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Effect of allopurinol or superoxide dismutase plus catalase on cardiovascular function after burn injury in the anaesthetized rat
Bums (1989)
15, (3), 147-151
147
F’ri’nted in Great Britain
Effect of allopurinol or superoxide dismutase plus catalase on cardiovascular function after burn injury in the anaesthetized rat Elisabeth A. Speakman and B. J. Chapman Department
of Physiology
and Pharmacology,
A scald burn injury was applied to 22-23
Southampton
per cent of the body surface area
of anaedhetized rats. lie cardac output was 38-60 per cent lower in the burn injury group than in thecontrol group; heart rate and mean arterial blood pressure were only slightly affected. but burn injury cad a signifcant increase in total petipheral resik~ance.The involvement oxygen-free radicals in this immediate fall in cardiac ouLputwas investigated.Retreatment with a blocker offree radical production, allopurinol or the infusion qf the free radical scavengers superoxide dismutase plus catalase caused no cardiovascular improvement, sugpting that oxygen free radicals are not involved in thefall in cardiac output a#er burn injuy. Allopurinol treatment, however, prevented fhe rise in total peripheral resistance stm after bum injury.
of
Introduction It is well documented that cardiac output falls sharply following a scald burn injury (Dobson and Warner, 1957; Wolfe and Miller, 1976; Chapman et al., 1979). Several factors, including a fall in cardiac contractility (Chapman and Speakman, 1988 a, b) and plasma volume loss (Hilton, 1980) have been implicated in this fall in cardiac output, but the underlying cause has not been identified. The highly reactive molecules, oxygen free radicals, have been implicated as the damaging species in many types of circulatory impairment including ischaemia/reperfusion damage (McCord, 1985), endotoxin shock (Manson and Hess, 1983), haemorrhagic shock (Allan et al., 1986) and in some changes following burn injury (Hilton, 1980; Bjlirk and Arturson, 1982; Saez et al., 1984). McCord (1985) has suggested that upon reperfusion, previously ischaemic tissues produce oxygen free radicals. He proposed a mechanism for the beneficial action of allopurinol (a xanthine oxidase inhibitor) in reducing the formation of oxygen free radicals in this type of injury. Allen et al. (1986) investigated the effect of allopurinol on the cardiovascular responses of dogs to haemorrhage/reinfusion. They found that pretreatment with allopurinol (50mg/kg) 60 min before haemorrhagelreinfusion prevented a subsequent gradual decline in haemodynamic function. Saez et al. (1984) observed that either allopurinol or superoxide dismutase 0 1989 Butterworth & Co (Publishers) Ltd 0305-4179/89/030147-05
$03.00
University,
UK
(SOD) pretreatment led to an increased survival rate of mice subjected to burn injury. They proposed that the generation of free radicals at the local tissue level and in underperfused organs may be one of the causes of death after thermal injury. Bjark and Arturson (1982) also reported that infusion of the free radical scavengers SOD and catalase (CAT) reduced oedema formation following burn injury. We therefore decided to investigate the possible involvement of oxygen free radicals in the immediate fall in cardiac output following burn injury. Two protocols were used: pretreatment with allopurinol, the xanthine oxidase inhibitor, or infusion of SOD, a specific scavenger of the superoxide anion radical (McCord and Fridovich, 1969) together with CAT which detoxifies hydrogen peroxide to water (Halliwell, 1974).
Methods Female Wistar albino rats (body wt 25&28Og) were anaesthetized with sodium pentobarbitone (Sagatal, 60 mg/kg i.p., plus supplementary doses as necessary). Body temperature was maintained at 37°C. Four stainless-steel wires were placed subcutaneously around the rat for the measurement of cardiac output by impedance cardiography as previously described (Chapman et al., 1977). Cannulae were placed in the trachea, carotid artery and vena cava close to the right atrium (via the jugular vein). The animals were shaved on the back before application of burn injury (BI) or mock bum injury (MBI). They were placed in a specially made plastic cradle with an opening below. The cradle was lowered into a water-bath at 90°C for 2Os, giving a full skin thickness bum injury to 22-23 per cent of the body surface area; the bum injured area was then cooled in room-temperature water for 1 min to prevent further burning. Control, i.e. MBI, animals were treated in exactly the same way, but were exposed to room temperature water only. Measurements were taken 60,3O and 5 min before and 5, 30, 60 and 90min after BI or MBI. Arterial blood samples (0.05 ml) were collected for the duplicate measurement of haematocrit.
Burns (1989) Vol. 15/No.
148
Three separate studies were performed. In the first study animals were divided into four groups: i.v. injection of allopurinol(50 mg/kg dissolved in I ml saline) 60 min before BI or MBI; i.v. injection of 1 ml saline 60 min before BI or MBI, six rats per group. In the second study, animals were divided into four groups, SOD (5 mg/kg) plus CAT (5 mg/kg) in I ml saline was infused for 60 min starting 45 min before BI or MBI; or saline alone was infused into rats subjected to BI or MBI, six rats per group. In the third study SOD plus CAT was infused for 60 min into six rats for the estimation of the plasma concentrations and half-lives of these enzymes. Blood samples (0.2 ml) were collected and centrifuged then SOD was assayed by the method of Marklund and Marklund (1974) and CAT by the method of Aebi (1984). Allopurinol MW 136.1 daltons (Wellcome), was dissolved in saline by addition of a small quantity of NaOH. Superoxide dismutase (MW 33 000 daltons, 3300 U/mg protein; Sigma) and catalase (MW 240 000, 11000 U/mg protein; Sigma) were dissolved in saline.
Results The cardiovascular measurements were taken before and after allopurinol injection, but before BI or MB1 were compared. It was observed that allopurinol alone caused statistically significant falls in cardiac output (P
Premock-burn
Pre-burn
Premock-burn
Pre-burn
3
In animals that were not pretreated with drugs to modify free radical concentrations (Figs. 2,3), MB1 caused no significant change in any cardiovascular measurement, while BI resulted in the following changes. Cardiac output and stroke volume fell significantly by 5 min postbum and remained low, but the heart rate remained unchanged. Mean arterial blood pressure changed little postbum although a transient, small but statistically significant fall was observed immediately after BI. Total peripheral resistance rose postbum, although this rise was not statistically significant in the SOD/CAT experiment. No significant change in central venous pressure was observed (Fig. 3). Fig. 2 shows the cardiovascular changes in rats subjected to BI or MB1 before and after saline or allopurinol pretreatment. The xanthine oxidase inhibitor did not significantly improve cardiac output or stroke volume but did reduce the rise in total peripheral resistance observed postbum. The following numbers of rats survived for 180 min: saline + mock-burn-6 rats saline + burn injury-5 rats allopurinol + mock-burn-6 rats allopurinol+ burn injury-l rat Thus, 17 rats which had not received the combined treatment of allopurinol and burn injury survived; only one rat survived the combined treatment of allopurinol and burn injury. These data are summarized in Tub/e I. Fisher’s exact test gave P (two-tailed test) = 0.0016. The incidence of death following the combined treatment of allopurinol plus BI could not, therefore, reasonably be thought to have occurred by chance alone. Fi& 3 shows the cardiovascular changes in rats subjected to BI or MB1 given either saline or SOD/CAT infusions. The SOD/CAT infusion did not significantly modify any response to BI. Plasma SOD/CAT levels were elevated throughout the experimental period (Fig. 4). The half-life of SOD in the rat when the infusion was begun was 1Omin and that of CAT 7.2 min. The SOD/CAT activity of the infusate before and after the infusion period was not significantly changed, i.e. there was no loss of activity of the enzyme in the syringe attached to the infusion pump.
Discussion Our data suggest that allopurinol may not be an ideal drug to use in rat studies since we found that allopurinol itself altered cardiovascular function. The allopurinol injectate had a pH of 11.0. An aliquot of this solution was titrated to pH 7.4 with dilute HCl. This indicated that O.17mmol of base was added to each rat. Comparison with the data of Swan et al. (1955) suggest that the plasma [HCO,- ] would therefore rise by about ImM, which would be of minor physiological significance. It is therefore likely that the changes observed were due to the allopurinol itself. Allan et al. (1986) also reported that a similar dose of allopurinol changed cardio-
Table I. Figure 1. Cardiovascular measurements taken after the injection of allopurinol(50 mg/kg) or saline (I ml), but before bum or mockbum injury. Results are shown as mean f s.e.m., n = 6, in each group and were evaluated statistically by analysis of variance. ‘PC 0.05.
Died
Survived
Total
% Died
Burn injury + allopurinol Burn or allopurinol or neither
5 1
1 17
6 18
83.8 5.6
Totals
6
18
24
88.9
Speakman
and Chapman:
100
function after burn injury
Cardiovascular
r
E
-z 5 a 5 0 x
E
sa
2 mu .-u k
60
c.E
.F
0
149
40
*
20
a ** 20
***
-
*** **+ P o
Allopurinol injection t ,
I -60
yrn/m,:k-burn,
f
OL
‘r
injury,
Burn/mock-burn
I
-3 -30
0
30
60
90
Time (min)
Figure 2. Cardiovascular changes after burn or mock-burn injury and the effects of allopurinol treatment (5OmgIkg). Results are shown as mean f s.e.m., n = 6, in each group, changes from prebum or pre mock-bum values were evaluated by Student’s paired t test. ‘PC 0.05, ‘*PC 0.01, ?? *‘P< 0.001. 0, Mock-bum + saline; ?? , mock-burn + allopurinol; ?,?burn injury + saline; ?? , burn injury + allopurinol.
--60
-30
injury
I I
I
I
I
0
30
60
90
Time (min)
Figure 3. Cardiovascular changes after burn or mock-burn injury and the effects of superoxide dismutase plus cataiase (SOD/CAT, 5 mg/kg/h). Results are shown as mean f s.e.m., n=6, in each group, changes from prebum or pre mock-bum values were evaluated by Student’s paired t test. ‘P-C 0.05, “P-C 0.01, ‘**P-C 0.001. 0, Mock-bum+ saline; ?? , mock-bum-t-SOD/CAT; ?,? burn injury + saline; ?? , burn injury + SOD/CAT.
Burns (1989) Vol. 15/No. 3
150
40
r
in liposomes. Elevation of SOD and CAT plasma levels have been shown to cause a slight reduction in postbum oedema formation (Bjiirk and Arturson, 1982) and to increase the survival rate of burn-injured mice (Saez et al., 1984). However, the present data show that elevation of these circulating oxygen free radical scavengers does not prevent the early cardiovascular changes observed postbum. In conclusion, these results do not support the concept that circulating oxygen free radicals are involved in the circulatory changes after bum injury, nor that cardiac impairment after bum injury is due to intracellular oxygen free radicals generated via the xanthine oxidase pathway.
A
= E
3 b
12y-/& 0
c
LII -15
0
Sod/cat
infusion
1
1
15
30
-m
I
I
I
I
60
90
120
Time (min)
Figure 4. The SOD/CAT enzyme activity in rat plasma, before, during and after infusion of SOD/CAT (5 mg/kg/h in 1 ml 0.9 per , Catalase cent saline). Results are shown as mean f s.e.m., n = 6. ?? activity; ??SOD activity.
Acknowledgements We are grateful to Dr F. Kelly and Mr G. Ricketts for teaching and helping with the SOD and CAT assays. E. A. Speakman is an SERC student.
References vascular function in the dog: they observed a fall in blood pressure and a rise in heart rate. Since allopurinol caused these changes, interpretation of the results is difficult, but it is clear that allopurinol pretreatment does not cause any improvement in cardiovascular function after burn injury. Furthermore, allopurinol pretreatment did not cause any improvement in survival rate. In fact allopurinol treatment resulted in a significant fall in the survival of BI rats. The cause of this is unImown, but Marldey and Smallman (1970) observed that pretreatment with uric acid or precursors of uric acid, increased the survival of mice exposed to burn injury, it is possible that allopurinol decreases survival rates by reducing endogenous uric acid formation, although allernative mechanisms can be envisaged. Saez et al. (1984) found that allopurinol pretreatment improved the 24-h survival of BI mice; this difference from the present result may be due to species difference in sensitivity to burn or to allopurinol or the fact that the rats in the present study were kept under anaesthesia throughout the study. In the second and third studies, the doses of SOD and CAT used were comparable to those used in investigations into ischaemia/reperfusion damage in the in sihr heart of dogs (Jolly et al., 1984; Przylenk and Kloner, 1986) and bum injury in the rat (Bjiirk and Arturson, 1982) and dog (Hilton, 1980). SOD has been reported to have a half-life of 6 min in the hamster (Ley and Arfors, 1982) and 8 min in the rat (Turrens et al., 1984), and a similar half-life was found in the present study. The half-life of CAT was 7.2 min which is, however, shorter than the value of 20 min reported by Turrens et al. (1984). The scavengers were infused because the half-lives were known to be short, and direct measurement showed that plasma SOD and CAT activities were markedly elevated by the doses given. The molecular weight of SOD is 33 000 dallons and CAT 240 000 daltons, and it has been reported Ihat molecules as large as 300 000 daltons may pass through capillary membranes after burn injury (Ganrot et al., 1974). Hence, it is probable that these circulating scavengers may have an action outside the circulation after burn injury. Allopurinol crosses cell membranes and probably crosses the blood/ brain barrier (Iansek et al. (1986)), but it is unlikely that SOD or CAT would cross either of these barriers unless enclosed
Aebi H. (1984) Catalase in vitro. Methods hzyrtd. 105, 121. Allan G., Cambridge D., Lee-Tsang-Tan L. et al. (1986) The protective action of ailopurinol in an experimental model of haemorrhagic shock and reperfusion. Br. J. Pharmacol. 89, 149. Bj6rk J. and Arturson G. (1982) Effect of cimetidine, hydrocortisone, superoxide dismutase and catalase on the development of oedema after thermal injury. Burns 9, 249. Chapman B. J. and Speakman E. A. (1988a) Changes in cardiac output and papillary muscle contraction in the rat after a scald bum injury. 1. Physiol. 400,51P. Chapman B. J. and Speakman E. A. (1988b) Cardiac function curves after a scald bum injury to the anaesthetised rat. J. Physiol. 407, 17P. Chapman B. J., Chen C. F. and Munday K. A. (1977) Measurement of the cardiac output in the rat by impedance cardiography. J. Physiol. 270, 3P. Chapman B. J., Munday K. A. and Philpot M. E. (1979) Effects of cimetidine on some cardiovascular responses to thermal injury. ]. Physiol. 301,66P. Dobson E. L. and Warner G. F. (1957) Factors concerned in the early stages of thermal shock. Circ. Res. 5,69. Ganrot K., Jacobson S. and Rothman V. (1974) Transcapillary passage of plasma proteins in experimental bums. Acta Physiol. &and. 91,297. Halliwell 8. (1974) SOD, CAT and glutathione peroxidase: solutions to the problems of living with oxygen. Nezu Phybol. 73, 1075. Hilton J. G. (1980) Effects of alterations of polyunsaturated fatty acid metabolism upon volume loss induced by thermal trauma. 1. Trauma 20,663. Iansek R. Packham D, Aspey B. S. et al. (1986) As assessment of the possible protective effect of allopurinol in acute stroke. J. Neurol. Neurosurg. Psychiatry 49, 585. Jolly S. R., Kane W. J., Bailie M. B. et al. (1984) Canine myocardial reperfusion injury. Its reduction by the combined administration of superoxide dismutase and catalase. Circ. Res. 54,277. Ley K. and Arfors K. E. (1982) Changes in macromolecular permeability by intravascular generation of oxygenderived free radicals. Microvasc. Res. 24,25. McCord J. M. (1985) Oxygen derived free radicals in postischaemic tissue injury. New Engl. J. Med. 312, 159. McCord J. M. and Fridovich I. (1969) SOD an enzymic function for erythrocuprein (hemocuprein). 1, Biol. Chw. 244, 6049.
151
Speakman and Chapman: Cardiovascular function after burn injury
i _,
Manson N. H. and Hess M. L. (1983) Interaction of oxygen free radicals and cardiac sarcoplasmic reticulum proposed role in the pathogenesis of endotoxin shock. Circ. Shock 10, P205. Markley K. and Smallman E. (1970) Protection against burn, tourniquet and endotoxin shock by purine compounds. 1. Trauma 10, 598. Markhmd S. and Marklund G. (1974) Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for SOD. Br. J Biochem. 47, 469. Przyklenk K. and Kloner R. A. (1986) SOD plus CAT improve contractile function in the the canine model of the ‘stunned myocardium’. Circ. Res. 58, 148. Saez J. C., Ward P. H., Gunther B. et al. (1984) Superoxide radical involvement in the pathogenesis of burn shock. Circ. Shock 12, 229. Swan R. C., Axelrod D. R., Seip M. et al. (1955) Distribution of
sodium bicarbonate infused into nephrectomized dogs. 1. C&I. Inu&. 34, 1795. Turrens J. F., Crap0 J. D. and Freeman B. A. (1984) Protection against toxicity by the injection of liposome entrapped catalase and superoxide dismutase. J. Qn. hwes~. 73,s~. Wolfe R. R. and Miller H. I. (1976) Burn shock in untreated and saline resuscitated guinea pigs. Development of a model. 1. Surg. Res. 21,269.
Paper accepted
3 December
1988.
Correspondence should be addressed to: Dr B. J. Chapman, Department of Physiology and Pharmacology, Southampton University, Southampton SO9 3TU, UK.
Award of the G. Whitaker International Bums Prize Palerrno, Italy for 19 89 In the course of a meeting
held on March
12th 1989 at the seat of the G. Whitaker
Foundation,
Italy, after examining the scientific activity in the field of research, teaching, clinical organization, prevention and cooperation among the nations, presented by various candidates, in the light of the consideration guiding the analysis of the high level of candidates, the Adjudicating committee unanimously decided to award the prize for 1989 to:
Palermo,
Shi Si-Xiang MD, Director of Shanghai Burns Institute, Shanghai Second Medical University, People’s Republic of China The prize is awarded for this motivation: “For having succeeded in a developing country, in creating a culture and a science of bums treatment. With traditional Chinese perseverance and in the face of many difficulties, and with an intuitive sense of new biological trends, he put forward bold technological innovations in the surgical treatment of the severely burned patient. When it was thought that burns covering 50-60 per cent of the body surface area were the maximum limit for possible survival, his technique led to the recovery of patients with bums extending to 90 per cent. He has thus established a new guideline in the surgical treatment of the bum patient, an indication that today fascinates and interests the entire scientific world.”
The official prize-giving of this prestigious award will be held on June 8th 1989 in PaJermo at the seat of the G. Whitaker Foundation in the course of a ceremony in the presence of authorities and representatives of the academic, scientific and cultural world.
15, (3), 147-151
147
F’ri’nted in Great Britain
Effect of allopurinol or superoxide dismutase plus catalase on cardiovascular function after burn injury in the anaesthetized rat Elisabeth A. Speakman and B. J. Chapman Department
of Physiology
and Pharmacology,
A scald burn injury was applied to 22-23
Southampton
per cent of the body surface area
of anaedhetized rats. lie cardac output was 38-60 per cent lower in the burn injury group than in thecontrol group; heart rate and mean arterial blood pressure were only slightly affected. but burn injury cad a signifcant increase in total petipheral resik~ance.The involvement oxygen-free radicals in this immediate fall in cardiac ouLputwas investigated.Retreatment with a blocker offree radical production, allopurinol or the infusion qf the free radical scavengers superoxide dismutase plus catalase caused no cardiovascular improvement, sugpting that oxygen free radicals are not involved in thefall in cardiac output a#er burn injuy. Allopurinol treatment, however, prevented fhe rise in total peripheral resistance stm after bum injury.
of
Introduction It is well documented that cardiac output falls sharply following a scald burn injury (Dobson and Warner, 1957; Wolfe and Miller, 1976; Chapman et al., 1979). Several factors, including a fall in cardiac contractility (Chapman and Speakman, 1988 a, b) and plasma volume loss (Hilton, 1980) have been implicated in this fall in cardiac output, but the underlying cause has not been identified. The highly reactive molecules, oxygen free radicals, have been implicated as the damaging species in many types of circulatory impairment including ischaemia/reperfusion damage (McCord, 1985), endotoxin shock (Manson and Hess, 1983), haemorrhagic shock (Allan et al., 1986) and in some changes following burn injury (Hilton, 1980; Bjlirk and Arturson, 1982; Saez et al., 1984). McCord (1985) has suggested that upon reperfusion, previously ischaemic tissues produce oxygen free radicals. He proposed a mechanism for the beneficial action of allopurinol (a xanthine oxidase inhibitor) in reducing the formation of oxygen free radicals in this type of injury. Allen et al. (1986) investigated the effect of allopurinol on the cardiovascular responses of dogs to haemorrhage/reinfusion. They found that pretreatment with allopurinol (50mg/kg) 60 min before haemorrhagelreinfusion prevented a subsequent gradual decline in haemodynamic function. Saez et al. (1984) observed that either allopurinol or superoxide dismutase 0 1989 Butterworth & Co (Publishers) Ltd 0305-4179/89/030147-05
$03.00
University,
UK
(SOD) pretreatment led to an increased survival rate of mice subjected to burn injury. They proposed that the generation of free radicals at the local tissue level and in underperfused organs may be one of the causes of death after thermal injury. Bjark and Arturson (1982) also reported that infusion of the free radical scavengers SOD and catalase (CAT) reduced oedema formation following burn injury. We therefore decided to investigate the possible involvement of oxygen free radicals in the immediate fall in cardiac output following burn injury. Two protocols were used: pretreatment with allopurinol, the xanthine oxidase inhibitor, or infusion of SOD, a specific scavenger of the superoxide anion radical (McCord and Fridovich, 1969) together with CAT which detoxifies hydrogen peroxide to water (Halliwell, 1974).
Methods Female Wistar albino rats (body wt 25&28Og) were anaesthetized with sodium pentobarbitone (Sagatal, 60 mg/kg i.p., plus supplementary doses as necessary). Body temperature was maintained at 37°C. Four stainless-steel wires were placed subcutaneously around the rat for the measurement of cardiac output by impedance cardiography as previously described (Chapman et al., 1977). Cannulae were placed in the trachea, carotid artery and vena cava close to the right atrium (via the jugular vein). The animals were shaved on the back before application of burn injury (BI) or mock bum injury (MBI). They were placed in a specially made plastic cradle with an opening below. The cradle was lowered into a water-bath at 90°C for 2Os, giving a full skin thickness bum injury to 22-23 per cent of the body surface area; the bum injured area was then cooled in room-temperature water for 1 min to prevent further burning. Control, i.e. MBI, animals were treated in exactly the same way, but were exposed to room temperature water only. Measurements were taken 60,3O and 5 min before and 5, 30, 60 and 90min after BI or MBI. Arterial blood samples (0.05 ml) were collected for the duplicate measurement of haematocrit.
Burns (1989) Vol. 15/No.
148
Three separate studies were performed. In the first study animals were divided into four groups: i.v. injection of allopurinol(50 mg/kg dissolved in I ml saline) 60 min before BI or MBI; i.v. injection of 1 ml saline 60 min before BI or MBI, six rats per group. In the second study, animals were divided into four groups, SOD (5 mg/kg) plus CAT (5 mg/kg) in I ml saline was infused for 60 min starting 45 min before BI or MBI; or saline alone was infused into rats subjected to BI or MBI, six rats per group. In the third study SOD plus CAT was infused for 60 min into six rats for the estimation of the plasma concentrations and half-lives of these enzymes. Blood samples (0.2 ml) were collected and centrifuged then SOD was assayed by the method of Marklund and Marklund (1974) and CAT by the method of Aebi (1984). Allopurinol MW 136.1 daltons (Wellcome), was dissolved in saline by addition of a small quantity of NaOH. Superoxide dismutase (MW 33 000 daltons, 3300 U/mg protein; Sigma) and catalase (MW 240 000, 11000 U/mg protein; Sigma) were dissolved in saline.
Results The cardiovascular measurements were taken before and after allopurinol injection, but before BI or MB1 were compared. It was observed that allopurinol alone caused statistically significant falls in cardiac output (P
Premock-burn
Pre-burn
Premock-burn
Pre-burn
3
In animals that were not pretreated with drugs to modify free radical concentrations (Figs. 2,3), MB1 caused no significant change in any cardiovascular measurement, while BI resulted in the following changes. Cardiac output and stroke volume fell significantly by 5 min postbum and remained low, but the heart rate remained unchanged. Mean arterial blood pressure changed little postbum although a transient, small but statistically significant fall was observed immediately after BI. Total peripheral resistance rose postbum, although this rise was not statistically significant in the SOD/CAT experiment. No significant change in central venous pressure was observed (Fig. 3). Fig. 2 shows the cardiovascular changes in rats subjected to BI or MB1 before and after saline or allopurinol pretreatment. The xanthine oxidase inhibitor did not significantly improve cardiac output or stroke volume but did reduce the rise in total peripheral resistance observed postbum. The following numbers of rats survived for 180 min: saline + mock-burn-6 rats saline + burn injury-5 rats allopurinol + mock-burn-6 rats allopurinol+ burn injury-l rat Thus, 17 rats which had not received the combined treatment of allopurinol and burn injury survived; only one rat survived the combined treatment of allopurinol and burn injury. These data are summarized in Tub/e I. Fisher’s exact test gave P (two-tailed test) = 0.0016. The incidence of death following the combined treatment of allopurinol plus BI could not, therefore, reasonably be thought to have occurred by chance alone. Fi& 3 shows the cardiovascular changes in rats subjected to BI or MB1 given either saline or SOD/CAT infusions. The SOD/CAT infusion did not significantly modify any response to BI. Plasma SOD/CAT levels were elevated throughout the experimental period (Fig. 4). The half-life of SOD in the rat when the infusion was begun was 1Omin and that of CAT 7.2 min. The SOD/CAT activity of the infusate before and after the infusion period was not significantly changed, i.e. there was no loss of activity of the enzyme in the syringe attached to the infusion pump.
Discussion Our data suggest that allopurinol may not be an ideal drug to use in rat studies since we found that allopurinol itself altered cardiovascular function. The allopurinol injectate had a pH of 11.0. An aliquot of this solution was titrated to pH 7.4 with dilute HCl. This indicated that O.17mmol of base was added to each rat. Comparison with the data of Swan et al. (1955) suggest that the plasma [HCO,- ] would therefore rise by about ImM, which would be of minor physiological significance. It is therefore likely that the changes observed were due to the allopurinol itself. Allan et al. (1986) also reported that a similar dose of allopurinol changed cardio-
Table I. Figure 1. Cardiovascular measurements taken after the injection of allopurinol(50 mg/kg) or saline (I ml), but before bum or mockbum injury. Results are shown as mean f s.e.m., n = 6, in each group and were evaluated statistically by analysis of variance. ‘PC 0.05.
Died
Survived
Total
% Died
Burn injury + allopurinol Burn or allopurinol or neither
5 1
1 17
6 18
83.8 5.6
Totals
6
18
24
88.9
Speakman
and Chapman:
100
function after burn injury
Cardiovascular
r
E
-z 5 a 5 0 x
E
sa
2 mu .-u k
60
c.E
.F
0
149
40
*
20
a ** 20
***
-
*** **+ P o
Allopurinol injection t ,
I -60
yrn/m,:k-burn,
f
OL
‘r
injury,
Burn/mock-burn
I
-3 -30
0
30
60
90
Time (min)
Figure 2. Cardiovascular changes after burn or mock-burn injury and the effects of allopurinol treatment (5OmgIkg). Results are shown as mean f s.e.m., n = 6, in each group, changes from prebum or pre mock-bum values were evaluated by Student’s paired t test. ‘PC 0.05, ‘*PC 0.01, ?? *‘P< 0.001. 0, Mock-bum + saline; ?? , mock-burn + allopurinol; ?,?burn injury + saline; ?? , burn injury + allopurinol.
--60
-30
injury
I I
I
I
I
0
30
60
90
Time (min)
Figure 3. Cardiovascular changes after burn or mock-burn injury and the effects of superoxide dismutase plus cataiase (SOD/CAT, 5 mg/kg/h). Results are shown as mean f s.e.m., n=6, in each group, changes from prebum or pre mock-bum values were evaluated by Student’s paired t test. ‘P-C 0.05, “P-C 0.01, ‘**P-C 0.001. 0, Mock-bum+ saline; ?? , mock-bum-t-SOD/CAT; ?,? burn injury + saline; ?? , burn injury + SOD/CAT.
Burns (1989) Vol. 15/No. 3
150
40
r
in liposomes. Elevation of SOD and CAT plasma levels have been shown to cause a slight reduction in postbum oedema formation (Bjiirk and Arturson, 1982) and to increase the survival rate of burn-injured mice (Saez et al., 1984). However, the present data show that elevation of these circulating oxygen free radical scavengers does not prevent the early cardiovascular changes observed postbum. In conclusion, these results do not support the concept that circulating oxygen free radicals are involved in the circulatory changes after bum injury, nor that cardiac impairment after bum injury is due to intracellular oxygen free radicals generated via the xanthine oxidase pathway.
A
= E
3 b
12y-/& 0
c
LII -15
0
Sod/cat
infusion
1
1
15
30
-m
I
I
I
I
60
90
120
Time (min)
Figure 4. The SOD/CAT enzyme activity in rat plasma, before, during and after infusion of SOD/CAT (5 mg/kg/h in 1 ml 0.9 per , Catalase cent saline). Results are shown as mean f s.e.m., n = 6. ?? activity; ??SOD activity.
Acknowledgements We are grateful to Dr F. Kelly and Mr G. Ricketts for teaching and helping with the SOD and CAT assays. E. A. Speakman is an SERC student.
References vascular function in the dog: they observed a fall in blood pressure and a rise in heart rate. Since allopurinol caused these changes, interpretation of the results is difficult, but it is clear that allopurinol pretreatment does not cause any improvement in cardiovascular function after burn injury. Furthermore, allopurinol pretreatment did not cause any improvement in survival rate. In fact allopurinol treatment resulted in a significant fall in the survival of BI rats. The cause of this is unImown, but Marldey and Smallman (1970) observed that pretreatment with uric acid or precursors of uric acid, increased the survival of mice exposed to burn injury, it is possible that allopurinol decreases survival rates by reducing endogenous uric acid formation, although allernative mechanisms can be envisaged. Saez et al. (1984) found that allopurinol pretreatment improved the 24-h survival of BI mice; this difference from the present result may be due to species difference in sensitivity to burn or to allopurinol or the fact that the rats in the present study were kept under anaesthesia throughout the study. In the second and third studies, the doses of SOD and CAT used were comparable to those used in investigations into ischaemia/reperfusion damage in the in sihr heart of dogs (Jolly et al., 1984; Przylenk and Kloner, 1986) and bum injury in the rat (Bjiirk and Arturson, 1982) and dog (Hilton, 1980). SOD has been reported to have a half-life of 6 min in the hamster (Ley and Arfors, 1982) and 8 min in the rat (Turrens et al., 1984), and a similar half-life was found in the present study. The half-life of CAT was 7.2 min which is, however, shorter than the value of 20 min reported by Turrens et al. (1984). The scavengers were infused because the half-lives were known to be short, and direct measurement showed that plasma SOD and CAT activities were markedly elevated by the doses given. The molecular weight of SOD is 33 000 dallons and CAT 240 000 daltons, and it has been reported Ihat molecules as large as 300 000 daltons may pass through capillary membranes after burn injury (Ganrot et al., 1974). Hence, it is probable that these circulating scavengers may have an action outside the circulation after burn injury. Allopurinol crosses cell membranes and probably crosses the blood/ brain barrier (Iansek et al. (1986)), but it is unlikely that SOD or CAT would cross either of these barriers unless enclosed
Aebi H. (1984) Catalase in vitro. Methods hzyrtd. 105, 121. Allan G., Cambridge D., Lee-Tsang-Tan L. et al. (1986) The protective action of ailopurinol in an experimental model of haemorrhagic shock and reperfusion. Br. J. Pharmacol. 89, 149. Bj6rk J. and Arturson G. (1982) Effect of cimetidine, hydrocortisone, superoxide dismutase and catalase on the development of oedema after thermal injury. Burns 9, 249. Chapman B. J. and Speakman E. A. (1988a) Changes in cardiac output and papillary muscle contraction in the rat after a scald bum injury. 1. Physiol. 400,51P. Chapman B. J. and Speakman E. A. (1988b) Cardiac function curves after a scald bum injury to the anaesthetised rat. J. Physiol. 407, 17P. Chapman B. J., Chen C. F. and Munday K. A. (1977) Measurement of the cardiac output in the rat by impedance cardiography. J. Physiol. 270, 3P. Chapman B. J., Munday K. A. and Philpot M. E. (1979) Effects of cimetidine on some cardiovascular responses to thermal injury. ]. Physiol. 301,66P. Dobson E. L. and Warner G. F. (1957) Factors concerned in the early stages of thermal shock. Circ. Res. 5,69. Ganrot K., Jacobson S. and Rothman V. (1974) Transcapillary passage of plasma proteins in experimental bums. Acta Physiol. &and. 91,297. Halliwell 8. (1974) SOD, CAT and glutathione peroxidase: solutions to the problems of living with oxygen. Nezu Phybol. 73, 1075. Hilton J. G. (1980) Effects of alterations of polyunsaturated fatty acid metabolism upon volume loss induced by thermal trauma. 1. Trauma 20,663. Iansek R. Packham D, Aspey B. S. et al. (1986) As assessment of the possible protective effect of allopurinol in acute stroke. J. Neurol. Neurosurg. Psychiatry 49, 585. Jolly S. R., Kane W. J., Bailie M. B. et al. (1984) Canine myocardial reperfusion injury. Its reduction by the combined administration of superoxide dismutase and catalase. Circ. Res. 54,277. Ley K. and Arfors K. E. (1982) Changes in macromolecular permeability by intravascular generation of oxygenderived free radicals. Microvasc. Res. 24,25. McCord J. M. (1985) Oxygen derived free radicals in postischaemic tissue injury. New Engl. J. Med. 312, 159. McCord J. M. and Fridovich I. (1969) SOD an enzymic function for erythrocuprein (hemocuprein). 1, Biol. Chw. 244, 6049.
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Manson N. H. and Hess M. L. (1983) Interaction of oxygen free radicals and cardiac sarcoplasmic reticulum proposed role in the pathogenesis of endotoxin shock. Circ. Shock 10, P205. Markley K. and Smallman E. (1970) Protection against burn, tourniquet and endotoxin shock by purine compounds. 1. Trauma 10, 598. Markhmd S. and Marklund G. (1974) Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for SOD. Br. J Biochem. 47, 469. Przyklenk K. and Kloner R. A. (1986) SOD plus CAT improve contractile function in the the canine model of the ‘stunned myocardium’. Circ. Res. 58, 148. Saez J. C., Ward P. H., Gunther B. et al. (1984) Superoxide radical involvement in the pathogenesis of burn shock. Circ. Shock 12, 229. Swan R. C., Axelrod D. R., Seip M. et al. (1955) Distribution of
sodium bicarbonate infused into nephrectomized dogs. 1. C&I. Inu&. 34, 1795. Turrens J. F., Crap0 J. D. and Freeman B. A. (1984) Protection against toxicity by the injection of liposome entrapped catalase and superoxide dismutase. J. Qn. hwes~. 73,s~. Wolfe R. R. and Miller H. I. (1976) Burn shock in untreated and saline resuscitated guinea pigs. Development of a model. 1. Surg. Res. 21,269.
Paper accepted
3 December
1988.
Correspondence should be addressed to: Dr B. J. Chapman, Department of Physiology and Pharmacology, Southampton University, Southampton SO9 3TU, UK.
Award of the G. Whitaker International Bums Prize Palerrno, Italy for 19 89 In the course of a meeting
held on March
12th 1989 at the seat of the G. Whitaker
Foundation,
Italy, after examining the scientific activity in the field of research, teaching, clinical organization, prevention and cooperation among the nations, presented by various candidates, in the light of the consideration guiding the analysis of the high level of candidates, the Adjudicating committee unanimously decided to award the prize for 1989 to:
Palermo,
Shi Si-Xiang MD, Director of Shanghai Burns Institute, Shanghai Second Medical University, People’s Republic of China The prize is awarded for this motivation: “For having succeeded in a developing country, in creating a culture and a science of bums treatment. With traditional Chinese perseverance and in the face of many difficulties, and with an intuitive sense of new biological trends, he put forward bold technological innovations in the surgical treatment of the severely burned patient. When it was thought that burns covering 50-60 per cent of the body surface area were the maximum limit for possible survival, his technique led to the recovery of patients with bums extending to 90 per cent. He has thus established a new guideline in the surgical treatment of the bum patient, an indication that today fascinates and interests the entire scientific world.”
The official prize-giving of this prestigious award will be held on June 8th 1989 in PaJermo at the seat of the G. Whitaker Foundation in the course of a ceremony in the presence of authorities and representatives of the academic, scientific and cultural world.