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Mitochondria-toxic activity in burned human skin: relation to severity of burn and period after burn
Burns,9.13-16
PrintedinGreatBritain
13
Mitochondria-toxic activity in burned human skin: relation to severity of burn and period after burn Hisashi Aoyarna,
Kohji Suzuki and Yohei lzawa
Department of Burns and Plastic Surgery, Chukyo Hospital, Nagoya, Japan
Masanao
Kobashashi
and Takayuki
Ozawa
Department of Biomedical Chemistry, Faculty of Medicine, University of Nago ya, Nagoya, Japan
Summary
Toxic activity in the extracts of burned human skins impairing respiratory control of rat-liver mitochondria was compared in terms of the severity of burn injury and period after burn. The toxic activity was detected in the extracts from skins of third-degree burn, but not in those from skins of deep-dermal burn or of burn ulcers, in the present experimental conditions. In the case of third-degree burn, the toxic activity was not evident during the first few days post burn, but was significantly increased after IO days. The results of this study suggest the effective period for early therapeutic debridement of the necrotized tissues in the management of severely burned patients.
lowering respiratory control index (RCI), exists in burned human skins (Aoyama et al., 1980). Another paper reported the presence of similar activity in burned skins experimentally induced on rats, its increase in the second week after burn and isolation, with some characterization, of the substance responsible for the toxic activity (Suzuki et al., 1981). Extending these observations, the present paper compares the toxic activity in burned skins from human patients in terms of the severity of bum injury and time after burn.
INTRODUCTION
MATERIALS AND METHODS Burned skin extracts
THE: presence of some toxic substances has been proposed to explain so-called burn death in extensive burn injury. Such substances are supposed to be produced in the burned skin, transferred into general circulation and cause generalized damages in other tissues. Several authors have investigated such toxic substances from human or animal skins, or sera, after burn, testing the toxic activities by various methods (Rosenthal et al., 1972; Allgijwer et al., 1973; Raffa and Trunkey, 1978; Moati et al., 1979; Schmidt et al., 1979). A previous paper from our institutes reported that toxic activity on mitochondrial function, as
Burned skins of third-degree burn, deep-dermal burn and burn ulcer were taken from bum patients at surgical operations using razor blades. Third-degree burn skins were taken between 11 and I8 days post burn, and deep dermal burn and burn ulcer skin were taken between 13 and I9 days and between 26 and 60 days post burn, respectively. When the toxic activity in burned skins at various periods after burn was compared, skins of third-degree burn were taken from 8 patients, using tangential excision, with a razor blade. For the controls, skins from unburned patients were taken at plastic surgery (split-skin grafting). Skins were
a
b
Fig. I. Appearance of typical examples of the three groups of burned skins studied. a, Third-degree burn (flame), 16 days post burn; b, deep-dermal burn (hot water), 14 days post burn; c, burn ulcer (flame), 60 days post burn. 4-f
0
i
3-t
8 t 0
8 8 t 8
with the skin extracts for 10 minutes on ice at 20°C using a Beckman 39550 oxygen-electrode in the presence ofsuccinate as the substrate. RCI was calculated as the ratio of the rate of oxygenconsumption in the presence of ADP (State 3) and the rate in its absence (State 4).
E
RESULTS
2-t
1*I ._ oeep derma) b”rn
Burn ulcer
Fig. 2. Effects of the extracts from normal and the three groups of burned skins on mitochondrial RCI. Means f standard deviations are indicated by vertical bars.
extracted with O-9 per cent NaCl as described previously (Aoyama et al., 1980), and the extracts were stored at - 30°C until use. Measurement
of the toxic activity
As in the previous papers (Aoyama et al., 1980; Suzuki et al., 198 l), the toxic activity in the skin extracts was measured by the decrease in respiratory control index (RCI) of mitochondria isolated from rat liver. Oxygen-consumption by mitochondria was recorded, after preincubation
The materials obtained at surgical operations were classified based on their appearances, reflecting the severity of burn injury and the time after burn, into three groups: the third-degree burn, deep-dermal burn and burn ulcer. Skins of the third-degree burn were covered with denatured, necrotized tissue turning into hard, thick eschar in the course of time. Burned skins in which blisters were formed and were without sufficient epithelization for two weeks, thus required plastic surgery and were classified as deep-dermal burn. These were sometimes associated with rather thin eschar. Burn ulcers could result from either of these two groups, in which eschar was either taken away or replaced by granulation tissue. The appearances of the typical cases of the third-degree burn, deepdermal bum and burn ulcer at the time of operation are shown in Fig. 1. RCI values of mitochondria preincubated with the extracts from these three groups of burned skins were compared in Fig. 2. RCI values of mitochondria preincubated with the extracts from skins of deep-dermal burn and of burn ulcers ranged from 2.8 to 3.9 (3.4kO.4,
Aoyama et al.: Mitochondria-toxic
Activity
5.01
2.0 :
10; ______-_-.--_____1 2 3 4
5
6
--_ 7 8 9 10 11 12 13 14 15 16 17 18 Tunepost burn (dl
Fig. 3. Post-burn change in RCI-impairing activity in the extracts from skins of third-degree bum. Each line
connected by the same symbol represents the effect of extracts from burned skins on RCI in the same patient. Burned skins were sampled from the patient at days marked by the symbols. mean _ts.d.) and from 2.7 to 3.6 (3.1+0.4), respectively, and were not significantly different from those of the controls preincubated with the extracts from normal skins (3.2 kO.3). However, mitochondria preincubated with the extracts from skins of the third-degree bum showed significantly lower RCI values, ranging from 1.O to 2.6 (2.2f0.5), than those of the controls (P
Production of some toxic substances, referred to as ‘burn toxins’, by burned skins has long been proposed in order to explain the pathogenesis of ‘burn shock’ and, recently of more importance, of the ‘late death’ in severely and extensively burned patients. Such substances are supposed to be transferred into general circulation and cause generalized damages in several other tissues. Burn toxins of various nature and with various toxic functions have been reported by several groups of investigators (Rosenthal et al., 1972; AllgGwer et al.. 1973; Raffa and Trunkey. 1978; Schmidt et al., 1979; Moati et al., 1979). The presence of the mitochondria-toxic activity in saline extracts of burned human skins was first reported in a previous paper from our institutes (Aoyama et al., 1980). The present report
15
has extended this observation, specifying the severity of bum and the time after burn required for its appearance in the extracts of the burned skins. According to the present results, only the extracts from skins of the third-degree burn accompanied by thick eschar contained the mitochondria-toxic activity; those from skins of deep-dermal bum and of bum ulcers, both lacking thick eschar, did not significantly affect mitochondrial RCI under the experimental conditions used (Fig. 2). These results appear to agree with the previous observations in experimentally induced burn of rats that most of the toxic activity was associated with the eschar but little with the residual dermis after surgical dtbridement (Suzuki et al., 198 1). In studies on burn toxins so far reported, the period for their appearance was not studied in detail. Most of them were on materials collected at a much shorter post-bum period than ours or on those produced in vitro. Despite rather wide scattering of data resulting from the difficulties in controlling the conditions of the human materials, the results of the time study shown in Fig. 3 appear to coincide well with the previous results of the animal experiments cited above showing that the toxic activity was increased significantly l-2 weeks after bum. Our results reported here and those in animal experiments reported previously (Suzuki et al., 1981) have clearly indicated that it takes 1-2 weeks for the mitochondria-toxic activity to be significantly increased in burned skins. This period appears to coincide with that of formation of eschar, which contains the most of the toxic activity in burned skins (Suzuki et al., 1981). These results are of practical interest in relation to the clinical experiences that early dlbridement of necrotized tissues greatly improves the prognoses of extensive burns (Burke et al., 1974). One of the most extensively studied burn toxins is reported to be a lipoprotein of a large molecular weight (Schoenenberger et al., 1975). The substance responsible for the mitochondriatoxic activity in burned rat skin was strongly suggested to be free fatty acid(s) (Suzuki et al., 198 1). Preliminary analyses of that from human skins of burned patients support this conclusion (unpublished results). It is of interest that mitochondrial damage, especially vacuolization, is morphologically observed in several tissues of animals with extensive burn or after injection of purified burn toxins (Rosenthal et al., 1972; Raffa et al., 1978; Kremer et al., 1979; Schmidt et al., 1979).
Burns Vol. g/No. 1
16
Acknowledgements
This study is supported in part by a grant from the Central Japan Burn Federation. The authors would like to express their thanks to Miss Yoshiko Takayama assistances.
for her
skillful
technical
REFERENCES
Aoyama H., Izawa Y. and Ozawa T. (1980)
Toxic effects of extracts from burned skin, serum and blister fluid of bum patients on mitochondrial function. Burns 7, 33. Allgower M., Cueni L. B., Stldtler K. et al. (1973) Burn toxin in mouse skin. J. Trauma 13, 95. Burke J. F., Bondoc C. C. and Quinby W. C. (1974) Primary burn excision and immediate grafting: A method shortening illness. J. Trauma 14, 389. Kremer B., Allgower M., Scheidegger A.-M. et al. (1979) Toxin-specific ultrastructural alterations of the mouse liver after burn injuries and the possibility of a specific antitoxic therapy. &and. J. Plast. Reconstr. Surg. 13,2 17.
Paper accepted I6 November I98 I
should be addressed 10: Professor T. Ozawa, Department 65, Showa-ku, Nagoya, 466 Japan.
Correspondence
Tsuruma-cho
Moati F., Sepulchre C., Miskulin M. et al. (1979) Biochemical and pharmacological properties of a cardiotoxic factor isolated from the blood serum of burned patients. J. Path. 127, 147. Raffa J. and Trunkev D. D. (1978) Mvocardial depression in acute thermal injury. J: Tr&na$, !$. Rosenthal S. R., Hawley P. L. and Hakim A. A. (1972) Purified burn toxic factor and its competition. Surgery 71, 527. Schmidt K., Scholmerich J., Kremer B. et al. (1979) Studies on the structure and biological effects of pyrotoxins purified from burned skin. World J. Surg. 3, 36 I. Schoenenberger Cl. A., Burkhardt G., Kalberer F. et al. (1975) Experimental evidence for a significant impairment of host defense for gram-negative organisms by a specific cutaneous toxin produced by severe burn injuries. Surg. Gynecol. Obset. 141,555: Suzuki K.. Aovama H.. Izawa Y. et al. C . I98 I), Isolation of a substance toxic to mitochondrial function from burned skin of rats. Burns 8, I IO.
of Biomedical Chemistry,
University of Nagoya,
PrintedinGreatBritain
13
Mitochondria-toxic activity in burned human skin: relation to severity of burn and period after burn Hisashi Aoyarna,
Kohji Suzuki and Yohei lzawa
Department of Burns and Plastic Surgery, Chukyo Hospital, Nagoya, Japan
Masanao
Kobashashi
and Takayuki
Ozawa
Department of Biomedical Chemistry, Faculty of Medicine, University of Nago ya, Nagoya, Japan
Summary
Toxic activity in the extracts of burned human skins impairing respiratory control of rat-liver mitochondria was compared in terms of the severity of burn injury and period after burn. The toxic activity was detected in the extracts from skins of third-degree burn, but not in those from skins of deep-dermal burn or of burn ulcers, in the present experimental conditions. In the case of third-degree burn, the toxic activity was not evident during the first few days post burn, but was significantly increased after IO days. The results of this study suggest the effective period for early therapeutic debridement of the necrotized tissues in the management of severely burned patients.
lowering respiratory control index (RCI), exists in burned human skins (Aoyama et al., 1980). Another paper reported the presence of similar activity in burned skins experimentally induced on rats, its increase in the second week after burn and isolation, with some characterization, of the substance responsible for the toxic activity (Suzuki et al., 1981). Extending these observations, the present paper compares the toxic activity in burned skins from human patients in terms of the severity of bum injury and time after burn.
INTRODUCTION
MATERIALS AND METHODS Burned skin extracts
THE: presence of some toxic substances has been proposed to explain so-called burn death in extensive burn injury. Such substances are supposed to be produced in the burned skin, transferred into general circulation and cause generalized damages in other tissues. Several authors have investigated such toxic substances from human or animal skins, or sera, after burn, testing the toxic activities by various methods (Rosenthal et al., 1972; Allgijwer et al., 1973; Raffa and Trunkey, 1978; Moati et al., 1979; Schmidt et al., 1979). A previous paper from our institutes reported that toxic activity on mitochondrial function, as
Burned skins of third-degree burn, deep-dermal burn and burn ulcer were taken from bum patients at surgical operations using razor blades. Third-degree burn skins were taken between 11 and I8 days post burn, and deep dermal burn and burn ulcer skin were taken between 13 and I9 days and between 26 and 60 days post burn, respectively. When the toxic activity in burned skins at various periods after burn was compared, skins of third-degree burn were taken from 8 patients, using tangential excision, with a razor blade. For the controls, skins from unburned patients were taken at plastic surgery (split-skin grafting). Skins were
a
b
Fig. I. Appearance of typical examples of the three groups of burned skins studied. a, Third-degree burn (flame), 16 days post burn; b, deep-dermal burn (hot water), 14 days post burn; c, burn ulcer (flame), 60 days post burn. 4-f
0
i
3-t
8 t 0
8 8 t 8
with the skin extracts for 10 minutes on ice at 20°C using a Beckman 39550 oxygen-electrode in the presence ofsuccinate as the substrate. RCI was calculated as the ratio of the rate of oxygenconsumption in the presence of ADP (State 3) and the rate in its absence (State 4).
E
RESULTS
2-t
1*I ._ oeep derma) b”rn
Burn ulcer
Fig. 2. Effects of the extracts from normal and the three groups of burned skins on mitochondrial RCI. Means f standard deviations are indicated by vertical bars.
extracted with O-9 per cent NaCl as described previously (Aoyama et al., 1980), and the extracts were stored at - 30°C until use. Measurement
of the toxic activity
As in the previous papers (Aoyama et al., 1980; Suzuki et al., 198 l), the toxic activity in the skin extracts was measured by the decrease in respiratory control index (RCI) of mitochondria isolated from rat liver. Oxygen-consumption by mitochondria was recorded, after preincubation
The materials obtained at surgical operations were classified based on their appearances, reflecting the severity of burn injury and the time after burn, into three groups: the third-degree burn, deep-dermal burn and burn ulcer. Skins of the third-degree burn were covered with denatured, necrotized tissue turning into hard, thick eschar in the course of time. Burned skins in which blisters were formed and were without sufficient epithelization for two weeks, thus required plastic surgery and were classified as deep-dermal burn. These were sometimes associated with rather thin eschar. Burn ulcers could result from either of these two groups, in which eschar was either taken away or replaced by granulation tissue. The appearances of the typical cases of the third-degree burn, deepdermal bum and burn ulcer at the time of operation are shown in Fig. 1. RCI values of mitochondria preincubated with the extracts from these three groups of burned skins were compared in Fig. 2. RCI values of mitochondria preincubated with the extracts from skins of deep-dermal burn and of burn ulcers ranged from 2.8 to 3.9 (3.4kO.4,
Aoyama et al.: Mitochondria-toxic
Activity
5.01
2.0 :
10; ______-_-.--_____1 2 3 4
5
6
--_ 7 8 9 10 11 12 13 14 15 16 17 18 Tunepost burn (dl
Fig. 3. Post-burn change in RCI-impairing activity in the extracts from skins of third-degree bum. Each line
connected by the same symbol represents the effect of extracts from burned skins on RCI in the same patient. Burned skins were sampled from the patient at days marked by the symbols. mean _ts.d.) and from 2.7 to 3.6 (3.1+0.4), respectively, and were not significantly different from those of the controls preincubated with the extracts from normal skins (3.2 kO.3). However, mitochondria preincubated with the extracts from skins of the third-degree bum showed significantly lower RCI values, ranging from 1.O to 2.6 (2.2f0.5), than those of the controls (P
Production of some toxic substances, referred to as ‘burn toxins’, by burned skins has long been proposed in order to explain the pathogenesis of ‘burn shock’ and, recently of more importance, of the ‘late death’ in severely and extensively burned patients. Such substances are supposed to be transferred into general circulation and cause generalized damages in several other tissues. Burn toxins of various nature and with various toxic functions have been reported by several groups of investigators (Rosenthal et al., 1972; AllgGwer et al.. 1973; Raffa and Trunkey. 1978; Schmidt et al., 1979; Moati et al., 1979). The presence of the mitochondria-toxic activity in saline extracts of burned human skins was first reported in a previous paper from our institutes (Aoyama et al., 1980). The present report
15
has extended this observation, specifying the severity of bum and the time after burn required for its appearance in the extracts of the burned skins. According to the present results, only the extracts from skins of the third-degree burn accompanied by thick eschar contained the mitochondria-toxic activity; those from skins of deep-dermal bum and of bum ulcers, both lacking thick eschar, did not significantly affect mitochondrial RCI under the experimental conditions used (Fig. 2). These results appear to agree with the previous observations in experimentally induced burn of rats that most of the toxic activity was associated with the eschar but little with the residual dermis after surgical dtbridement (Suzuki et al., 198 1). In studies on burn toxins so far reported, the period for their appearance was not studied in detail. Most of them were on materials collected at a much shorter post-bum period than ours or on those produced in vitro. Despite rather wide scattering of data resulting from the difficulties in controlling the conditions of the human materials, the results of the time study shown in Fig. 3 appear to coincide well with the previous results of the animal experiments cited above showing that the toxic activity was increased significantly l-2 weeks after bum. Our results reported here and those in animal experiments reported previously (Suzuki et al., 1981) have clearly indicated that it takes 1-2 weeks for the mitochondria-toxic activity to be significantly increased in burned skins. This period appears to coincide with that of formation of eschar, which contains the most of the toxic activity in burned skins (Suzuki et al., 1981). These results are of practical interest in relation to the clinical experiences that early dlbridement of necrotized tissues greatly improves the prognoses of extensive burns (Burke et al., 1974). One of the most extensively studied burn toxins is reported to be a lipoprotein of a large molecular weight (Schoenenberger et al., 1975). The substance responsible for the mitochondriatoxic activity in burned rat skin was strongly suggested to be free fatty acid(s) (Suzuki et al., 198 1). Preliminary analyses of that from human skins of burned patients support this conclusion (unpublished results). It is of interest that mitochondrial damage, especially vacuolization, is morphologically observed in several tissues of animals with extensive burn or after injection of purified burn toxins (Rosenthal et al., 1972; Raffa et al., 1978; Kremer et al., 1979; Schmidt et al., 1979).
Burns Vol. g/No. 1
16
Acknowledgements
This study is supported in part by a grant from the Central Japan Burn Federation. The authors would like to express their thanks to Miss Yoshiko Takayama assistances.
for her
skillful
technical
REFERENCES
Aoyama H., Izawa Y. and Ozawa T. (1980)
Toxic effects of extracts from burned skin, serum and blister fluid of bum patients on mitochondrial function. Burns 7, 33. Allgower M., Cueni L. B., Stldtler K. et al. (1973) Burn toxin in mouse skin. J. Trauma 13, 95. Burke J. F., Bondoc C. C. and Quinby W. C. (1974) Primary burn excision and immediate grafting: A method shortening illness. J. Trauma 14, 389. Kremer B., Allgower M., Scheidegger A.-M. et al. (1979) Toxin-specific ultrastructural alterations of the mouse liver after burn injuries and the possibility of a specific antitoxic therapy. &and. J. Plast. Reconstr. Surg. 13,2 17.
Paper accepted I6 November I98 I
should be addressed 10: Professor T. Ozawa, Department 65, Showa-ku, Nagoya, 466 Japan.
Correspondence
Tsuruma-cho
Moati F., Sepulchre C., Miskulin M. et al. (1979) Biochemical and pharmacological properties of a cardiotoxic factor isolated from the blood serum of burned patients. J. Path. 127, 147. Raffa J. and Trunkev D. D. (1978) Mvocardial depression in acute thermal injury. J: Tr&na$, !$. Rosenthal S. R., Hawley P. L. and Hakim A. A. (1972) Purified burn toxic factor and its competition. Surgery 71, 527. Schmidt K., Scholmerich J., Kremer B. et al. (1979) Studies on the structure and biological effects of pyrotoxins purified from burned skin. World J. Surg. 3, 36 I. Schoenenberger Cl. A., Burkhardt G., Kalberer F. et al. (1975) Experimental evidence for a significant impairment of host defense for gram-negative organisms by a specific cutaneous toxin produced by severe burn injuries. Surg. Gynecol. Obset. 141,555: Suzuki K.. Aovama H.. Izawa Y. et al. C . I98 I), Isolation of a substance toxic to mitochondrial function from burned skin of rats. Burns 8, I IO.
of Biomedical Chemistry,
University of Nagoya,