JOURNAI.
OF SURGICAL
27, 168-174
RESEARCH
(1979)
Zinc Sulfate
and Wound
JOHN V. HALEY, Depurtment
of Behavioral
Submitted
Science, Lexington,
University Kentucky
for publication
Healing
PH.D. of Kentucky 40506
October
College
qf
Medicine,
24, 1978
The assumption that wounds heal at a constant rate over time, when measured by volume or area, was questioned. Data from two studies indicate that healing rate changes over time. When this bias was taken into consideration, the conflicting findings of studies of zinc sulfate and wound healing support the hypothesis that zinc therapy is effective for patients with low serum zinc. It was concluded that in order to study the effect of zinc therapy, lesion size and zinc intake should be controlled and serum zinc should be monitored.
INTRODUCTION
analysis was reported. If it were not known that the probability of rejecting a null The first studies exploring the effectivehypothesis is less than 0.05, these findings ness of zinc sulfate in accelerating the healwould look like the results of a coin toss. ing of wounds in humans were by Pories Attempts to resolve the conflicting findings and his colleagues [ 16- 191. Since that time a on the basis of etiology, dose level, difnumber of reports with confusing and conferences in age, or health status of the patradictory results have appeared. Thirteen tient samples are equally frustrating. For exstudies have been reported which dealt with ample, in the five controlled studies which directly observable wounds in humans. Six used patients with leg ulcers, the ages of of these involved ulcers of the lower limbs, the patients in the samples and the dose three with wounds resulting from total levels were similar. Yet, two of these excision of pilonidal sinuses, two with pastudies found zinc sulfate beneficial, one tients having decubitus ulcers, and one each found that it retarded healing and two found involved patients whose ulcers were assono difference. Subdividing the studies into ciated with sickle cell anemia and wounds other categories results in a similar lack of resulting from an adrenalectomy. pattern. Three of the studies were reports of There is a possible explanation that is clinical impressions based on observations more basic and may resolve these apparent of patients treated with zinc sulfate and had contradictions. In one of their earlier studies, no control groups. Of the remaining 10 Pories et al. [18, 191 suggested that straight studies, 4 had control groups without a lines would result if the logarithm of wound placebo and 6 were double-blind studies. volume were plotted against time. This is an Four of the ten controlled studies conexponential function of the form y = aebZ, cluded that the treated group healed more where y is wound volume, x is time, e is the quickly than the control group, four studies base of the natural logarithm, and b is a found no differences between the two negative constant reflecting healing rate. groups, and one study found that the control group healed at a faster rate than the If this formula is a valid representation it implies that y (wound volume) changes at a treated group. The authors of the remaining study concluded that the treated group constant rate over time. In a series of studies, healed more quickly, but there were only du Noiiy [6-81 used a variety of mathematical formulas to describe cicatrization of three patients in each group and no statistical 0022-4804/79/090
168-07$0
1.00/O
Copyright 0 1979 by Academtc Press, Inc. All rights of reproduction in any form reserved.
168
JOHN
V. HALEY:
ZINC
SULFATE
wounds. He found that an exponential function was the most satisfactory [8]. But in his equation y represented the proportion of the wound that healed rather than wound area or volume. Although he plotted wound area against time, his calculations were based on the proportion of the wound that healed. He concluded that the rate of healing was not constant over time, but rather the fractional rate of healing was constant. An alternate approach in describing how wounds heal would be to follow Pories’ lead and relate the size of the lesion to time directly. Since wounds are multidimensional, a power function would be expected. This would take the form y = uxb, where y is wound size, x is time, a is the antilogarithm of the intercept of log y vs log X, and b is the slope. If the logarithm of wound size were plotted against the logarithm of time, a straight line would result. One of the problems that arises in this comparison is the arbitrariness of the time scale. Zero is assigned to the point in time when the first observation was made. So it is absolute relative to the beginning of the experiment but arbitrary relative to the lesion which may have existed months or even years prior to the start of the experiment. A constant is, therefore, necessary to correct for the arbitrariness of the time scale. So the equation relating wound area or volume to time would take the form y = a(x + c)~, where c is the constant that corrects the time scale. Data were presented for patients in two studies which can be used to test how well the power function describes wound healing. Pories and Strain [19] give the results of eight patients, presumably the same patients as in their earlier report [18]. They state that accurate measures were no longer possible when the wound volume was around 1 ml. Using this as an approximate cutting point, there are four patients that can be evaluated. The other four patients had too few observation points to determine if the function was curvilinear. Myers and Cherry [141 give data for two patients that
AND
WOUND
HEALING
169
were used as their own control. The data of these six patients are presented in Table 1 and the corresponding graphs in Fig. 1. Estimates of the constant were made by the least squares method using logarithmic transformations. In every case, the power function gives a better fit than the exponential function. It is even able to account for Pories’ patient PS 1, whom he discounted because of surgery that had taken place several years prior to the study. Myers and Cherry 1141 state that “healing rate curves tended to be asymptotic, and when plotted on semilog paper often came close to being straight lines.” They presented a semilog graph of their patient MC 1. It did come close to being a straight line, but deviated consistently in a curvilinear fashion. Their patient MC 2 followed a straight line until Week 20, when the values were below the straight line. This occurred during the treatment period. A power function appears to be more appropriate for describing how wounds heal, emphasizing further that healing rate changes over time as a function of the wound size. Studies that use patients as their own control and make pre- and postcomparisons assume that healing rate is constant which is erroneous. Also, if the treatment and placebo groups do not have average wound sizes that are approximately equal, a bias is introduced favoring the group with the largest mean. In order to adequately evaluate the effectiveness of zinc sulfate, independent groups should be used, one placebo and one treatment, and the mean lesion size should be the same for both groups. If area or volume is used, a logarithmic transformation might be necessary depending upon the assumptions and the robustness of the statistical test used. When these possible biases are taken into consideration, a less conflicting picture emerges from a review of the literature. Brief comments of each study will be made and final conclusions will be drawn. The studies by Cohen [5] and Greaves and Skillen [ll] will not be considered be-
170
JOURNAL
OF SURGICAL
RESEARCH: TABLE
HEALING
OF WOUNDS
OVER TIME
VOL.
27, NO.
3, SEPTEMBER
1979
1 FOR SIX PATIENTS
[ 11, 161”
MC1 Wound volume (ml)
Days
Days
PS 1 0 4 7 10 14 18 22 26 33
PS 2 65 21 16 16.5 12 9.5 9 6 6
0 2 6 9 12 15
16:5 7.5 3.0 3.0 2.5 2.0
0 4 8 11 15 18
PS 3 0 4 8 11 15 19
Wound volume (ml)
42 24 15 13 8 6
PS 4 25.5 9.5 8.5 4.7 3.0 2.0
MC2
Weeks
Wound area (cm’)
0 1 2 3 4 6 13 15 16 18 20 21 22 23
11.7 10.7 11.2 9.1 7.2 6.8 4.7 4.3 3.6 3.7 2.9 3.5 2.8 2.7
Wound area Days 0 1 2 3 4 5 7 8 10 11 12 13 14 15 17 20 21 22 26 29 30
(cm*) 267 211 178 222 172 150 136 147 128 119 106 103 106 100 100 72 67 72 61 50 44
u Data from W. J. Pories and W. H. Strain, In (A. S. Prasad, Ed.), Zinc and Wound Healing, Zinc Metabolism. Courtesy of Charles C Thomas, Springfield, Ill., and M. B. Myers and G. Cherry, Zinc and the Healing of Chronic Leg Ulcers. Courtesy of Dun Donnelley, New York.
cause they did not use control groups. Pories
lesions were smaller. The fact that days for complete healing were significantly less for healthy males with wounds formed by excis- the treatment group, in spite of the bias, ing pilonidal sinuses. They were randomly supports the conclusions of the study that assigned to control and treatment groups zinc therapy was effective. Ignoring the fact and observed at approximately 5-day in- that this was not a double-blind study the tervals. The study was not double-blind conclusions seem valid. Husain [13] studied 104 patients with nor was a placebo given to the control group. Two measures were evaluated, days for ulcers of the lower limb. He started with a complete healing and healing rate (ml/day). double-blind study but was able to identify Both were significant. The significance of the treatment group because he monitored healing rate is suspect because the mean of serum zinc levels. Two measures were used, the treatment group is greater than the mean days required for healing and surface area of of the control group (54.5 vs 32.3). Since ulcers (mm*). Both measures were sighealing rate is not constant over time and nificant. The mean initial area of the placebo the treatment group had further to go, a group was slightly larger than the mean for the treatment group (4830 vs 42!90), introgreater healing rate would be expected. Days for complete healing was biased in ducing a possible bias in favor of the placebo favor of the control group since their group. In spite of this, the treated group et al. [16, 171 studied 20 young otherwise
JOHN V. HALEY:
ZINC SULFATE
AND WOUND HEALING
171
ml.
ml.
cm2
weeks
weeks
FIG. 1. Graph of logarithmic transformations for six patients.
healed more quickly than the placebo group (P < 0.001) supporting his conclusion that zinc sulfate can be considered a useful adjuvant therapy in the management of chronic leg ulcers. Barcia [2] attempted to replicate the study by Pories et al. [16, 171. His sample consisted of 20 young and otherwise healthy patients with chronic pilonidal disease, whom he assigned to control and treatment groups on an odd-even basis. This was not a double-blind study. The initial wound volume was approximately the same for the control and treatment groups (43.4 vs 46.0). There was no significant difference between
the two groups in days for complete healing, and the healing rate (ml/day) was essentially the same. Barcia suggests that his study does not agree with Pories et al. either because zinc sulfate is not a significant factor or that neither study could be controlled sufficiently to cover all variations in individual patients. Carruthers [3] in his review of oral zinc and cutaneous healing suggests that dietary factors may account for this discrepancy. To check this or to control for it he recommends the measurement of zinc levels. Sergeant et al. [20] did a double-blind study using 34 patients with active leg ulcers
172
JOURNAL
OF SURGICAL
RESEARCH:
associated with sickle cell anemia. He evaluated the effectiveness of zinc by the average rate of healing (mm*/day) and by the number of cases showing improvement. The average initial ulcer area was 855 mm* in the treatment group and 689 mm2 in the placebo group. The larger treatment group mean could introduce a bias that would account for the significant difference in healing rate. But the larger number of improved patients in the treatment group and the differences in serum zinc levels between the two groups supports their conclusion that zinc sulfate may prove a useful adjunct to the therapy of sickle cell ulcers. Myers and Cherry [14] used 16 leg ulcer patients as their own control. They observed these patients for a variable period of time and then started them on zinc therapy for an additional period of time which also varied from patient to patient. They did not use a placebo or double-blind. They also observed a control group of 12 additional patients who never received zinc. The measure that they evaluated was healing rate (cm?week). They made two comparisons, serum zinc levels of good healers vs poor healers and healing rate of treatment vs control group. The erroneous assumption of constant healing rates over time is especially crucial in a study of this type. This is supported by the fact that healing rate was much lower during the second observation period for both the treated and the nontreated groups. Also, the ulcer size was larger for the control group than for the treated group which would tend to bias the results in favor of the control group. A final source of bias is the variable observation period. The treated group received the drug and was observed from 2.5 to 21.5 weeks (mean = 13.3 weeks) while everyone in the control group was observed for 9 weeks. Since healing rates reduce over time this variable observation period would favor the control group. Myers and Cherry also present graphs for two patients. These correspond to patients MC 1 and MC 2 in Fig. 1. Patient MC 2, whose initial serum zinc level was 52 pg/
VOL. 27, NO. 3, SEPTEMBER
1979
. Control
Y=875(x-5.7)-*7'5
o Treatment
I
Y=4?1(X-9.5)-.7'5
250, 200. 150.
100 75
50. 40, I
5
10
FIG. 2. Graph of logarithmic patient MC 2.
15
20
25
transformation
for
100 ml was observed for a 15-week control period and for a 15-week zinc therapy period. Figure 2 shows the results when a power function is assumed and the control and treatment periods are analyzed separately. For this patient, there does seem to be a difference between the two periods. The slope of the function is the same, but the additive constant which corrects for the arbitrary time scale is different. For this case, zinc therapy had the effect of placing the patient in an earlier time period when the healing rate was higher without affecting the other parameters of the function. There was no difference between the control and treatment period for patient MC 1 who had normal serum zinc levels (126 pgl 100 ml). Norris and Reynolds [lS] studied 14 chronic patients with decubitus ulcers. They used a double-blind cross over design in which the patients served as their own control. Half of the patients received zinc and half received placebo for 12 weeks. This was reversed for the last 12 weeks. The measure evaluated was volume using the method of Pories et al. [ 181. Because of the type of patients used in the study, seven died during the course of the study. Only three patients completed the 24-week study.
JOHN
V. HALEY:
ZINC
SULFATE
Since 10 patients received zinc for at least 4 weeks, and eight patients received a placebo for at least 4 weeks, Norris and Reynolds considered that they had 18 patient-exposure periods. They analyzed their data with ar test for independent measures which was inappropriate because four patients were in both groups (dependent measures), and only six zinc patients and four placebo patients were independent. Because of the missing data on paired observations, it is not possible to analyze or interpret the results. Greaves and Ive [lo] reported a doubleblind study of 38 patients with chronic leg ulceration. Ulcer area was determined by the product of two radii measured at right angles, one of which lay along the long axis of the ulcer. Treatment was continued until the ulcer had healed or until 4 months had elapsed, whichever occurred first. The zinc group had a greater healing rate, but it did not reach statistical significance. Ulcer size was approximately the same for the zinc and placebo groups, so no bias is evident. However, their method for determining area was not as accurate as in the other studies. Clayton [4] did a double-blind study of 10 patients with leg ulcers. The patients were observed for an average of 4 weeks. His criteria for healing were mean days to 50 and 90% healing, and mean percentage of the ulcer healed in 7 and 14 days. The average ulcer area was 44.9 cm2 for the zinc group and 65.8 cm2 for the placebo group. The placebo group healed faster than the zinc group for all criteria. Considering that healing rates vary as a function of lesion size and the placebo group mean was 47% larger than the zinc group mean, these results are not surprising. The short time period of the study also enhances the bias of assuming a constant rate of healing over time. Finally, studies indicate that at least 4 weeks are required for serum zinc levels to show a significant increase and 7 to 8 weeks for clinical manifestations [ 11. Hallbook and Lanner [12] studied 27 patients with venous stasis leg ulcers. In addition to doing a double-blind study, they
AND
WOUND
HEALING
173
monitored serum zinc concentrations and further subdivided the patients into two groups on the basis of this determination. Group 1 consisted of 14 patients with serum zinc less than 110 pg/lOO ml. Seven of these patients were given zinc sulfate and seven were given a placebo. There were 13 patients in Group II with serum zinc levels of 110 j&100 ml or more. Six were given zinc sulfate and seven were given a placebo. The ulcer area was larger for the placebo treated patients than for the zinc-sulfatetreated patients in both groups. In spite of this, for patients with low serum zinc levels, those treated with zinc sulfate healed at a faster rate than those treated with a placebo (P = 0.02). The unique feature of this study is that the authors subdivided their groups into high and low levels of serum zinc. They concluded that zinc sulfate increases the rate of healing for patients with low serum zinc. Flynn et al. [9] in a study of adrenalectomized patients followed six patients on long-term corticosteroid therapy from 6 months to 2 years. They all had serum zinc deficiency and delayed healing. Three patients were given zinc supplements and three were not. A double-blind or placebo was not used. The data were presented in a case history format. The treated patients had healed completely by the eighth week. The control patients had little or no healing by the 14th or 16th week. Rate of healing was positively associated with serum zinc levels. No statistical analysis was performed. DISCUSSION
Four of the ten controlled studies which concluded that zinc sulfate treatment is effective seem to be supported by the foregoing analysis. Two of these [ 16,17,20] had treatment groups whose average size of lesion was larger than the control group. This introduced a bias in favor of the treatment group healing at a faster rate than the control group. However, both studies used additional measures such as days to- com-
174
JOURNAL
OF SURGICAL
RESEARCH:
plete healing. For these measures, the bias would be in favor of the control group. So one may conclude that all four studies support the hypothesis that zinc therapy is effective. There were four studies that found no difference between the zinc treatment and control groups. The control group in the Myers and Cherry study [14] had larger lesions than the treatment group. This bias could easily account for the lack of significant findings. Because of missing data, it was not possible to analyze the results of the Norris and Reynolds study adequately [ 151. Greaves and Ive [lo] failed to find significant differences, but their measure of area was less accurate than in other studies. Barcia’s study [2] did not have any biases and did not find any significant results. The Clayton study [4] found that the placebo group had a greater healing rate than the zinc group, but the placebo group had much larger lesions and the study did not allow enough time for zinc therapy to be effective. A more consistent pattern emerges when two factors are taken into consideration. First, wounds do not heal at a constant rate over time. The second factor is that zinc therapy is effective only for patients who have low serum zinc levels. In order to study the effect of zinc therapy systematically, it is necessary to monitor serum zinc levels and to control for lesion size and zinc intake. With one exception, the studies that failed to demonstrate a significant effect for zinc therapy had problems in their design, in their ability to measure the lesions, or to control for lesion size. REFERENCES 1. Abdulla, M., Norden, A., and Haeger-Aronsen, B. Serum levels after oral zinc. Lance? 2: 154, 1973.
VOL. 27, NO. 3, SEPTEMBER
1979
2. Barcia, B. J. Lack of acceleration of healing with zinc sulfate. Ann. Surg. 172: 1048, 1970. 3. Carruthers, R. Oral zinc in cutaneous healing. Drugs 6: 161, 1973. 4. Clayton, R. J. Double-blind trial of zinc sulfate in patients with leg ulcers. grit. J. C/in. Pratt. 26: 368, 1972. 5. Cohen, C. Zinc sulfate and bedsores. Brit. Med. J. 2: 561, 1968. 6. du Noily, P. L. Cicatrization of wounds, II. J. Exp. Med. 24: 451, 1916. 7. du Noiiy, P. L. Cicatrization of wounds, III. J. EXD. Med. 24: 461. 1916. 8. du’ Nobly, P. L. Cicatrization of wounds, X. J. Exp. Med. 29: 329, 1919. 9. Flynn, A., Strain, W. H., Pories, W. J., et al. Zinc deficiency with altered adrenocortical function and its relation to delayed healing. Lancet 1: 789, 1973. 10. &eaves, M. W., and Ive, F. A. Double-blind trial of zinc sulfate in the treatment of chronic venous leg ulceration. Brir. J. Dermatol. 87: 632, 1972. 11. &eaves, M. W., and Skillen, A. W. Effects of long-continued ingestion of zinc sulfate in patients with venous leg ulcerations. Lancer 2: 889, 1970. 12. Hallbook, T., and Lanner, E. Serum-zinc and healing of venous leg ulcers. Lancer 2: 780, 1972. 13. Husain, S. L. Oral zinc sulfate in leg ulcers. Lancer 1: 1069, 1969. 14. Myers, M. B., and Cherry, G. Zinc and the healing of chronic leg ulcers. Amer. J. Surg. 120: 77, 1970. 1.5. Norris, J. R., and Reynolds, R. E. The effect of oral zinc sulfate therapy on decubitus ulcers. J. Amer. Geriaf. Sot. 19: 793, 1971. 16. Pories, W. J., Henzel, J. H., Rob, C. G., et al. Acceleration of wound healing in man with zinc sulfate given by mouth. Lancer 1: 121, 1967. 17. Pories, W. J., Henzel, J. H., Rob, C. G., et al. Acceleration of healing with zinc sulfate. Ann. Surg. 165: 432, 1967. 18. Pories, W. J., Schear, E. W., Jordan, D. R., er al. Measurement of human wound healing. Surgev 59: 821, 1966. 19. Pories, W. J., and Strain, W. H. In A. S. Prasad (Ed.), Zinc and Wound Healing, Zinc Merabohm. Springfield, 111.: Charles C Thomas, 1966. Pp. 378-391. 20. Sejeant, G. R., Galloway, R. E., and Gueri, M. C.: Oral zinc sulfate in sickle cell ulcers. Lance? 2: 891, 1970.
OF SURGICAL
27, 168-174
RESEARCH
(1979)
Zinc Sulfate
and Wound
JOHN V. HALEY, Depurtment
of Behavioral
Submitted
Science, Lexington,
University Kentucky
for publication
Healing
PH.D. of Kentucky 40506
October
College
qf
Medicine,
24, 1978
The assumption that wounds heal at a constant rate over time, when measured by volume or area, was questioned. Data from two studies indicate that healing rate changes over time. When this bias was taken into consideration, the conflicting findings of studies of zinc sulfate and wound healing support the hypothesis that zinc therapy is effective for patients with low serum zinc. It was concluded that in order to study the effect of zinc therapy, lesion size and zinc intake should be controlled and serum zinc should be monitored.
INTRODUCTION
analysis was reported. If it were not known that the probability of rejecting a null The first studies exploring the effectivehypothesis is less than 0.05, these findings ness of zinc sulfate in accelerating the healwould look like the results of a coin toss. ing of wounds in humans were by Pories Attempts to resolve the conflicting findings and his colleagues [ 16- 191. Since that time a on the basis of etiology, dose level, difnumber of reports with confusing and conferences in age, or health status of the patradictory results have appeared. Thirteen tient samples are equally frustrating. For exstudies have been reported which dealt with ample, in the five controlled studies which directly observable wounds in humans. Six used patients with leg ulcers, the ages of of these involved ulcers of the lower limbs, the patients in the samples and the dose three with wounds resulting from total levels were similar. Yet, two of these excision of pilonidal sinuses, two with pastudies found zinc sulfate beneficial, one tients having decubitus ulcers, and one each found that it retarded healing and two found involved patients whose ulcers were assono difference. Subdividing the studies into ciated with sickle cell anemia and wounds other categories results in a similar lack of resulting from an adrenalectomy. pattern. Three of the studies were reports of There is a possible explanation that is clinical impressions based on observations more basic and may resolve these apparent of patients treated with zinc sulfate and had contradictions. In one of their earlier studies, no control groups. Of the remaining 10 Pories et al. [18, 191 suggested that straight studies, 4 had control groups without a lines would result if the logarithm of wound placebo and 6 were double-blind studies. volume were plotted against time. This is an Four of the ten controlled studies conexponential function of the form y = aebZ, cluded that the treated group healed more where y is wound volume, x is time, e is the quickly than the control group, four studies base of the natural logarithm, and b is a found no differences between the two negative constant reflecting healing rate. groups, and one study found that the control group healed at a faster rate than the If this formula is a valid representation it implies that y (wound volume) changes at a treated group. The authors of the remaining study concluded that the treated group constant rate over time. In a series of studies, healed more quickly, but there were only du Noiiy [6-81 used a variety of mathematical formulas to describe cicatrization of three patients in each group and no statistical 0022-4804/79/090
168-07$0
1.00/O
Copyright 0 1979 by Academtc Press, Inc. All rights of reproduction in any form reserved.
168
JOHN
V. HALEY:
ZINC
SULFATE
wounds. He found that an exponential function was the most satisfactory [8]. But in his equation y represented the proportion of the wound that healed rather than wound area or volume. Although he plotted wound area against time, his calculations were based on the proportion of the wound that healed. He concluded that the rate of healing was not constant over time, but rather the fractional rate of healing was constant. An alternate approach in describing how wounds heal would be to follow Pories’ lead and relate the size of the lesion to time directly. Since wounds are multidimensional, a power function would be expected. This would take the form y = uxb, where y is wound size, x is time, a is the antilogarithm of the intercept of log y vs log X, and b is the slope. If the logarithm of wound size were plotted against the logarithm of time, a straight line would result. One of the problems that arises in this comparison is the arbitrariness of the time scale. Zero is assigned to the point in time when the first observation was made. So it is absolute relative to the beginning of the experiment but arbitrary relative to the lesion which may have existed months or even years prior to the start of the experiment. A constant is, therefore, necessary to correct for the arbitrariness of the time scale. So the equation relating wound area or volume to time would take the form y = a(x + c)~, where c is the constant that corrects the time scale. Data were presented for patients in two studies which can be used to test how well the power function describes wound healing. Pories and Strain [19] give the results of eight patients, presumably the same patients as in their earlier report [18]. They state that accurate measures were no longer possible when the wound volume was around 1 ml. Using this as an approximate cutting point, there are four patients that can be evaluated. The other four patients had too few observation points to determine if the function was curvilinear. Myers and Cherry [141 give data for two patients that
AND
WOUND
HEALING
169
were used as their own control. The data of these six patients are presented in Table 1 and the corresponding graphs in Fig. 1. Estimates of the constant were made by the least squares method using logarithmic transformations. In every case, the power function gives a better fit than the exponential function. It is even able to account for Pories’ patient PS 1, whom he discounted because of surgery that had taken place several years prior to the study. Myers and Cherry 1141 state that “healing rate curves tended to be asymptotic, and when plotted on semilog paper often came close to being straight lines.” They presented a semilog graph of their patient MC 1. It did come close to being a straight line, but deviated consistently in a curvilinear fashion. Their patient MC 2 followed a straight line until Week 20, when the values were below the straight line. This occurred during the treatment period. A power function appears to be more appropriate for describing how wounds heal, emphasizing further that healing rate changes over time as a function of the wound size. Studies that use patients as their own control and make pre- and postcomparisons assume that healing rate is constant which is erroneous. Also, if the treatment and placebo groups do not have average wound sizes that are approximately equal, a bias is introduced favoring the group with the largest mean. In order to adequately evaluate the effectiveness of zinc sulfate, independent groups should be used, one placebo and one treatment, and the mean lesion size should be the same for both groups. If area or volume is used, a logarithmic transformation might be necessary depending upon the assumptions and the robustness of the statistical test used. When these possible biases are taken into consideration, a less conflicting picture emerges from a review of the literature. Brief comments of each study will be made and final conclusions will be drawn. The studies by Cohen [5] and Greaves and Skillen [ll] will not be considered be-
170
JOURNAL
OF SURGICAL
RESEARCH: TABLE
HEALING
OF WOUNDS
OVER TIME
VOL.
27, NO.
3, SEPTEMBER
1979
1 FOR SIX PATIENTS
[ 11, 161”
MC1 Wound volume (ml)
Days
Days
PS 1 0 4 7 10 14 18 22 26 33
PS 2 65 21 16 16.5 12 9.5 9 6 6
0 2 6 9 12 15
16:5 7.5 3.0 3.0 2.5 2.0
0 4 8 11 15 18
PS 3 0 4 8 11 15 19
Wound volume (ml)
42 24 15 13 8 6
PS 4 25.5 9.5 8.5 4.7 3.0 2.0
MC2
Weeks
Wound area (cm’)
0 1 2 3 4 6 13 15 16 18 20 21 22 23
11.7 10.7 11.2 9.1 7.2 6.8 4.7 4.3 3.6 3.7 2.9 3.5 2.8 2.7
Wound area Days 0 1 2 3 4 5 7 8 10 11 12 13 14 15 17 20 21 22 26 29 30
(cm*) 267 211 178 222 172 150 136 147 128 119 106 103 106 100 100 72 67 72 61 50 44
u Data from W. J. Pories and W. H. Strain, In (A. S. Prasad, Ed.), Zinc and Wound Healing, Zinc Metabolism. Courtesy of Charles C Thomas, Springfield, Ill., and M. B. Myers and G. Cherry, Zinc and the Healing of Chronic Leg Ulcers. Courtesy of Dun Donnelley, New York.
cause they did not use control groups. Pories
lesions were smaller. The fact that days for complete healing were significantly less for healthy males with wounds formed by excis- the treatment group, in spite of the bias, ing pilonidal sinuses. They were randomly supports the conclusions of the study that assigned to control and treatment groups zinc therapy was effective. Ignoring the fact and observed at approximately 5-day in- that this was not a double-blind study the tervals. The study was not double-blind conclusions seem valid. Husain [13] studied 104 patients with nor was a placebo given to the control group. Two measures were evaluated, days for ulcers of the lower limb. He started with a complete healing and healing rate (ml/day). double-blind study but was able to identify Both were significant. The significance of the treatment group because he monitored healing rate is suspect because the mean of serum zinc levels. Two measures were used, the treatment group is greater than the mean days required for healing and surface area of of the control group (54.5 vs 32.3). Since ulcers (mm*). Both measures were sighealing rate is not constant over time and nificant. The mean initial area of the placebo the treatment group had further to go, a group was slightly larger than the mean for the treatment group (4830 vs 42!90), introgreater healing rate would be expected. Days for complete healing was biased in ducing a possible bias in favor of the placebo favor of the control group since their group. In spite of this, the treated group et al. [16, 171 studied 20 young otherwise
JOHN V. HALEY:
ZINC SULFATE
AND WOUND HEALING
171
ml.
ml.
cm2
weeks
weeks
FIG. 1. Graph of logarithmic transformations for six patients.
healed more quickly than the placebo group (P < 0.001) supporting his conclusion that zinc sulfate can be considered a useful adjuvant therapy in the management of chronic leg ulcers. Barcia [2] attempted to replicate the study by Pories et al. [16, 171. His sample consisted of 20 young and otherwise healthy patients with chronic pilonidal disease, whom he assigned to control and treatment groups on an odd-even basis. This was not a double-blind study. The initial wound volume was approximately the same for the control and treatment groups (43.4 vs 46.0). There was no significant difference between
the two groups in days for complete healing, and the healing rate (ml/day) was essentially the same. Barcia suggests that his study does not agree with Pories et al. either because zinc sulfate is not a significant factor or that neither study could be controlled sufficiently to cover all variations in individual patients. Carruthers [3] in his review of oral zinc and cutaneous healing suggests that dietary factors may account for this discrepancy. To check this or to control for it he recommends the measurement of zinc levels. Sergeant et al. [20] did a double-blind study using 34 patients with active leg ulcers
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associated with sickle cell anemia. He evaluated the effectiveness of zinc by the average rate of healing (mm*/day) and by the number of cases showing improvement. The average initial ulcer area was 855 mm* in the treatment group and 689 mm2 in the placebo group. The larger treatment group mean could introduce a bias that would account for the significant difference in healing rate. But the larger number of improved patients in the treatment group and the differences in serum zinc levels between the two groups supports their conclusion that zinc sulfate may prove a useful adjunct to the therapy of sickle cell ulcers. Myers and Cherry [14] used 16 leg ulcer patients as their own control. They observed these patients for a variable period of time and then started them on zinc therapy for an additional period of time which also varied from patient to patient. They did not use a placebo or double-blind. They also observed a control group of 12 additional patients who never received zinc. The measure that they evaluated was healing rate (cm?week). They made two comparisons, serum zinc levels of good healers vs poor healers and healing rate of treatment vs control group. The erroneous assumption of constant healing rates over time is especially crucial in a study of this type. This is supported by the fact that healing rate was much lower during the second observation period for both the treated and the nontreated groups. Also, the ulcer size was larger for the control group than for the treated group which would tend to bias the results in favor of the control group. A final source of bias is the variable observation period. The treated group received the drug and was observed from 2.5 to 21.5 weeks (mean = 13.3 weeks) while everyone in the control group was observed for 9 weeks. Since healing rates reduce over time this variable observation period would favor the control group. Myers and Cherry also present graphs for two patients. These correspond to patients MC 1 and MC 2 in Fig. 1. Patient MC 2, whose initial serum zinc level was 52 pg/
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. Control
Y=875(x-5.7)-*7'5
o Treatment
I
Y=4?1(X-9.5)-.7'5
250, 200. 150.
100 75
50. 40, I
5
10
FIG. 2. Graph of logarithmic patient MC 2.
15
20
25
transformation
for
100 ml was observed for a 15-week control period and for a 15-week zinc therapy period. Figure 2 shows the results when a power function is assumed and the control and treatment periods are analyzed separately. For this patient, there does seem to be a difference between the two periods. The slope of the function is the same, but the additive constant which corrects for the arbitrary time scale is different. For this case, zinc therapy had the effect of placing the patient in an earlier time period when the healing rate was higher without affecting the other parameters of the function. There was no difference between the control and treatment period for patient MC 1 who had normal serum zinc levels (126 pgl 100 ml). Norris and Reynolds [lS] studied 14 chronic patients with decubitus ulcers. They used a double-blind cross over design in which the patients served as their own control. Half of the patients received zinc and half received placebo for 12 weeks. This was reversed for the last 12 weeks. The measure evaluated was volume using the method of Pories et al. [ 181. Because of the type of patients used in the study, seven died during the course of the study. Only three patients completed the 24-week study.
JOHN
V. HALEY:
ZINC
SULFATE
Since 10 patients received zinc for at least 4 weeks, and eight patients received a placebo for at least 4 weeks, Norris and Reynolds considered that they had 18 patient-exposure periods. They analyzed their data with ar test for independent measures which was inappropriate because four patients were in both groups (dependent measures), and only six zinc patients and four placebo patients were independent. Because of the missing data on paired observations, it is not possible to analyze or interpret the results. Greaves and Ive [lo] reported a doubleblind study of 38 patients with chronic leg ulceration. Ulcer area was determined by the product of two radii measured at right angles, one of which lay along the long axis of the ulcer. Treatment was continued until the ulcer had healed or until 4 months had elapsed, whichever occurred first. The zinc group had a greater healing rate, but it did not reach statistical significance. Ulcer size was approximately the same for the zinc and placebo groups, so no bias is evident. However, their method for determining area was not as accurate as in the other studies. Clayton [4] did a double-blind study of 10 patients with leg ulcers. The patients were observed for an average of 4 weeks. His criteria for healing were mean days to 50 and 90% healing, and mean percentage of the ulcer healed in 7 and 14 days. The average ulcer area was 44.9 cm2 for the zinc group and 65.8 cm2 for the placebo group. The placebo group healed faster than the zinc group for all criteria. Considering that healing rates vary as a function of lesion size and the placebo group mean was 47% larger than the zinc group mean, these results are not surprising. The short time period of the study also enhances the bias of assuming a constant rate of healing over time. Finally, studies indicate that at least 4 weeks are required for serum zinc levels to show a significant increase and 7 to 8 weeks for clinical manifestations [ 11. Hallbook and Lanner [12] studied 27 patients with venous stasis leg ulcers. In addition to doing a double-blind study, they
AND
WOUND
HEALING
173
monitored serum zinc concentrations and further subdivided the patients into two groups on the basis of this determination. Group 1 consisted of 14 patients with serum zinc less than 110 pg/lOO ml. Seven of these patients were given zinc sulfate and seven were given a placebo. There were 13 patients in Group II with serum zinc levels of 110 j&100 ml or more. Six were given zinc sulfate and seven were given a placebo. The ulcer area was larger for the placebo treated patients than for the zinc-sulfatetreated patients in both groups. In spite of this, for patients with low serum zinc levels, those treated with zinc sulfate healed at a faster rate than those treated with a placebo (P = 0.02). The unique feature of this study is that the authors subdivided their groups into high and low levels of serum zinc. They concluded that zinc sulfate increases the rate of healing for patients with low serum zinc. Flynn et al. [9] in a study of adrenalectomized patients followed six patients on long-term corticosteroid therapy from 6 months to 2 years. They all had serum zinc deficiency and delayed healing. Three patients were given zinc supplements and three were not. A double-blind or placebo was not used. The data were presented in a case history format. The treated patients had healed completely by the eighth week. The control patients had little or no healing by the 14th or 16th week. Rate of healing was positively associated with serum zinc levels. No statistical analysis was performed. DISCUSSION
Four of the ten controlled studies which concluded that zinc sulfate treatment is effective seem to be supported by the foregoing analysis. Two of these [ 16,17,20] had treatment groups whose average size of lesion was larger than the control group. This introduced a bias in favor of the treatment group healing at a faster rate than the control group. However, both studies used additional measures such as days to- com-
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plete healing. For these measures, the bias would be in favor of the control group. So one may conclude that all four studies support the hypothesis that zinc therapy is effective. There were four studies that found no difference between the zinc treatment and control groups. The control group in the Myers and Cherry study [14] had larger lesions than the treatment group. This bias could easily account for the lack of significant findings. Because of missing data, it was not possible to analyze the results of the Norris and Reynolds study adequately [ 151. Greaves and Ive [lo] failed to find significant differences, but their measure of area was less accurate than in other studies. Barcia’s study [2] did not have any biases and did not find any significant results. The Clayton study [4] found that the placebo group had a greater healing rate than the zinc group, but the placebo group had much larger lesions and the study did not allow enough time for zinc therapy to be effective. A more consistent pattern emerges when two factors are taken into consideration. First, wounds do not heal at a constant rate over time. The second factor is that zinc therapy is effective only for patients who have low serum zinc levels. In order to study the effect of zinc therapy systematically, it is necessary to monitor serum zinc levels and to control for lesion size and zinc intake. With one exception, the studies that failed to demonstrate a significant effect for zinc therapy had problems in their design, in their ability to measure the lesions, or to control for lesion size. REFERENCES 1. Abdulla, M., Norden, A., and Haeger-Aronsen, B. Serum levels after oral zinc. Lance? 2: 154, 1973.
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2. Barcia, B. J. Lack of acceleration of healing with zinc sulfate. Ann. Surg. 172: 1048, 1970. 3. Carruthers, R. Oral zinc in cutaneous healing. Drugs 6: 161, 1973. 4. Clayton, R. J. Double-blind trial of zinc sulfate in patients with leg ulcers. grit. J. C/in. Pratt. 26: 368, 1972. 5. Cohen, C. Zinc sulfate and bedsores. Brit. Med. J. 2: 561, 1968. 6. du Noily, P. L. Cicatrization of wounds, II. J. Exp. Med. 24: 451, 1916. 7. du Noiiy, P. L. Cicatrization of wounds, III. J. EXD. Med. 24: 461. 1916. 8. du’ Nobly, P. L. Cicatrization of wounds, X. J. Exp. Med. 29: 329, 1919. 9. Flynn, A., Strain, W. H., Pories, W. J., et al. Zinc deficiency with altered adrenocortical function and its relation to delayed healing. Lancet 1: 789, 1973. 10. &eaves, M. W., and Ive, F. A. Double-blind trial of zinc sulfate in the treatment of chronic venous leg ulceration. Brir. J. Dermatol. 87: 632, 1972. 11. &eaves, M. W., and Skillen, A. W. Effects of long-continued ingestion of zinc sulfate in patients with venous leg ulcerations. Lancer 2: 889, 1970. 12. Hallbook, T., and Lanner, E. Serum-zinc and healing of venous leg ulcers. Lancer 2: 780, 1972. 13. Husain, S. L. Oral zinc sulfate in leg ulcers. Lancer 1: 1069, 1969. 14. Myers, M. B., and Cherry, G. Zinc and the healing of chronic leg ulcers. Amer. J. Surg. 120: 77, 1970. 1.5. Norris, J. R., and Reynolds, R. E. The effect of oral zinc sulfate therapy on decubitus ulcers. J. Amer. Geriaf. Sot. 19: 793, 1971. 16. Pories, W. J., Henzel, J. H., Rob, C. G., et al. Acceleration of wound healing in man with zinc sulfate given by mouth. Lancer 1: 121, 1967. 17. Pories, W. J., Henzel, J. H., Rob, C. G., et al. Acceleration of healing with zinc sulfate. Ann. Surg. 165: 432, 1967. 18. Pories, W. J., Schear, E. W., Jordan, D. R., er al. Measurement of human wound healing. Surgev 59: 821, 1966. 19. Pories, W. J., and Strain, W. H. In A. S. Prasad (Ed.), Zinc and Wound Healing, Zinc Merabohm. Springfield, 111.: Charles C Thomas, 1966. Pp. 378-391. 20. Sejeant, G. R., Galloway, R. E., and Gueri, M. C.: Oral zinc sulfate in sickle cell ulcers. Lance? 2: 891, 1970.
