SURGICAL RESEARCH
THE ROLE OF BRADYKININ IN ENDOTOXIC SHOCK OF INTESTINAL ORIGIN R. L.W. FINK,M. L. MASHFORD AND D. T. CARIDIS Departments of Surgery and Medicine, University of Melbourne, St Vincent's Hospital, Melbourne A relationship was looked fo r between blood levels of bradykinin and endotox&mia produced by superior mesenteric artery 'occlusion in the rabbit. The levels of bradykinin were unrelated to and unaltered by the degree of endotoxzemia observed in the systemic and portal circulations. These findings suggest that bradykinin is not responsible for the release of endotoxin from the gut, or for the circulatory c'ollapse which follows.
WILSONET ALII (1965) and Mason and Melmon (1966) found that the hamodynamic effects of infused bradykinin parallel some of the events observed in septic shock in man and experimental animals, and this observation led to speculation that the kinin release might mediate some of the effects of endotoxic shock. Nies et alii (1968) showed that infusion of endotoxin into non-anaesthetized monkeys produced an elevation in whole blood kinin concentration and a decrease in plasma kininogen. Kimbal et a& (1972) infused purified bacterial endotoxin into human volunteers and observed circulatory changes similar to those produced by kinins and also elevated blood bradykinin levels. These elevated blood bradykinin levels were found as early as 30 minutes after infusion of endotoxin, reached a peak at one hour and returned to normal by four hours. Recently, Cuevas and Fine (1973) infused vasoactive amines into rabbits and observed endotoxzemia which they speculated was of intestinal oriqin. The present study was undertaken with the object of studying a possible relationship between endotoxamia of Reprints: Mr R. L. W. Fink, Melbourne, Vic. 3000. I02
20
Collins Street,
intestinal origin produced by temporary occlusion of the superior mesenteric artery and bradykinin release.
MATERIALS AND METHODS Both New Zealand and French rabbits of varying weight and sex were anaesthetized with nitrous oxide, oxygen and Fluothane. Under sterile conditions both femoral arteries were cannulated, and the cannulz advanced into the aorta. One was used for blood sampling and blood volume replacement, while continuous monitoring of the blood pressure was carried out through the other. The abdomen was then opened, and a sling passed around the superior mesenteric artery in such a way that it could be tightened and released after closure of the abdomen. Seven groups, each comprising six rabbits, were studied. The characteristics of each group are given in Table I . In Group I, superior mesenteric artery occlusion (SMAO) was maintained for one hour, and then released until the end of the experiment at 23 hours. Severe hypotension was observed after release of the arterial occlusion. I n Group I1 a sham operation was performed. I n an effort to combat the hypotension noted in Group I, and to correct the AUST.N.Z. J. SURG.,VOL.47-No.
I,
FEBRUARY, 1977
FINK ET ALII
BRADYKININ I N ENDOTOXIC SHOCK
hypovolzmia, the animals in Groups I11 and I V received dextran 70 infusion, while those in Groups V, V I and V I I were infused with pooled rabbit blood. I n those animals in which portal vein blood was sampled, a cannula was passed through a gastric vein into the portal vein without impeding portal vein flow. Samples from either the aorta or the portal vein were taken at times -IO’, 0,+IS’, +45’,+75‘,e o ’ , +I~o’, relative to occlusion at time o and release at time +60’. Two rnillilitres of blood were used for endotoxin estimation, and 5 ml for estimation of bradykinin. Endotoxin TABLEI Groups Studied with Relevant Studies Indicated All Operations Lasted One Hour Group Operation
Sample site
Assay
Volume replacement
I
SMAO
Femoralartery
End.
Nil
I1
Sham
Femoralartery
End.
Nil
ShlAO
Femoral artery
End.
Dextran 7 0
111
-~
~~~~~
IV
SM40
Femqral artery
End.+Brad.
Dextran 70
V
SMAO
Femoral artery
End.+Brad.
Pooled blood
VI
Sham
Femoral artery
End.+Brad.
Pooled blood
VII
SMAO
Portal vein
End.+Brad.
Pooled blood
and in all instances the highest levels occurred after release of occlusion at one hour. As the limulus lysate test is based on a twofold dilution technique, these levels are not absolute and represent quantitative approximates. For this reason no statistical analysis of the endotoxin results can be made. TABLE2 Endotoxamia Occurs and Persists A p e r Superior Mesenteric Artery Occlusion o
+75’
Time:
-10‘
Endotoxin (ngiml) Endotoxin (ngiml) Endotoxin fneiml) Enzotoxin (ngiml) Endotoxin (rigid Endotoxin (nsiml)
o
8
16
8
o
120
120
250
o
30
60
o
30
IZO
o
30
30
8
8
o
60
120
120
60
+15’
+45’
60 250
16 500
30 500
4-90‘
16 500
+150’ Rabbit
32
I
500
2
60
3
500
4
30
60
5
250
250
6
120
1000
As can be seen in Figure I , marked hypotension followed S M A O release (Group I ) . This phenomenon was uniformly observed and could not be corrected by either dextran 70 or pooled blood infusion (Groups 111, IV, V and VII). Table 3 shows the results of bradykinin and endotoxin estimation after S M A O and pooled blood replacement in Group V. It can be seen 4
SMAO =superior mesenteric artery occlusion, Sham=laparotomy only without arterial occlusion. End. = Endotoxin. Brad. = Bradykinin.
levels were measured by the limulus lysate technique of Reinhold and Fine (1971). Bradykinin was measured by a radioimmunoassay using the technique of Mashford and Roberts (197z), and at times in order to assess the accuracy of this method, a known amount of bradykinin was added to the collecting syringe as an internal standard.
RESULTS Table z shows that soon after occlusion of the superior mesenteric artery (Group I ) , endotoxzmia occurred, and that it persisted until the end of the experiment hours after SMAO. Whilst the levels of endotoxin varied, in general they rose with the passage of time, AUST. N.Z. J. SURG., VOL.47-No.
I,
FEBRUARY, 1977
(3
I \
I I
w
a:
m
_I
T
” o
60
TIME
120
150
(MIN.)
FIGUREI : Graph showing the development of hypotension after one hour of superior mesenteric occlusion.
I03
BRADYKININ I N ENDOTOXIC SHOCK
that although endotoxmiia followed the previously observed pattern, the bradykinin levels were not altered by the presence of endotoxin of intestinal origin. Similar results were obtained with the experiments in Group I V where the animals were infused with dextran 70. TABLE3 Endooloxin and Bradykanin Levels t n Systemic Circulation after Superior Mcscnterzc Artery Occluston
Time
- 10' 0
Endotoxin (nglml) 0
32
+IS'
I20
f45'
500 500 500
+7S' +90', +I50
Bradykinin (ng/ml&S.D.)
500
In the animals subjected to sham operations (Groups I1 and VI), where no S M A O took piace, no hyoptension and no endotoxzmia were observed, and the bradykinin levels were no different from those in all other groups. I t was thought possible that bradykinin released from the gut might have been destroyed before reaching the sampling site on the arterial side of the general circulation, and a final group (Group V I I ) of experiments were performed, in the course of which samples were collected from the portal vein, for assay TABLE4 Endotoxin and Bradykinin Levels i n Portal Circulation After Superior Mesenteric Artery Occlusion
Time
Endotoxin (ngiml)
- 10'
0
0 +1q' ,
8 R 16
-k 90:
+75'
1000 I000
+IS0
500
~+45'
Bradykinin (ng/ml& S.D.)
of bradykinin and endotoxin levels after SMAO. Table 4 shows that the endotoxin levels followed the previously observed pattern, namely, gradual increase and subsequent stabilization following SMAO, and again the bradykinin levels were not altered by the presence of endotoxin.
DISCUSSION Until now, experimental studies directed to investigation of the possible role of kinins in I04
FINK ET ALII
endotoxic shock have involved the infusion of purified bacterial endotoxin with subsequent measurement of a kinin response (Nies et a&, 1968; Fal, 19%; Kimball et alii, 1972; Griffiths, 1973). I t was previously shown (Caridis et alii, 1973), and we were able to prove again in this study, that temporary occlusion of the superior artery in the rabbit ( S M A O ) regularly produced endotoxxmia and death. The endotoxin which is released following S M A O is of intestinal origin (Caridis et alii, 1973). W e have further shown that one hour of temporary occlusion of the superior mesenteric artery produces a marked hypotension which does not respond to dextran 70 or pooled blood replacement. Two possible roles have been suggested for the kinins in endotoxic shock. First, they may act as chemical mediators responsible for some of the hanodynamic events (Melmon, 1967; Nies et ulii, 1968). Second, they may increase gut permeability to intestinal endotoxins, thus contributing to the initiation and continuation of endotoxxmia (Cuevas and Fine, 1973). Continuous formation of kinins would be required for such roles, and raised levels would be expected in both the systemic and the portal circulations. I n our experiments we failed to demonstrate any significantly elevated levels of bradykinin in the femoral artery and the portal vein corresponding with endotoxzmia, suggesting that, at least in the model that we used, kinins have no such role. There are difficulties in measuring circulating kinins, as they are rapidly formed by activation or release of kallikinins, and rapidly removed by kininases. T h e radioimmunoassay employed, however, has been shown to satisfy requirements of sensitivity and recovery in the measurement of circulatine. kinins in man and dogs, so that it should have detected a siqnificant bradykinin response ( Mashford and Roberts, 1972). That these results are at variance with those showing a kinin response to infused purified bacterial endotoxin (Nies et alii, 1968; Kimball et alii, 1972) may be exdained by either a dose or a species difference. Several workers have shown that species difference is important in endotoxamia and bradykinin response (Urbanitz et a& 1070 ; Nies and Melmon, 197.1; Griffiths, 1973). Also, when endotoxin is infused, there is generally an immediate high endotoxEmia, and AUST. N.Z. J. SURC., VOL. 47-NO.
I,
FEBRUARY, 1977
SYSTEMIC ANTIBIOTIC ADMINISTRATION
gradually the endotoxin levels decrease. We have shown that the opposite occurs with SMAO endotommia, when endotoxin levels gradually increase after occlusion of the superior mesenteric artery, but this is unaccompanied by any change in blood kinin levels.
REFERENCES CARIDIS,D. T., ISHIYAMA,M., WOODRUFF, P. W. H. and FINE, J. (I973), 1. reticuloendoth. Soc., 14: 513. CUEVAS,P. and FINE,J. (1g73), Gastroenterology, 64: 285. FAL, W. . (1969), A r c h i v u m Immunologice r t Therapta Experimentalis, 17 : 406. GRIFFITHS, J. (1973), 1. Trauma, 13 : 67.
L A W ET ALII KIMBALL,H. R., MELMON,K. L. and WOLFF,S. M. ( ~ g p ) PTOC. , SOC.exp. Biol. (N.Y.), 139: 1078. MASHFORD,M. L. and ROBERTS,M. L. (1g72), Biochem. Pharmacol., 21 : 2727. MASON,D. T . and MELMON,K. L. (I@), J . clin. Invest., 45: 1685. MELMON,I
THE ROLE OF BRADYKININ IN ENDOTOXIC SHOCK OF INTESTINAL ORIGIN R. L.W. FINK,M. L. MASHFORD AND D. T. CARIDIS Departments of Surgery and Medicine, University of Melbourne, St Vincent's Hospital, Melbourne A relationship was looked fo r between blood levels of bradykinin and endotox&mia produced by superior mesenteric artery 'occlusion in the rabbit. The levels of bradykinin were unrelated to and unaltered by the degree of endotoxzemia observed in the systemic and portal circulations. These findings suggest that bradykinin is not responsible for the release of endotoxin from the gut, or for the circulatory c'ollapse which follows.
WILSONET ALII (1965) and Mason and Melmon (1966) found that the hamodynamic effects of infused bradykinin parallel some of the events observed in septic shock in man and experimental animals, and this observation led to speculation that the kinin release might mediate some of the effects of endotoxic shock. Nies et alii (1968) showed that infusion of endotoxin into non-anaesthetized monkeys produced an elevation in whole blood kinin concentration and a decrease in plasma kininogen. Kimbal et a& (1972) infused purified bacterial endotoxin into human volunteers and observed circulatory changes similar to those produced by kinins and also elevated blood bradykinin levels. These elevated blood bradykinin levels were found as early as 30 minutes after infusion of endotoxin, reached a peak at one hour and returned to normal by four hours. Recently, Cuevas and Fine (1973) infused vasoactive amines into rabbits and observed endotoxzemia which they speculated was of intestinal oriqin. The present study was undertaken with the object of studying a possible relationship between endotoxamia of Reprints: Mr R. L. W. Fink, Melbourne, Vic. 3000. I02
20
Collins Street,
intestinal origin produced by temporary occlusion of the superior mesenteric artery and bradykinin release.
MATERIALS AND METHODS Both New Zealand and French rabbits of varying weight and sex were anaesthetized with nitrous oxide, oxygen and Fluothane. Under sterile conditions both femoral arteries were cannulated, and the cannulz advanced into the aorta. One was used for blood sampling and blood volume replacement, while continuous monitoring of the blood pressure was carried out through the other. The abdomen was then opened, and a sling passed around the superior mesenteric artery in such a way that it could be tightened and released after closure of the abdomen. Seven groups, each comprising six rabbits, were studied. The characteristics of each group are given in Table I . In Group I, superior mesenteric artery occlusion (SMAO) was maintained for one hour, and then released until the end of the experiment at 23 hours. Severe hypotension was observed after release of the arterial occlusion. I n Group I1 a sham operation was performed. I n an effort to combat the hypotension noted in Group I, and to correct the AUST.N.Z. J. SURG.,VOL.47-No.
I,
FEBRUARY, 1977
FINK ET ALII
BRADYKININ I N ENDOTOXIC SHOCK
hypovolzmia, the animals in Groups I11 and I V received dextran 70 infusion, while those in Groups V, V I and V I I were infused with pooled rabbit blood. I n those animals in which portal vein blood was sampled, a cannula was passed through a gastric vein into the portal vein without impeding portal vein flow. Samples from either the aorta or the portal vein were taken at times -IO’, 0,+IS’, +45’,+75‘,e o ’ , +I~o’, relative to occlusion at time o and release at time +60’. Two rnillilitres of blood were used for endotoxin estimation, and 5 ml for estimation of bradykinin. Endotoxin TABLEI Groups Studied with Relevant Studies Indicated All Operations Lasted One Hour Group Operation
Sample site
Assay
Volume replacement
I
SMAO
Femoralartery
End.
Nil
I1
Sham
Femoralartery
End.
Nil
ShlAO
Femoral artery
End.
Dextran 7 0
111
-~
~~~~~
IV
SM40
Femqral artery
End.+Brad.
Dextran 70
V
SMAO
Femoral artery
End.+Brad.
Pooled blood
VI
Sham
Femoral artery
End.+Brad.
Pooled blood
VII
SMAO
Portal vein
End.+Brad.
Pooled blood
and in all instances the highest levels occurred after release of occlusion at one hour. As the limulus lysate test is based on a twofold dilution technique, these levels are not absolute and represent quantitative approximates. For this reason no statistical analysis of the endotoxin results can be made. TABLE2 Endotoxamia Occurs and Persists A p e r Superior Mesenteric Artery Occlusion o
+75’
Time:
-10‘
Endotoxin (ngiml) Endotoxin (ngiml) Endotoxin fneiml) Enzotoxin (ngiml) Endotoxin (rigid Endotoxin (nsiml)
o
8
16
8
o
120
120
250
o
30
60
o
30
IZO
o
30
30
8
8
o
60
120
120
60
+15’
+45’
60 250
16 500
30 500
4-90‘
16 500
+150’ Rabbit
32
I
500
2
60
3
500
4
30
60
5
250
250
6
120
1000
As can be seen in Figure I , marked hypotension followed S M A O release (Group I ) . This phenomenon was uniformly observed and could not be corrected by either dextran 70 or pooled blood infusion (Groups 111, IV, V and VII). Table 3 shows the results of bradykinin and endotoxin estimation after S M A O and pooled blood replacement in Group V. It can be seen 4
SMAO =superior mesenteric artery occlusion, Sham=laparotomy only without arterial occlusion. End. = Endotoxin. Brad. = Bradykinin.
levels were measured by the limulus lysate technique of Reinhold and Fine (1971). Bradykinin was measured by a radioimmunoassay using the technique of Mashford and Roberts (197z), and at times in order to assess the accuracy of this method, a known amount of bradykinin was added to the collecting syringe as an internal standard.
RESULTS Table z shows that soon after occlusion of the superior mesenteric artery (Group I ) , endotoxzmia occurred, and that it persisted until the end of the experiment hours after SMAO. Whilst the levels of endotoxin varied, in general they rose with the passage of time, AUST. N.Z. J. SURG., VOL.47-No.
I,
FEBRUARY, 1977
(3
I \
I I
w
a:
m
_I
T
” o
60
TIME
120
150
(MIN.)
FIGUREI : Graph showing the development of hypotension after one hour of superior mesenteric occlusion.
I03
BRADYKININ I N ENDOTOXIC SHOCK
that although endotoxmiia followed the previously observed pattern, the bradykinin levels were not altered by the presence of endotoxin of intestinal origin. Similar results were obtained with the experiments in Group I V where the animals were infused with dextran 70. TABLE3 Endooloxin and Bradykanin Levels t n Systemic Circulation after Superior Mcscnterzc Artery Occluston
Time
- 10' 0
Endotoxin (nglml) 0
32
+IS'
I20
f45'
500 500 500
+7S' +90', +I50
Bradykinin (ng/ml&S.D.)
500
In the animals subjected to sham operations (Groups I1 and VI), where no S M A O took piace, no hyoptension and no endotoxzmia were observed, and the bradykinin levels were no different from those in all other groups. I t was thought possible that bradykinin released from the gut might have been destroyed before reaching the sampling site on the arterial side of the general circulation, and a final group (Group V I I ) of experiments were performed, in the course of which samples were collected from the portal vein, for assay TABLE4 Endotoxin and Bradykinin Levels i n Portal Circulation After Superior Mesenteric Artery Occlusion
Time
Endotoxin (ngiml)
- 10'
0
0 +1q' ,
8 R 16
-k 90:
+75'
1000 I000
+IS0
500
~+45'
Bradykinin (ng/ml& S.D.)
of bradykinin and endotoxin levels after SMAO. Table 4 shows that the endotoxin levels followed the previously observed pattern, namely, gradual increase and subsequent stabilization following SMAO, and again the bradykinin levels were not altered by the presence of endotoxin.
DISCUSSION Until now, experimental studies directed to investigation of the possible role of kinins in I04
FINK ET ALII
endotoxic shock have involved the infusion of purified bacterial endotoxin with subsequent measurement of a kinin response (Nies et a&, 1968; Fal, 19%; Kimball et alii, 1972; Griffiths, 1973). I t was previously shown (Caridis et alii, 1973), and we were able to prove again in this study, that temporary occlusion of the superior artery in the rabbit ( S M A O ) regularly produced endotoxxmia and death. The endotoxin which is released following S M A O is of intestinal origin (Caridis et alii, 1973). W e have further shown that one hour of temporary occlusion of the superior mesenteric artery produces a marked hypotension which does not respond to dextran 70 or pooled blood replacement. Two possible roles have been suggested for the kinins in endotoxic shock. First, they may act as chemical mediators responsible for some of the hanodynamic events (Melmon, 1967; Nies et ulii, 1968). Second, they may increase gut permeability to intestinal endotoxins, thus contributing to the initiation and continuation of endotoxxmia (Cuevas and Fine, 1973). Continuous formation of kinins would be required for such roles, and raised levels would be expected in both the systemic and the portal circulations. I n our experiments we failed to demonstrate any significantly elevated levels of bradykinin in the femoral artery and the portal vein corresponding with endotoxzmia, suggesting that, at least in the model that we used, kinins have no such role. There are difficulties in measuring circulating kinins, as they are rapidly formed by activation or release of kallikinins, and rapidly removed by kininases. T h e radioimmunoassay employed, however, has been shown to satisfy requirements of sensitivity and recovery in the measurement of circulatine. kinins in man and dogs, so that it should have detected a siqnificant bradykinin response ( Mashford and Roberts, 1972). That these results are at variance with those showing a kinin response to infused purified bacterial endotoxin (Nies et alii, 1968; Kimball et alii, 1972) may be exdained by either a dose or a species difference. Several workers have shown that species difference is important in endotoxamia and bradykinin response (Urbanitz et a& 1070 ; Nies and Melmon, 197.1; Griffiths, 1973). Also, when endotoxin is infused, there is generally an immediate high endotoxEmia, and AUST. N.Z. J. SURC., VOL. 47-NO.
I,
FEBRUARY, 1977
SYSTEMIC ANTIBIOTIC ADMINISTRATION
gradually the endotoxin levels decrease. We have shown that the opposite occurs with SMAO endotommia, when endotoxin levels gradually increase after occlusion of the superior mesenteric artery, but this is unaccompanied by any change in blood kinin levels.
REFERENCES CARIDIS,D. T., ISHIYAMA,M., WOODRUFF, P. W. H. and FINE, J. (I973), 1. reticuloendoth. Soc., 14: 513. CUEVAS,P. and FINE,J. (1g73), Gastroenterology, 64: 285. FAL, W. . (1969), A r c h i v u m Immunologice r t Therapta Experimentalis, 17 : 406. GRIFFITHS, J. (1973), 1. Trauma, 13 : 67.
L A W ET ALII KIMBALL,H. R., MELMON,K. L. and WOLFF,S. M. ( ~ g p ) PTOC. , SOC.exp. Biol. (N.Y.), 139: 1078. MASHFORD,M. L. and ROBERTS,M. L. (1g72), Biochem. Pharmacol., 21 : 2727. MASON,D. T . and MELMON,K. L. (I@), J . clin. Invest., 45: 1685. MELMON,I
