound, 7.0 cm. in length, was made with a scalpel down through the subcutaneous tissue and panniculus carnosus to the underlying deep muscle sheath. The wound was then undermined at three points to approximately 5 cm. lateral to the skin edge by inserting Metzenbaum scissors at the upper, middle, and lower poles, spreading the blades and tearing pockets between the superficial and deep muscles. A soft, water-moistened, sterile, porous polyvinyl sponge* was then inserted into each pocket about 5 cm. away from the incision. Each sponge was discshaped, approximately 1 cm. in diameter and 0.3 cm. in thickness. The dry weight of each of the upper and lower sponges was determined and recorded. The middle sponge, for histopathological examination, was unweighed but was of the same general size. The purpose of the sponge implants was to obtain relatively pure samples of reparative wound tissue for histological and biochemical analysis [3]. The * Ivalon,
Clay-Adams,
Inc.,
New
York,
N.Y.
PENICILLAMINE
AND
WOUND
HEALING
IN
GUINEA
PIGS
wound was then closed with five interrupted, evenly spaced 35gauge stainless steel wire sutures. No dressing was applied. Sutures were removed on the seventh day. Forty-eight animals were operated upon; 43 survived for study. Determination
of Wound-Breaking
Strength
The animals were killed by carbon dioxide asphyxiation using dry ice. The length of the paravertebral skin wound after careful clipping of the hair was measured in situ and then excised together with a rectangular margin of normal skin approximately 3 cm. on each side of the wound and about 1 cm. beyond each pole. The pelt was mounted on a special multibladed cutting device designed in our laboratory [S] with the wound exactly perpendicular to the blades and the skin surface down. The midpoint of the wound was centered on the base of the cutter and the pelt gently stretched to smooth out wrinkles. It was then sectioned into seven strips each 0.6 cm. wide. Four strips were tested in a fresh state for their breaking strength on an apparatus also designed in our laboratory [8]. The essential parts consist of a strain gauge connected to a recorder. Each wound strip is attached to and suspended from vertically positioned jaws, the upper connected with the strain gauge, the lower pulled down slowly by a constant speed motor operating slowly revolving gears. The instrument is calibrated with known weights before each operational period and recalibrated several times during each “run.” The variation with known weights was approximately 1%. Preparation of the strips for testing required about 15 minutes from the moment of sacrifice. The breaking strength of the strips was then tested sequentially starting with the cephalad end of the wound. Strips numbered 1, 3, 5, and 7 were tested fresh, as mentioned above. In another study we found that the various strips, from 1 to 7, healed at similar rates so that any value could be considered as representative for breaking strength or histological evaluation [7]. Strip No. 2 was tested for its breaking strength after immersion for 48 hours in 10% unbuffered formalin. Strip No. 6 was im161
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mersed for 48 hours in a saturated mercuric chloride solution. Strip No. 4 was fixed in buffered 10yO formalin for histological examination. Hydroxyproline
Determination
in Sponges
The polyvinyl sponges were excised carefully and most of the surface tissue dissected off, using good light and an ophthalmologist’s loupe as visual aids. Collagen was isolated as gelatin by twice extracting the upper and lower sponges individually with 2 to 3 ml. of water at 22 lbs. per sq. inch in the autoclave for 3 hours. The extracts were evaporated to dryness and hydrolyzed in sealed tubes with 6N hydrochloric acid at 130°C. for 3 hours. The hydrolysates were brought to dryness and aliquots were taken; hydroxyproline was determined according to Woessner [ 121. Histopathological
Investigation
The middle sponge was fixed in buffered 10% formalin and then prepared for histopathological study. Serial sections were stained routinely with hematoxylin and eosin and modified Van Gieson’s stains. The Muller-Mowry modification of the latter provides collagen fiber and ground substance detail [9].
RESULTS A small percentage of complications was found in the healing incisions despite strict aseptic operative technique. This was reported by us before and is apparently unavoidable [6, 71. Some animals were able to produce focal irritation by rubbing the wound on the sides of the cage or possibly by rubbing the wound with their hind paws. Of the 43 survivors 18 were available for study of 14-day wounds and 25 with 2I-day wounds. Calculation of the food intake revealed that the average daily intake of penicillamine per animal in Groups B and C sacrificed at 14 days had been 48 mg. In Group C the average daily intake of L-cysteine, apart from that contained in the Reid-Briggs diet, had been 73 mg. The comparable values for the animals killed at 21 days were 49 mg. per day penicillamine in 162
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1967
Group B and 50 mg. per day in Group C. In Group C the average daily intake of separately added L-cysteine had been 74 mg. The mean body weights for the animals sacrificed I4 days postoperatively were: Group A, 320 gm., Group B, 317 gm., and Group C, 336 gm. The weights in the 21-day postoperative groups were: Group A, 357 gm., Group B, 352 gm., and Group C, 322 gm. Clinical evaluation at sacrifice revealed that all 6 of the 14-day control wounds in Group A had healed without complication. Five of Group B and 4 of Group C were also well healed. One wound in Group B showed focal ulceration and 1 in Group C revealed focal fresh bleeding and another focal surface blood clot. Histopathological examination of the wounds revealed uncomplicated healing in the 6 controls of Group A, focal ulceration in 1 of the 6 penicillamine-treated Group B animals, and other complications in 3 of the Group C animals: 1 with central cystic change in the wound and 2 with disruption during technical preparation of the slides. The latter could be a reflection of some qualitative change despite the absence of gross wound disruption. Comparative studies of the amount and caliber of wound collagen revealed no evidence of inhibition in Groups B and C. On the contrary the evaluation seemed to indicate more collagen in Group B than Group A: In 3 wounds it was about the same and in another 3 it was considered to be more abundant in Group B. Group C was more difficult to compare quantitatively in the 3 complicated wounds. Two of the second 3 were the same as in Group A and 1 of the 3 was more abundant than in Group A. Clearly, there was no suppression of collagen in either penicillamine-treated group. Clinical evaluation of the 21-day wounds revealed focal crusting on the wound surface by old blood clot in 3 wounds of Group A, in 1 of Group B and in 2 of Group C. There were 7 survivors in Group A, 8 in Group B and 10 in Group C. Histopathological examination of the 21-day wounds showed focal ulceration in 1 of the 7 controls in Group A, one gaping wound out of a total of 8 animals in Group B and two gaping wounds among the wounds of 10 animals in Group C. Comparative study of
GEEVER
ET
AL.:
PENICILLAMINE
1.
14-Day
Group A Controls
MEANS S.D. i S.E. _t
Breaking
WOUND
Strength
in Grams-Fresh
Wound
Wounds*
Group B Penicillamine 48 mg./day
HEALING
IN
GUINEA
Strips
21-Day Group C Penicillamine 48 mg. and L-Cysteine 73 mg./day
Group A Controls
Wounds*
Group B Penicillamine 49 mg./day
Group C Penicillamine 50 mg. and L-Cysteine 74 mg./day
485 425 358 289 249 323
323 355 331 251 239 273
295 329 319 255 128 179
557 641 741 608 490 590 491
336 218 293 219 207 139 164 242
171 171 163 200 186 185 110 168 94 215
355 87.7 35.8
295 47.6 19.4
251 76.1 31.1
588 88.1 33.3
227f 64.1 22.7
1sst 37.6 11.8
* Each value f Statistically
represents significant
the mean differences
of 4 strips from from controls.
PIGS
breaking strength more than penicillamine alone. The difference was statistically significant (P between 0.02 and 0.025). Furthermore, the wounds of both treated groups were weaker at 21 days than at 14 days, whereas the controls showed an increase of approximately 65 percent over the 1Cday values. Chemical treatment of one strip from each wound with 10yO unbuffered formalin for 48 hours revealed a striking increase of breaking strength over the fresh specimen’s value. In the 14-day specimens the means of the breaking strengths in the various groups were as follows: Group A, 1,621 gm., Group B, 1,219 gm., and Group C, 1,078 gm. The difference between Group A and Group C was of borderline significance (P between 0.1 and 0.05) but was not significant between Group A and Group B (P, 0.2). Chemical treatment of one strip from each wound with saturated mercuric chloride solution for 48 hours also caused an increase in breaking strength over the fresh strip value: the mean of Group A, 1,383 gm., Group B, 1,050 gm., and Group C, 1,193 gm. These differences were not statistically significant (P greater than 0.1 and 0.4, respectively). Similarly in the 21-day specimens the corresponding means following 4%hour formalin
the amount of wound collagen between groups revealed: about the same amount in 3 wounds of Group A and Group B; in 3 the amount was greater in Group A, and in 1 of Group B the reverse was true. Comparison of Group A with Group C showed equal amounts of wound collagen in 3, more collagen in the wounds of 3 animals in Group A, and the reverse in 1 animal of Group C. The breaking strength values of the fresh strips of 14- and 21-day wounds are listed in Table 1, which includes means, standard deviations, and standard errors. The penicillaminetreated groups showed a decrease in wound strength as compared to their controls, but this w-as statistically significant only in the 21-day group. The P values of the 14-day fresh strips were as follows: in comparing controls with the penicillamine-treated group, P was found to be between 0.1 and 0.2; in controls versus the penicillamine plus cysteine group, P was between 0.05 and 0.10. The latter level of significance might be questioned. However, in the 21-day experiment comparison of fresh strips of the controls with either penicillamine alone or penicillamine plus cysteine revealed the P value to be less than 0.001. Penicillamine plus added cysteine (Group C) depressed the Table
AND
each
wound.
163
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solution were: Group 1,881 gm., and Group
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4,
A, 2,317 gm., Group B, C, 1,928 gm. These dif-
ferences were not statistically significant (P greater than 0.3). And following 48-hour treatment with saturated mercuric chloride solution the mean values were: Group A, 1,866 gm., Group B, 1,613 gm., and Group C, 2,028 gm. These differences were not statistically significant (P greater than 0.4). The
hydroxyproline
findings
in the sponge
implants are listed in Table 2. In the 14-day implants there were no statistically significant differences between the groups. The test for the significance of the difference between the Group A controls and the Group B penicillamine treated showed the P value to be between 0.25 and 0.3. Groups A and C are obviously similar. However, in the 21-day implants there was significantly more hydroxyproline in Group B than in the controls (P between 0.001 and 0.005). Added cysteine in both 14- and 21-day sponge implants, Group C, had no significant effect on comparison with
the control
Groups
A. The values
for the
14-day specimens are obviously similar. When the difference between Groups A and C in the 2l-day sponge implants was subjected to statistical test, P was found to be between 0.1 and 0.2. Histopathological examination of the sponge Table
2.
Hydroxyproline
Values,
1967
APRIL
implants was carried out and the amount of collagen graded + to ++++. Focal peripheral collagen only was graded + and diffuse collagen deposition in all porous areas as observed in a cross section through the middle third of the sponge was evaluated ++++. Intermediate quantitations were + + and -I++. The findings in the 14-day specimens were: Group A controls, +++ (2), ++ (3) and f ( 1); Group B, penicillamine-treated, +++ (11, ++ (4) and + (1); Group C, penicillamine plus L-cysteine, ++++ ( 1)) +++ (11, ++ (3), and + (1) (Table 3). Histopathological examination of the sponge implants in the 21-day groups revealed: Group
4 ++++ (51, +++ Cl), and ++ (1); Group B, ++++ (41, +++ (31, and ++ (1); Group C, ++++ ++ (1).
(4), +++
DISCUSSION The findings in this experiment are suggestive
of some
qualitative
abnormality
wg./lOO
mg. Implanted
Sponge
21-Day Implants
Group C Penicillamine
999 1113 898 1003 1023 1208
MEANS S.D. f S.E. f 164
1041 105.6 43.2
in the
reparative collagen of the penicillamine-treated guinea pigs. There was no evidence of a quantitative suppression of reparative collagen as determined by histopathological examination or biochemical (hydroxyproline) analysis.
14-Day Implants
Group A Controls
(51, and
Group A Controls
Group B Penicillamine
and L-Cysteine
999 1030 950 937 713 1122
722 700 977 876 1742 1346
990 915 840 1495 1485 1327 1643
1104 1074 1549 1386 1643 1643 1891 1891
1008 990 827 893 1465 1356 861 1218 1317 1376
1060 403.3 164.6
1242 322.1 121.8
1523 315.4 111.5
1131 240.6 76.0
958 135.8 55.5
Group B Penicillamine
Group C Penicillamine and L-Cysteine
GEEVER
Table
3.
Histopathological
ET
Findings
AL.:
PENICILLAMINE
in Sponge
Implants:
+++ +++ ++ ++ ++ +
Grading
Group B Penicillamine 48 mg. /day +++ ++ ++ ++ ++ +
WOUND
Reparative
HEALING
Collagen
21 Days
14 Days
Group A Controls
AND
Group C Penicillamine 48 mg. and L-Cysteine 73 mg. daily ++++ +++ ++ ++ ++ +
Group A Controls ++++ ++++ ++++ ++++ ++++ +++ ++
Group B Penicillamine 49 mg. /day ++++ ++++ ++-t+ ++++ +-t+ +++ +++ -t+
IN
GUINEA
PIGS
Deposition
-Group C Penicillamine 50 mg. and L-Cysteine 74 mg. daily ++++ ++++ ++++ ++++ +++ +++ +++ +++ +++ ++
+ to ++++.
Thus, our findings differ from those reported in rats by Nimni and Bavetta who described significant inhibition of collagen synthesis in subcutaneous sponge implants [lo]. Chemical treatment in vitro with 10% unbuffered formalin for 48 hours or with saturated mercuric chloride solution for the same period brought the breaking-strength values of the wound strips back to a nearly normal level in the 21day wounds. In the l4-day specimenssimilarly treated in vitro there was corresponding increase in breaking strength except for the formalin-treated wound strips in Group C, penicillamine plus L-cysteine. The difference between this group and the controls remained statistically significant. These findings are somewhat similar to those reported by us in experimental lathyrism in rats [Z]. They suggest that penicillamine interferes with the cross-linking of the wound collagen. Another possible explanation for the decreased breaking strength is that penicillamine exerted its effect on the noncollagenous proteins and the cell membranes within the wound. Although collagen is considered to be the sine qua non of a wound’s breaking strength, we demonstrated [6] that the mesh of cells and noncollagenous proteins in a 3-day wound has the capability of contributing significantly to its breaking strength. This is at a time when no collagen can be detected by
histopathological examination or by hydroxyproline determinations of simultaneously implanted polyvinyl sponges. Cysteine was added to the diet of the guinea pigs in Group C to determine if penicillamine acted by decreasing the availability of cysteine. It had been shown [4] that penicillamine induced urinary excretion of cysteine. It is obvious that in the dose used cysteine did not reverse or reduce the penicillamine effect. In the 21-day wounds it appeared to potentiate the penicillamine action. Both cysteine and penicillamine form complexes with copper and this introduces a possible role of this metal in wound healing. Aposhian [l] reported that cysteine is catabolized at a high rate by copper-containing enzymes. If the latter are inactivated by penicillamine, then cysteine should be available for additional copper complexing. Thus, penicillamine may in fact be preserving cysteine. The penicillamine effect has possible human clinical implications in the treatment of patients with large dosesof penicillin since penicillamine is a derivative of penicillin. A dose of 1.5 million units of penicillin G would yield, in vivo, approximately 400 mg. of penicilloic acid and 50 mg. of penicillamine. Surgical patients often receive much higher doses, i.e., 30 or more million units daily. This could yield penicillamine in quantities sufficient to cause 165
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impaired wound healing. Scheinberg [ll] reported skin friability in patients with Wilson’s disease treated by penicillamine with a daily dosage of 1.0 gm.
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1967
2.
3.
4. SUMMARY
1. Young guinea pigs given penicillamine by mouth in a milk powder diet showed impaired wound healing as manifested by decreased breaking strength of 14- and 21-day wounds; added cysteine did not reverse these findings. 2. Histopathological and biochemical studies showed no quantitative impairment of reparative collagen synthesis; thus, some type of qualitative change is postulated. 3. A possible clinical implication of these findings for surgical patients on high-dosage penicillin therapy was presented.
5. 6.
7.
8.
9. ACKNOWLEDGMENT This paper was supported in part by Army Research Office Grant No. NA-ARO-49-092-66G132 and National Institutes of Health Research Career Award (to S.M.L.) 5K6-GM14, 208-05.
10.
11. REFERENCES 12. 1.
166
Aposhian, H. V. Symposium in Medicine. Philadelphia: 290.
on Metal Lippincott,
Binding 1960. P.
Berberian, G. M., Kuyama, T., Geever, E. F., and Levenson, S. M. Wound healing defect in lathyrism. Surg. Forum 15:50, 1964. Boucek, R. J., and Noble, N. L. Connective tissue: A technique for its isolation and study. Arch. Path. 59:553, 1955. Crawhall, J. C., and Thompson, C. J. Cystinuria: Effect of n-penicillamine on plasma and urinary cysteine concentrates. Science 147: 1459, 1965. Croxton, F. E. Elementary Statistics. New York: Dover, 1959. P. 236. Geever, E. F., Levenson, S. M., and Manner, G. The role of noncollagenous substances in the breaking strength of experimental wounds. SUTgery 60:343, 1966. Geever, E. F., Stein, J. M., and Levenson, S. M. Variations in breaking strength in healing wounds of young guinea pigs. J. Trauma 5:624, 1965. Levenson, S. M., Crowley, L. V., Geever, E. F., Rosen, H., and Berard, C. W. Some studies of wound healing: Experimental methods, effect of ascorbic acid, and effect of deuterium oxide. J. Trauma 4:543, 1964. Mowry, R. W. Manual of Histologic and Special Staining Technics, Armed Forces Institute of Pathology, 2d Ed. New York: Blakiston Div., McGraw-Hill, 1960. P. 138. Nimni, M. E., and Bavetta, L. A. Collagen defect induced by penicillamine. Science 150:905, 1966. Scheinberg, I. H. n-Penicillamine with particular relation to Wilson’s disease. J. Chronic Dis. 17:293, 1964. Woessner, J. F., Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. 93:440, 1961.
-
PENICILLAMINE IN
E. E.
SEIFTER,
F.
YOUNG
GEEVER, PH.D.,
AND WOUND HEALING GUINEA PIGS
M.D., AND
IN a recent report of experimental wound healing in guinea pigs [6] we described increased breaking strength of wound strips following in vitro treatment of the wound strips with mercuric chloride solution. Since mercuric ions readily react with mercaptans, it seemed possible to us that mercury was acting by cross-linking two mercaptans. The role of sulfur-containing proteins (i.e., noncollagenous) in contributing to the strength of experimental wounds is not clear, and the finding regarding the action of mercury stimulated our interest in that direction. Recently, Nimni and Bavetta [lo] reported impaired wound healing in young rats after ingestion of penicillamine, 6,6-dimethylcysteine, a mercapto amino acid of interest because of its metal-chelating properties. In this report we describe our experience with penicillamine and its effect on wound healing in young guinea pigs. Since Crawhall and Thompson had postulated earlier [4] that penicillamine decreased the availability of cysteine to the animal, we also tested the ability of cysteine fed simultaneously with penicillamine to neutralize or otherwise affect the latter’s action on wound healing. From the Departments of Surgery, Pathology and Biochemistry of The Albert Einstein College of Medicine, New York, N.Y. Submitted for publication June 30, 1966. 160
S.
YOUSSEF,
S.
M.
M.D.,
LEVENSON,
MATERIAL Wounding Sponges
M.D.
AND and
METHODS
Implantation
of
Polyvinyl
In two experiments spaced six weeks apart standard dermal incisions and porous polyvinyl sponge implantations were performed on young male guinea pigs of the Hartley strain* and the wounds excised and sponges removed at 14 and 21 days postoperatively, at which time the animals were killed. The average body weight at operation was 298 gm. and the range from 227 to 362 gm. All animals were maintained on a special guinea pig test diett fed ad libitum and to which vitamin C was added separately in an amount of 0.1% for 10 days prior to operation to permit the animals to become accustomed to the diet. One day prior to operation they were separated into three groups according to body weight: Group A controls continued on the same dietary regimen to which 0.01% pyridoxine HCl (vitamin Be)$ was added; Group B with 0.25y0 nr-penicillamine$ and 0.01% pyridoxine HCl added daily to the basic diet; and Group C with 0.25% m-penicillamine, 0.375$ L-cysteine and 0.01% pyridoxine HCl * Tumblebrook Farm, Brant Lake, N.Y. ) General Biochemicals, Chagrin Falls, Ohio; ReidBriggs vitamin C-deficient guinea pig diet. Ohio. t Nutritional Biochemicals, Cleveland,
GEEVER
ET
AL.:
added daily to the basic diet. Extra pyridoxine HCI was added to all groups because of the belief that penicillamine might induce a pyridoxine deficiency. Food consumption was measured throughout the experiment. In the first 2 days the animals in Group C ate somewhat less than the animals in Groups A and B. Therefore, the intake of the latter groups was controlled by modified pair-feeding: the average intake per animal in Group C for 2 days was given for the succeeding 2 days to each animal in Groups A and B. Each guinea pig was housed in a separate cage. Under general anesthesia with 6 to 8 mg. of intraperitoneal pentobarbital all animals were subjected to paravertebral skin incision and subcutaneous implantation of three porous polyvinyl sponges. The operations were performed by the same individual under strictly aseptic conditions. The operative site parallel to and lateral to the vertebral column was clipped, depilated with Nair, washed off with gauze and warm water, prepared with Wescodyne solution (approx. 1.6% available iodine) and covered with a sterile cloth drape with a centrally located 8.0 X 1.5 cm. opening. A dermal wound, 7.0 cm. in length, was made with a scalpel down through the subcutaneous tissue and panniculus carnosus to the underlying deep muscle sheath. The wound was then undermined at three points to approximately 5 cm. lateral to the skin edge by inserting Metzenbaum scissors at the upper, middle, and lower poles, spreading the blades and tearing pockets between the superficial and deep muscles. A soft, water-moistened, sterile, porous polyvinyl sponge* was then inserted into each pocket about 5 cm. away from the incision. Each sponge was discshaped, approximately 1 cm. in diameter and 0.3 cm. in thickness. The dry weight of each of the upper and lower sponges was determined and recorded. The middle sponge, for histopathological examination, was unweighed but was of the same general size. The purpose of the sponge implants was to obtain relatively pure samples of reparative wound tissue for histological and biochemical analysis [3]. The * Ivalon,
Clay-Adams,
Inc.,
New
York,
N.Y.
PENICILLAMINE
AND
WOUND
HEALING
IN
GUINEA
PIGS
wound was then closed with five interrupted, evenly spaced 35gauge stainless steel wire sutures. No dressing was applied. Sutures were removed on the seventh day. Forty-eight animals were operated upon; 43 survived for study. Determination
of Wound-Breaking
Strength
The animals were killed by carbon dioxide asphyxiation using dry ice. The length of the paravertebral skin wound after careful clipping of the hair was measured in situ and then excised together with a rectangular margin of normal skin approximately 3 cm. on each side of the wound and about 1 cm. beyond each pole. The pelt was mounted on a special multibladed cutting device designed in our laboratory [S] with the wound exactly perpendicular to the blades and the skin surface down. The midpoint of the wound was centered on the base of the cutter and the pelt gently stretched to smooth out wrinkles. It was then sectioned into seven strips each 0.6 cm. wide. Four strips were tested in a fresh state for their breaking strength on an apparatus also designed in our laboratory [8]. The essential parts consist of a strain gauge connected to a recorder. Each wound strip is attached to and suspended from vertically positioned jaws, the upper connected with the strain gauge, the lower pulled down slowly by a constant speed motor operating slowly revolving gears. The instrument is calibrated with known weights before each operational period and recalibrated several times during each “run.” The variation with known weights was approximately 1%. Preparation of the strips for testing required about 15 minutes from the moment of sacrifice. The breaking strength of the strips was then tested sequentially starting with the cephalad end of the wound. Strips numbered 1, 3, 5, and 7 were tested fresh, as mentioned above. In another study we found that the various strips, from 1 to 7, healed at similar rates so that any value could be considered as representative for breaking strength or histological evaluation [7]. Strip No. 2 was tested for its breaking strength after immersion for 48 hours in 10% unbuffered formalin. Strip No. 6 was im161
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mersed for 48 hours in a saturated mercuric chloride solution. Strip No. 4 was fixed in buffered 10yO formalin for histological examination. Hydroxyproline
Determination
in Sponges
The polyvinyl sponges were excised carefully and most of the surface tissue dissected off, using good light and an ophthalmologist’s loupe as visual aids. Collagen was isolated as gelatin by twice extracting the upper and lower sponges individually with 2 to 3 ml. of water at 22 lbs. per sq. inch in the autoclave for 3 hours. The extracts were evaporated to dryness and hydrolyzed in sealed tubes with 6N hydrochloric acid at 130°C. for 3 hours. The hydrolysates were brought to dryness and aliquots were taken; hydroxyproline was determined according to Woessner [ 121. Histopathological
Investigation
The middle sponge was fixed in buffered 10% formalin and then prepared for histopathological study. Serial sections were stained routinely with hematoxylin and eosin and modified Van Gieson’s stains. The Muller-Mowry modification of the latter provides collagen fiber and ground substance detail [9].
RESULTS A small percentage of complications was found in the healing incisions despite strict aseptic operative technique. This was reported by us before and is apparently unavoidable [6, 71. Some animals were able to produce focal irritation by rubbing the wound on the sides of the cage or possibly by rubbing the wound with their hind paws. Of the 43 survivors 18 were available for study of 14-day wounds and 25 with 2I-day wounds. Calculation of the food intake revealed that the average daily intake of penicillamine per animal in Groups B and C sacrificed at 14 days had been 48 mg. In Group C the average daily intake of L-cysteine, apart from that contained in the Reid-Briggs diet, had been 73 mg. The comparable values for the animals killed at 21 days were 49 mg. per day penicillamine in 162
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Group B and 50 mg. per day in Group C. In Group C the average daily intake of separately added L-cysteine had been 74 mg. The mean body weights for the animals sacrificed I4 days postoperatively were: Group A, 320 gm., Group B, 317 gm., and Group C, 336 gm. The weights in the 21-day postoperative groups were: Group A, 357 gm., Group B, 352 gm., and Group C, 322 gm. Clinical evaluation at sacrifice revealed that all 6 of the 14-day control wounds in Group A had healed without complication. Five of Group B and 4 of Group C were also well healed. One wound in Group B showed focal ulceration and 1 in Group C revealed focal fresh bleeding and another focal surface blood clot. Histopathological examination of the wounds revealed uncomplicated healing in the 6 controls of Group A, focal ulceration in 1 of the 6 penicillamine-treated Group B animals, and other complications in 3 of the Group C animals: 1 with central cystic change in the wound and 2 with disruption during technical preparation of the slides. The latter could be a reflection of some qualitative change despite the absence of gross wound disruption. Comparative studies of the amount and caliber of wound collagen revealed no evidence of inhibition in Groups B and C. On the contrary the evaluation seemed to indicate more collagen in Group B than Group A: In 3 wounds it was about the same and in another 3 it was considered to be more abundant in Group B. Group C was more difficult to compare quantitatively in the 3 complicated wounds. Two of the second 3 were the same as in Group A and 1 of the 3 was more abundant than in Group A. Clearly, there was no suppression of collagen in either penicillamine-treated group. Clinical evaluation of the 21-day wounds revealed focal crusting on the wound surface by old blood clot in 3 wounds of Group A, in 1 of Group B and in 2 of Group C. There were 7 survivors in Group A, 8 in Group B and 10 in Group C. Histopathological examination of the 21-day wounds showed focal ulceration in 1 of the 7 controls in Group A, one gaping wound out of a total of 8 animals in Group B and two gaping wounds among the wounds of 10 animals in Group C. Comparative study of
GEEVER
ET
AL.:
PENICILLAMINE
1.
14-Day
Group A Controls
MEANS S.D. i S.E. _t
Breaking
WOUND
Strength
in Grams-Fresh
Wound
Wounds*
Group B Penicillamine 48 mg./day
HEALING
IN
GUINEA
Strips
21-Day Group C Penicillamine 48 mg. and L-Cysteine 73 mg./day
Group A Controls
Wounds*
Group B Penicillamine 49 mg./day
Group C Penicillamine 50 mg. and L-Cysteine 74 mg./day
485 425 358 289 249 323
323 355 331 251 239 273
295 329 319 255 128 179
557 641 741 608 490 590 491
336 218 293 219 207 139 164 242
171 171 163 200 186 185 110 168 94 215
355 87.7 35.8
295 47.6 19.4
251 76.1 31.1
588 88.1 33.3
227f 64.1 22.7
1sst 37.6 11.8
* Each value f Statistically
represents significant
the mean differences
of 4 strips from from controls.
PIGS
breaking strength more than penicillamine alone. The difference was statistically significant (P between 0.02 and 0.025). Furthermore, the wounds of both treated groups were weaker at 21 days than at 14 days, whereas the controls showed an increase of approximately 65 percent over the 1Cday values. Chemical treatment of one strip from each wound with 10yO unbuffered formalin for 48 hours revealed a striking increase of breaking strength over the fresh specimen’s value. In the 14-day specimens the means of the breaking strengths in the various groups were as follows: Group A, 1,621 gm., Group B, 1,219 gm., and Group C, 1,078 gm. The difference between Group A and Group C was of borderline significance (P between 0.1 and 0.05) but was not significant between Group A and Group B (P, 0.2). Chemical treatment of one strip from each wound with saturated mercuric chloride solution for 48 hours also caused an increase in breaking strength over the fresh strip value: the mean of Group A, 1,383 gm., Group B, 1,050 gm., and Group C, 1,193 gm. These differences were not statistically significant (P greater than 0.1 and 0.4, respectively). Similarly in the 21-day specimens the corresponding means following 4%hour formalin
the amount of wound collagen between groups revealed: about the same amount in 3 wounds of Group A and Group B; in 3 the amount was greater in Group A, and in 1 of Group B the reverse was true. Comparison of Group A with Group C showed equal amounts of wound collagen in 3, more collagen in the wounds of 3 animals in Group A, and the reverse in 1 animal of Group C. The breaking strength values of the fresh strips of 14- and 21-day wounds are listed in Table 1, which includes means, standard deviations, and standard errors. The penicillaminetreated groups showed a decrease in wound strength as compared to their controls, but this w-as statistically significant only in the 21-day group. The P values of the 14-day fresh strips were as follows: in comparing controls with the penicillamine-treated group, P was found to be between 0.1 and 0.2; in controls versus the penicillamine plus cysteine group, P was between 0.05 and 0.10. The latter level of significance might be questioned. However, in the 21-day experiment comparison of fresh strips of the controls with either penicillamine alone or penicillamine plus cysteine revealed the P value to be less than 0.001. Penicillamine plus added cysteine (Group C) depressed the Table
AND
each
wound.
163
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solution were: Group 1,881 gm., and Group
VOL.
7
NO.
4,
A, 2,317 gm., Group B, C, 1,928 gm. These dif-
ferences were not statistically significant (P greater than 0.3). And following 48-hour treatment with saturated mercuric chloride solution the mean values were: Group A, 1,866 gm., Group B, 1,613 gm., and Group C, 2,028 gm. These differences were not statistically significant (P greater than 0.4). The
hydroxyproline
findings
in the sponge
implants are listed in Table 2. In the 14-day implants there were no statistically significant differences between the groups. The test for the significance of the difference between the Group A controls and the Group B penicillamine treated showed the P value to be between 0.25 and 0.3. Groups A and C are obviously similar. However, in the 21-day implants there was significantly more hydroxyproline in Group B than in the controls (P between 0.001 and 0.005). Added cysteine in both 14- and 21-day sponge implants, Group C, had no significant effect on comparison with
the control
Groups
A. The values
for the
14-day specimens are obviously similar. When the difference between Groups A and C in the 2l-day sponge implants was subjected to statistical test, P was found to be between 0.1 and 0.2. Histopathological examination of the sponge Table
2.
Hydroxyproline
Values,
1967
APRIL
implants was carried out and the amount of collagen graded + to ++++. Focal peripheral collagen only was graded + and diffuse collagen deposition in all porous areas as observed in a cross section through the middle third of the sponge was evaluated ++++. Intermediate quantitations were + + and -I++. The findings in the 14-day specimens were: Group A controls, +++ (2), ++ (3) and f ( 1); Group B, penicillamine-treated, +++ (11, ++ (4) and + (1); Group C, penicillamine plus L-cysteine, ++++ ( 1)) +++ (11, ++ (3), and + (1) (Table 3). Histopathological examination of the sponge implants in the 21-day groups revealed: Group
4 ++++ (51, +++ Cl), and ++ (1); Group B, ++++ (41, +++ (31, and ++ (1); Group C, ++++ ++ (1).
(4), +++
DISCUSSION The findings in this experiment are suggestive
of some
qualitative
abnormality
wg./lOO
mg. Implanted
Sponge
21-Day Implants
Group C Penicillamine
999 1113 898 1003 1023 1208
MEANS S.D. f S.E. f 164
1041 105.6 43.2
in the
reparative collagen of the penicillamine-treated guinea pigs. There was no evidence of a quantitative suppression of reparative collagen as determined by histopathological examination or biochemical (hydroxyproline) analysis.
14-Day Implants
Group A Controls
(51, and
Group A Controls
Group B Penicillamine
and L-Cysteine
999 1030 950 937 713 1122
722 700 977 876 1742 1346
990 915 840 1495 1485 1327 1643
1104 1074 1549 1386 1643 1643 1891 1891
1008 990 827 893 1465 1356 861 1218 1317 1376
1060 403.3 164.6
1242 322.1 121.8
1523 315.4 111.5
1131 240.6 76.0
958 135.8 55.5
Group B Penicillamine
Group C Penicillamine and L-Cysteine
GEEVER
Table
3.
Histopathological
ET
Findings
AL.:
PENICILLAMINE
in Sponge
Implants:
+++ +++ ++ ++ ++ +
Grading
Group B Penicillamine 48 mg. /day +++ ++ ++ ++ ++ +
WOUND
Reparative
HEALING
Collagen
21 Days
14 Days
Group A Controls
AND
Group C Penicillamine 48 mg. and L-Cysteine 73 mg. daily ++++ +++ ++ ++ ++ +
Group A Controls ++++ ++++ ++++ ++++ ++++ +++ ++
Group B Penicillamine 49 mg. /day ++++ ++++ ++-t+ ++++ +-t+ +++ +++ -t+
IN
GUINEA
PIGS
Deposition
-Group C Penicillamine 50 mg. and L-Cysteine 74 mg. daily ++++ ++++ ++++ ++++ +++ +++ +++ +++ +++ ++
+ to ++++.
Thus, our findings differ from those reported in rats by Nimni and Bavetta who described significant inhibition of collagen synthesis in subcutaneous sponge implants [lo]. Chemical treatment in vitro with 10% unbuffered formalin for 48 hours or with saturated mercuric chloride solution for the same period brought the breaking-strength values of the wound strips back to a nearly normal level in the 21day wounds. In the l4-day specimenssimilarly treated in vitro there was corresponding increase in breaking strength except for the formalin-treated wound strips in Group C, penicillamine plus L-cysteine. The difference between this group and the controls remained statistically significant. These findings are somewhat similar to those reported by us in experimental lathyrism in rats [Z]. They suggest that penicillamine interferes with the cross-linking of the wound collagen. Another possible explanation for the decreased breaking strength is that penicillamine exerted its effect on the noncollagenous proteins and the cell membranes within the wound. Although collagen is considered to be the sine qua non of a wound’s breaking strength, we demonstrated [6] that the mesh of cells and noncollagenous proteins in a 3-day wound has the capability of contributing significantly to its breaking strength. This is at a time when no collagen can be detected by
histopathological examination or by hydroxyproline determinations of simultaneously implanted polyvinyl sponges. Cysteine was added to the diet of the guinea pigs in Group C to determine if penicillamine acted by decreasing the availability of cysteine. It had been shown [4] that penicillamine induced urinary excretion of cysteine. It is obvious that in the dose used cysteine did not reverse or reduce the penicillamine effect. In the 21-day wounds it appeared to potentiate the penicillamine action. Both cysteine and penicillamine form complexes with copper and this introduces a possible role of this metal in wound healing. Aposhian [l] reported that cysteine is catabolized at a high rate by copper-containing enzymes. If the latter are inactivated by penicillamine, then cysteine should be available for additional copper complexing. Thus, penicillamine may in fact be preserving cysteine. The penicillamine effect has possible human clinical implications in the treatment of patients with large dosesof penicillin since penicillamine is a derivative of penicillin. A dose of 1.5 million units of penicillin G would yield, in vivo, approximately 400 mg. of penicilloic acid and 50 mg. of penicillamine. Surgical patients often receive much higher doses, i.e., 30 or more million units daily. This could yield penicillamine in quantities sufficient to cause 165
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impaired wound healing. Scheinberg [ll] reported skin friability in patients with Wilson’s disease treated by penicillamine with a daily dosage of 1.0 gm.
4,
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1967
2.
3.
4. SUMMARY
1. Young guinea pigs given penicillamine by mouth in a milk powder diet showed impaired wound healing as manifested by decreased breaking strength of 14- and 21-day wounds; added cysteine did not reverse these findings. 2. Histopathological and biochemical studies showed no quantitative impairment of reparative collagen synthesis; thus, some type of qualitative change is postulated. 3. A possible clinical implication of these findings for surgical patients on high-dosage penicillin therapy was presented.
5. 6.
7.
8.
9. ACKNOWLEDGMENT This paper was supported in part by Army Research Office Grant No. NA-ARO-49-092-66G132 and National Institutes of Health Research Career Award (to S.M.L.) 5K6-GM14, 208-05.
10.
11. REFERENCES 12. 1.
166
Aposhian, H. V. Symposium in Medicine. Philadelphia: 290.
on Metal Lippincott,
Binding 1960. P.
Berberian, G. M., Kuyama, T., Geever, E. F., and Levenson, S. M. Wound healing defect in lathyrism. Surg. Forum 15:50, 1964. Boucek, R. J., and Noble, N. L. Connective tissue: A technique for its isolation and study. Arch. Path. 59:553, 1955. Crawhall, J. C., and Thompson, C. J. Cystinuria: Effect of n-penicillamine on plasma and urinary cysteine concentrates. Science 147: 1459, 1965. Croxton, F. E. Elementary Statistics. New York: Dover, 1959. P. 236. Geever, E. F., Levenson, S. M., and Manner, G. The role of noncollagenous substances in the breaking strength of experimental wounds. SUTgery 60:343, 1966. Geever, E. F., Stein, J. M., and Levenson, S. M. Variations in breaking strength in healing wounds of young guinea pigs. J. Trauma 5:624, 1965. Levenson, S. M., Crowley, L. V., Geever, E. F., Rosen, H., and Berard, C. W. Some studies of wound healing: Experimental methods, effect of ascorbic acid, and effect of deuterium oxide. J. Trauma 4:543, 1964. Mowry, R. W. Manual of Histologic and Special Staining Technics, Armed Forces Institute of Pathology, 2d Ed. New York: Blakiston Div., McGraw-Hill, 1960. P. 138. Nimni, M. E., and Bavetta, L. A. Collagen defect induced by penicillamine. Science 150:905, 1966. Scheinberg, I. H. n-Penicillamine with particular relation to Wilson’s disease. J. Chronic Dis. 17:293, 1964. Woessner, J. F., Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. 93:440, 1961.