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Effect of oxygen free radicals on cardiovascular function at organ and cellular levels
Effect of oxygen free radkats on cardiovascular function at organ and cellular levels Oxygen free radicals (OFR) have been implicated as a causative factor of cell damage in several pathologic condltlons. It is possible that OFR could have effects on cardiac function and contractlllty. The present investigation deals with the effects of OFR in the absence and in the presence of scavangers of OFR (superoxide dismutase and catalase) on cardiac function, index of cardiac contractility, serum creatine kinase (CK), and blood lactate, PO* and pli in the anesthetized dogs. The hemodynamic measurements and collection of blood samples for measurement of CK, lactate, PO, and pH were made before and at various intervals after admlnistratlon of OFR for 1 hour. Xanthine and xanthine oxidase were used to generate OFR. OFR produced a decrease in cardiac function and indices of myocardial contractility and an increase in the serum CK. OFR produced an increase in the systemic and pulmonary vascular resistance. Although there was a tendency for an increase in the blood lactate, the increase was not significant. The blood PO, and pH were not affected. Superoxlde dlsmutase (SOD), alone or in combination with catalase, tended to protect cardiac function against the deleterious effects of OFR. Scavengers of OFR prevented the OFR-Induced rise in serum CK. Although the protective effect of SOD plus catalase was slightly better than SOD alone, the results were not significantly different from each other. These results suggest that OFR are cardiac depressant and increase the peripheral vascular resistance besides causing cellular damage. Scavangers of OFR may be beneficial in counteracting the deleterious effects of OFR on hemodynamic parameters and cellular integrity. (AM HEART J 1989; 117: 1196.)
K. Prasad, MD, PhD, J. Kalra, MD, PhD, W. P. Chan, BSc, and A. K. Chaudhary, MPharm, PhD. Saskatoon, Saskatchewan, Canada Oxygen free radicals have been implicated as inducers of tissue injury in several pathologic conditions such as inflammation, irradiation, ischemia/reperfusion, circulatory shock injury, and respiratory disfree radicals tress syndrome.l-s Oxygen-derived include superoxide anion (O;), hydrogen peroxide (H,O,), and the hydroxyl radical (-OH). These radicals are generated by a number of cellular reactions.2,g 0; is catalyzed to H202 by superoxide dismutase (SOD) present in the biological system. H,O, is then catalytically reduced in the cell to H,O by catalase or glutathione peroxidase. There are various sources of oxygen free radicals including xanthine-xanthine oxidase,lO polymorphonuclear leukocytes,” auto-oxidation of catecholamines,12 and arachidonic acid metabolism.13 During tissue ischemia, it is not only that the production of xanthine and xanthine oxidase is increased,14z15 but that there
From the Departments of Physiology University of Saskatchewan. This
work
Received
was supported for publication
and Pathology,
by the Saskatchewan Sept. 2, 1988; accepted
Heart
College of Medicine, Foundation.
Jan. 6, 1989.
Reprint requests: K. Praaad, MD, Dept. of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N owo.
1196
is also a loss of both SOD and glutathione peroxidase,16 which would result in an increase in the level of oxygen free radicals. Oxygen free radicals have been suggested to exert their cytotoxic effect by causing peroxidation of membrane phospholipids, which can result in an increase in membrane fluidity, increasing permeability, and loss of membrane integrity.17-lg Also, oxygen free radicals have been reported to affect excitation-contraction coupling.18 The effect of oxygen free radicals on cardiovascular function and cardiac contractility is not clear. It is possible that such radicals might influence these properties. The present investigation therefore deals with the effects of exogenous oxygen free radicals on cardiac function, indices of cardiac contractility, serum creatine kinase, and blood lactate in anesthetized dogs. METHODS The experiments were carried out on mongrel dogs of either sex, weighing between 18 and 25 kg. The dogs were anesthetized with sodium pentobarbital(30 mg/kg) intravenously and intubated with a closed-cuff endotracheal tube. The lungs were ventilated with room air using a Harvard respiratory pump (Harvard Apparatus Co. Inc., S. Natick, Mass.) with a volume of 20 ml/kg and a respiratory rate of 20Jmin. Additional amounts of sodium
Volume 117 Number 6
pentobarbital (5 mg/kg) were administered intravenously when needed. Hemodynamic measurements. The femoral artery and vein were exposed. A 7F gauge Cournand (Cordis GF, Cordis Corp., Miami, Fla.) catheter was positioned at the aortic arch through the femoral artery to record aortic pressure. The same catheter was pushed into the left ventricle to record left ventricular pressure. Another 7F gauge Cournand (Cordis GF) catheter was positioned in the right atrium through the femoral vein to record right atria1 pressure and to collect blood samples for biochemical measurements. A Swan-Ganz triple lumen catheter (7F gauge) equipped with a thermistor tip (Baxter Healthcare Corp., Edwards Division, Santa Ana, Calif.) was passed into the pulmonary artery through the external jugular vein to measure pulmonary arterial pressure, pulmonary capillary wedge pressure, and cardiac output. Cardiac output was measured in triplicate with Edwards cardiac output computer (COM-1, Baxter Healthcare Corp., Scientific Division, McGaw Park, Ill.). Lead II of the electrocardiogram (ECG) was monitored throughout the experiment. The first derivative of left ventricular pressure (dp/dt) was recorded with a differentiating device coupled to left ventricular pressure at a frequency response of 100 Hz. All pressures were recorded with a Gould pressure transducer (Spectramed Inc., Cardiovascular Products Division, Oxnard, Calif.) and a Beckman R611 dynograph recorder (Beckman Instruments Inc., Brea, Calif.). The ratio of (dp/dt)/IIP was used as one index of myocardial contractility because it is not affected by preload or by a small change in ,the heart rate.20p21The dp/dt is not only affected by contractility but also by preload, afterload, and heart rate.21 The integrated isovolumetric pressure (IIP) was calculated by the method described previously by Prasad et a1.251 The other index of myocardial contractility that is not affected by preload and that is used in this experiment was (dp/dt)/PAW, where PAW is the pulmonary arterial wedge pressure. The left ventricular work index (LVWI), total systemic vascular resistance (TSVR), and cardiac index (CI) were calculated by previously described nnethods.23s24 The body surface area of the dogs was determined according to the method of Ettinger and Suter.25 Pulmonary vascular resistance (PVR) was calculated by the formula: PVR = PA&O, where PA,,, = mean pulmonary arterial pressure (in millimeters of mercury) and CO = cardiac output (in liters per minute). Biochemical measurements. Mixed venous blood was collected for the measurements of creatine kinase (CK) and lactate. Arterial blood was collected for measurement of PO, and pH. Blood lactate was measured using the lactic dehydrogenase system on an ACA Du Pont Discrete clinical analyzer (Du Pont Company, Wilmington, De1.)26 The method for measurement of CK was that described by Prasad et a1.27Two milliliters of blood was collected in a test tube containing 0.1 ml ethyleneglycol-bis @-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (20pM solution) and was centrifuged to collect plasma. N-acetylcysteine (1 mmol) was added at 0.1 ml/ml of plasma as a reducing agent. The CK determination was made using a
Oxygen
free radicals
and cardiovascular
function
1197
Fig. 1. A typical tracing of the effect of xanthine plus xanthine oxidase (X + X0) in the absence and in the presence of superoxide dismutase (SOD) or SOD plus catalase on the left ventricular pressure (LIP) and its first derivative (dp/dt). The control tracing before administration of any drug is designated as “0” minutes. The arrow marks the beginning of the bolus administration of xanthine plus xanthine oxidase and the subsequent strips of tracings are shown at intervals at the top of each strip of tracing. SOD or SOD plus catalase were administered continuously throughout the observation period. Top tracing shows the effect of X + X0. Middle tracing shows the effect of X + X0 in the presence of SOD. Bottom tracing shows the effect of X + X0 in the presence of SOD plus catalase. Vertical axis indicates the magnitude of various hemodynamic parameters. Zero point in the LVP and dpldt tracings is the base line. Positive dpldt is the rate of pressure development and the (-) dp/dt is the rate of pressure drop (rate of relaxation) of the ventricle. Note the marked decrease in the LVP and its dpldt with X + X0. Also note the ineffectiveness of X + X0 in decreasing the LVP and its dpldt in the presence of SOD or SOD + catalase.
creatine kinase reagent test kit (SVR CK test kit, Behrmg Diagnostics, Hoechst Canada Inc., Montreal, Quebec, Canada). PO, and pH were measured with Corning 175 automatic pH/blood gas system (Corning Medical, Medfield, Mass.).
1190
June 1999 American Heart Journal
Prasad et al.
_ ” ‘E 0 .
6000
r *
6000
5: ul* I-
LI 05
’ 15 TIME
1 30
l
gl E r
4000
z
2000
I 00
L
1 15
1 30
~~ II 05
(mln)
’
TIME
J 60
(min)
2. Effects of xanthine plus xanthine oxidase (X + X0) in the absence and presence of superoxide dismutase (SOD) or SOD plus catalase on the cardiac index, pulmonary vascular resistance (PVR), mean pulmonary arterial pressure (mPAP), and total systemic vascular resistance (TSR). The results are expressed as mean + SE. *p < 0.05, comparison of the values at different time courses with respect to the values before any treatment (0 minutes) in the respective groups (intragroup).
Fig.
Xanthine plus xanthine oxidase were used to generate oxygen free radicals, These substances have been used by various investigators in the past to generate oxygen free radica1s.10~28 SOD and catalase were used as scavengers of oxygen free radicals. The dogs were divided into three groups. Group I (n = 5) dogs received xanthine (0.14 mg/kg) plus xanthine oxidase (0.14 U/mg protein, Sigma Chemical Company, St. Louis, MO.) in the dose of 2 U/kg intravenously after initial hemodynamic and biochemical measurements. The effects of xanthine plus xanthine oxidase on the various hemodynamic and biochemical parameters were observed for a period of 1 hour. Group II (n = 5) dogs received SOD (3050 U/mg protein, Sigma Chemical Company) 5 minutes prior to the administration of xanthine plus xanthine oxidase in doses similar to dogs in group I. SOD in a dose of 1.4 mg/kg was administered intravenously in the bolus form followed immediately by a continuous infusion in a dose of 5 mg/kg/hr for the duration of study. Group III (n = 5) received SOD plus catalase (11000 U/mg, Sigma Chemical Company) 5 minutes prior to xanthine plus xanthine oxidase. Catalase and SOD each in the dose of 1.4 mg/kg intravenously were administered in a bolus form followed by a continuous infusion at a dose of 5 mg/kg/hr each for the entire duration of the study. The hemodynamic measurements and collections of blood samples for the measurement of lactate, CK, PO,, and pH were made before administration of any drug and after 5, 15, 30, and 60 minutes of xanthine plus xanthine oxidase administration in all three groups. Data analysis. The statistical analysis was made using paired t test for comparison in the same group and unpaired t test for comparison between two groups. Statistical significance was considered as p < 0.05. Protocol
of studies.
RESULTS Hemodynamics. A typical tracing of the effect of xanthine plus xanthine oxidase in the presence and absence of SOD and of SOD plus catalase on the left ventricular pressure and its dp/dt is shown in Fig. 1. Xanthine plus xanthine oxidase produced a marked decrease in the left ventricular pressure (LVP) and its dp/dt within 1 minute, reaching its lowest value within 5 minutes. The LVP and dp/dt then started to increase at about 10 to 15 minutes till the observation period (60 minutes). However, the recovery was not complete at the end of the observation period. The decreases in the LVP and its dp/dt with xanthine plus xanthine oxidase were less marked in the presence of SOD or SOD plus catalase. The changes in the hemodynamic parameters with exogenous oxygen free radicals (xanthine plus xanthine oxidase) in the absence and in the presence of SOD, and SOD plus catalase are summarized in Fig. 2 and 3 and Tables I and II. Xanthine plus xanthine oxidase. Oxygen free radicals produced a marked decrease in the mean arterial pressure (MAP), mean pulmonary arterial pressure (MPAP), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), left ventricular (+) dp/dt and (-) dp/dt, (dp/dt)/IIP, (dp/dt)/PAW, LVWI, CI, heart rate, and cardiac effort within 5 minutes. PVR and systemic TSVR increased with oxygen free radicals. No change was observed in mean right atria1 pressure (MRAP) and PAW. Most of the hemodynamics
Volume 117 Number
Oxygen free radicals and cardiovascular function
6
o-
150
F-;
140
4 ‘a5
130
I-
a 2 =-
2 m z
I
x-x0;
1199
700 600 0. -A 500 -.I 5: E
l - x-x0
+ SOD;
-
n -
x-x0
+ SOD + CATALASE
400
120 110
-
100
-
*
Ik
200
*
90’ -
300
,“’
100
L’
’
I
I
05
15
30
60
TIME
05
15 TIME
(min)
60
30 (min)
3. EBects of xanthine plus xanthine oxidase (X + X0) in the absence and presence of superoxide dismutase (SOD) or SOD plus catalase on the mean right atrial pressure (MRAP), heart rate, (dp/dt)/IIP, and cardiac effort. The results are expressed as mean + SE. *p < 0.05, comparison of the values at various time courses with respect to the values before any drug administration (0 minutes) in the respective groups.
Fig.
I. Changes in hemodynamics with various treatments
Table
Time (min) 0
__ Z 19.2
15 30 60
MAP (mm Hd
LVWZ (kg . MJminlM2)
PAW (mm Z-W
Group
Group
Group
Group
zz
zzz
Z
zz
zzz
Z
zz
zzz
Z
zz
zzz
4.1 +0.6
7.6 + 0.7
10.2 k 1.2
9.7 +0.7
10.5 It 1.1
12.5
25.6
156.0
119.2
151.3
7.5
7.0 ?c 1.2
zi 3.8 9.8
k4.0 10.0
k 11.2 67.5*
k6.8 81.0
k8.0 92.5
+1.6 0.9*
+ 1.9
k 1.8
+ 10.1
+8.1
+ 5.3
kO.3
kO.5
+0.5
AZ1.5
kO.3
+ 0.4
11.7 k4.4
8.8 + 0.7
12.5 +1.8
98.3 k 1.7
98.8 +6.2
2.5* kO.2
2.7 kO.5
3.3 kO.8
10.9 + 0.7
9.8 + 0.8
8.2 +0.6
3.3
2.9 20.3
4.5 r 0.8
9.2 kO.1
10.9 iz 1.5
8.5 kO.7
k9.2 5
L VEDP (mm fig)
96.7* k1.7
11.7
6.7
6.8
110.0
106.7
112.5
* 1.7
* 1.7 10.0
+ 1.2 10.0
+8.7 108.3
+ 1.7 100.0
zk 1.8 80.5
20.5 3.3
k1.4
k2.5
f 13.0
+ 2.9
27.4
f 0.8
8.3 ir0.8
1.7*
2.9 LO.4
2.3
2.2 +0.1
9.0
9.4
6.5
9.1
9.8
9.0
k 1.2
f 1.4
r 1.4
Results are expressetl BSmean+ SE. LVEDP, Left ventricular end-diastolic pressure; MAP, mean aortic pressure; LVWI, left ventricular work index; PAW, pulmonary arterial wedge pressure. Group I, Xanthine + xanthine oxidase; Group II, xanthine + xanthine oxidase in the presence of superoxide dismutase (SOD); Group III, xanthine + xanthine oxidase in the presence of SOD and catalase. *p < 0.05, comparison of the values at different time intervals with respect to the values before any treatment (0 minutes) in the respective groups.
changes tended to return toward control values at the end of the l-hour period of observation. However, the MAP, heart rate, LVEDP, LVWI, CI, cardiac effort, (-) dp/dt, and LVSP were still depressed at the end of the l-hour observation period. Xanthine plus xanthine oxidase and SOD. Left ventricular work index, (+) dp/dt, (-) dp/dt, (dp/ dt)lPAW, and cardiac effort decreased significantly.
TSVR increased significantly. No changes were observed in the PVR, MPAP, LVEDP, MAP, PAW, LVSP, heart rate, and MRAP. Xanthine plus xanthine oxidase in the presence of SOD produced changes in the hemodynamic parameters similar to that of xanthine plus xanthine oxidase. Although these changes with xanthine plus xanthine oxidase in the presence of SOD were less marked than in its
1200
Table
Time (min) 0 5 15 30 60
Prasad
et al.
American
June 1989 Heart Journal
II. Changes in hemodynamics with various treatments 1, VSP (mm Hd
i+)dp/dt (mm Hglsec)
Group
Group
__---.--. ___--
__ I
II
190.0 + 18.0 85.8* 19.1 132.5 k3.8 147.2 224.9 137.5 + 8.0
157.5 k 2.9 111.3 + 10.8 136.7 i4.4 145.0 i 2.9 137.5 t3.8
III 197.5 f 1.8 115.0 27.1 143.8 k 13.3 157.5 k5.3 125.0 27.1
__---
i-ldpldt (mm Hglsec) ---~__ --.-~-
I
II
III
2166.7 i 272.8 1100.0 Il15.5 1866.7 t 120.2 2300.0 + 173.2 2266.7 i 266.7
3133.3t i 176.4 1580.0* + 334.9 2333.3 + 185.6 2800.0 + 200.0 2433.3 + 338.3
2080.0 + 510.6 1600.0 k 141.8 2650.0 -t 390.0 3250.0 t 177.3 2200.0 + 141.8
I 2733.3 + 371.2 800.0* * 115.5 1833.3 k 152.8 1866.7 k 176.4 1533.3 k 352.8
+(dpldt)lPAW (Set- ‘)
___.
Group -____
.____-__
II
III
I
2233.3 k 166.7 930.0* + 155.9 1466.7t 266.7 1733.3 i: 266.7 1266.7 + 176.4
3000.0 ?I 709.2 900.0* * 70.9 1400.0 + 424.5 1750.0 z!I 319.0 1070.0 * 191.5
221.9 rf: 48.7 115.8 k4.3 174.6 k8.7 255.4 -+ 34.8 262.5 t 55.1
Group II 329.3 + 37.9 164.8’ IL 29.6 241.5 k31.6 263.6 -t 35.5 257.7 k41.4
III 192.2 i29.1 250.0 i 35.5 319.7 k25.5 352.9 + 28.5 262.3 + 57.1
Results are expressed as mean k SE. LVSP, Left ventricular systolic pressure; (+) dp/dt, rate of pressure development; (-) dp/dt, rate of relaxation; PAW, pulmonary arterial wedgle pressure. Notations of the groups are same as described in Table I. *p < 0.05, comparison of the values at different time intervals with respect to the values before any treatment (0 minutes) in the respective groups. tb < 0.05, group I versus group II or group III.
absence, they were not significant. Decreases in the heart rate and cardiac effort were greater in the presence of SOD than in its absence.
changes in the PO, and pH with xanthine plus xanthine oxidase in the presence and in the absence of SOD or SOD plus catalase were not significant.
Xanthine plus xanthine oxidase and SOD plus catalase. Xanthine plus xanthine oxidase in the
DISCUSSION
presence of SOD and catalase produced very little change in the measured hemodynamic parameters. Catalase and SOD protected deleterious effects of exogenous oxygen free radicals produced by xanthine plus xanthine oxidase on the hemodynamic parameters. Lactate and creatine kinase. The summary of the results of blood lactate and CK is given in Fig. 4. Blood lactate before the administration of drugs (0 minutes) was 1.33 -t 0.56 mmol/L. Although there was a tendency for an increase in the blood lactate after administration of xanthine plus xanthine oxidase, this increase was not significant. The changes in the blood lactate with xanthine plus xanthine oxidase in the presence of SOD or SOD and catalase were not significantly different from those with xanthine plus xanthine oxidase alone. The value of blood CK before drug treatment was 137 f 60 IU/L. There was a tendency for an increase in the serum CK with xanthine plus xanthine oxidase treatment. However, these changes were not significant. Serum CK did not change with xanthine plus xanthine oxidase treatment in the presence of SOD or SOD plus catalase. The serum CK was significantly lower in the group of dogs treated with SOD or SOD plus catalase. PO* and pH. The changes in the PO, and pH with various interventions are summarized in Fig. 4. The
In the present study we used xanthine plus xanthine oxidase for generation of oxygen free radicals. Xanthine plus xanthine oxidase have been used by various investigators to generate oxygen free radica1s.‘“~2sO;, however, has also been generated by purine and xanthine oxidase.28 Oxygen free radicals generated by xanthine plus xanthine oxidase produced a decrease in the cardiac function and indices of myocardial contractility. This decrease in the myocardial contractility might be due to the depressant effect of oxygen free radicals on the excitation contraction coupling mechanism in the cardiac muscle. As such, it has been reported by Hess et al.‘O that oxygen free radicals generated by xanthine plus xanthine oxidase depressed the Ca++ accumulation by sarcoplasmic reticulum (SR) and Ca++ ATPase (adenosine triphosphatase) of SR. These effects of oxygen free radicals on SR would produce a decrease in the cardiac contractility and rate of relaxation of cardiac muscle. Our results showed that there was a decrease in the index of cardiac contractility [(-t) dp/dt, (dp/dt)/IIP, (dp/dt)/PAW] and rate of cardiac relaxation [(-) dp/dt]. The other mechanism for a decrease in the myocardial contractility might be the cell damage produced by oxygen free radicals. Xanthine plus xanthine oxidase has been shown to produce O;.2*g, 2g 0; is catalyzed to H,O, by SOD present in the
Volume 117 Number
Oxygen free radicals and cardiovascular function
6
l
- X-X0
o-
x-x0
.-
x-x0
0
+ SOD + SOD
30 TIME
+ CATALASE
60 (min)
m
- X-XO+SOD+
l-l
-
x-x0
m
-
X-X0
1201
CATALASE + SOD
-I 0 min
60
min
Fig. 4. Effects of xanthine plus xanthine oxidase (X + X0) in the absence or in the presence of superoxide dismutase (SOD) or SOD plus catalase on the blood lactate, serum creatine kinase (CK), PO,, and nH. The results are exnressed as mean rt SE. tr, < 0.05, X + X0 versus X + X0 in the absence and in the presence of SOD or SbD plus catalase. -
biologic system. H202 is then catalytically reduced to H,O by catalase or glutathione peroxidase. The major danger of H,O, accumulation is the production of . OH by Haber-Weiss reactio+ or the Fenton reaction.31 *OH is more toxic than 0; and H,0,.2,g These radicals produce lipid peroxidation of cell membrane, resulting in the loss of membrane integrity.2,gs1g This loss of cell integrity and cell damage would then affect the contractility. The fall in the blood pressure observed with oxygen free radicals might be due to a decrease in the CI. The TSVR and PVR increased with the administration of xanthine plus xanthine oxidase. It appears that oxygen free radicals depress the myocardial contractility and increase the tone in the peripheral vasculature. Decrease in the CI appears to be due mainly to a decrease in the cardiac contractility and to an increase in TSVR. The hemodynamic parameters were better preserved in SOD or SOD plus catalase treated dogs than in the dogs without these treatments. However, the protective effect of SOD or SOD plus catalase against the ca:rdiac depressant effect of oxygen free radicals was not complete. The protective effect of SOD plus catalase is probably due to their oxygen free radical sc:avenging property.2~5~g~32The protective effect of SOD and catalase against the injurious effect of oxygen free radicals on the myocardium is well established.5* 33,34Although the protective effect of SOD plus catalase was slightly better than that of SOD alone, the results were not significantly differ-
ent from each other. The decrease in the cardiac contractility and cardiac function might be due to 0, and aOH. There was a tendency for an increase in the blood lactate, but this increase was not significant. The increase in the blood lactate might be due to a decrease in the capillary perfusion. This decrease in the capillary perfusion might be due to an increase in the TSVR and PVR that were evident in this study. The increase in blood lactate might also be due to a decrease in the capillary perfusion as a result of vascular damage produced by oxygen free radicals. As such, exogenous oxygen free radicals have been reported to produce structural alterations in the vascular endothelium including vacuoles, blebs, and edema.2* It is surprising that SOD and catalase were unable to affect this slight but not significant increase in the oxygen free radicalsinduced blood lactate. The serum CK increased markedly in the dogs with exogenous oxygen free radicals. This increase in serum CK suggests the tissue damage. As has been discussed earlier, oxygen free radicals are known to damage the biologic tissue through lipid peroxidation. The damage would produce release of CK in the blood through various tissues. The CK level in the blood did not rise in the groups of dogs treated with SOD or SOD plus catalase. This is to be expected, because SOD and cat&se are scavengers of oxygen free radicals. There was no change in the arterial PO, and pH of the arterial blood.
1202
Prasad
et al.
These results show that oxygen free radicals depressed the myocardial contractility and cardiac function and increased the serum CK. SOD and catalase, the scavengers of oxygen free radicals, attenuated the effects of oxygen free radicals on the hemodynamic parameters and biochemical changes. The cardiac depression with oxygen free radicals might be due to the myocardial cell damage and/or its depressing effect on the Ca++ uptake by SR and Cat+ ATPase of SR. These results suggest that oxygen free radicals have a cardiac depressant effect and this effect can be prevented by the administration of scavengers of oxygen free radicals. This work forms a part of the thesis for the MSc degree of Mr. W.P. Chan. The authors acknowledge the technical assistance of Mr. P.K. Chattopadhyay and Mr. Dennis Duncan. REFERENCES
1. Del Maestro RF, Thaw HH, Bjiirk J, Planker M, Arfors KE. Free radicals as mediators of tissue injury. Acta Physiol &and 1980;492(Suppl):43-57. 2. Del Maestro RF. An approach to free radicals in medicine and biology. Acta Physiol Stand 1980;492(Suppl):153-68. 3. Rowlev D. Gutteridse JMC. Blake D. Farr M. Ha&well B. Lipid “peroxidation In rheumatoid arthritis: thiobarbituric acid-reactive material and catalytic iron salts in synovial fluid from rheumatoid patients. Clin Sci 1984;66:691-5. 4. Petkau A, Chelack WS, Plekash SD. Protection by superoxide dismutase of white blood cells in x-irradiated mice. Life Sci 1978;22:867-82. 5. Jolly SR, Kane WJ, Bailie MB, Abrams GD, Lucchesi BR. Canine myocardial reperfusion injury. Its reduction by the combined administration of superoxide dismutase and catalase. Circ Res 1984;54:277-85. 6. McKechnie K, Furman BL, Parratt JR. Modification by oxygen free radical scavengers of the metabolic and cardiovascular effects of endotoxin infusion in conscious rats. Circ Shock 1986;19:429-39. I. Perkowski SZ, Havill AN, Flynn JT, Gee MH. Role of intra-pulmonary release of eicosanoids and superoxide anion as mediators of pulmonary dysfunction of endothelial injury in sheep with intermittent complement activation. Circ Res 1983;53:574-83. 8. Hammond B, Kontos HA, Hess ML. Oxygen radicals in the adult respiratory distress syndrome in myocardial ischaemia and reperfusion injury, and in cerebral vascular damage. Can J Phvsiol Pharmacol 1985;63:173-87. 9. Fridovich I. The biology ‘of oxygen radicals. Science 1978; 201:875-80. 10. Hess ML, Okabe E, Kontos HA. Proton and free oxygen radical interaction with the calcium transport system of cardiac sarcoplasmic reticulum. J Mol Cell Cardiol 1981; 13:767-72. 11. Fantone JC, Ward PA. Role of oxygen-derived free radicals and metabolites in leukocyte dependent inflammatory reactions. Am J Path01 1982;107:397-418. 12. Graham DG, Tiffany SM, Bell WR, Gutknecht WF. Autooxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine and related compounds toward CI300 neuroblastoma cells in vitro. Mol Pharmacol 1978;14:644-53. 13. Dzau VJ, Packer M, Lilly LS, Swartz SL, Hollenberg NK, Williams GH. Prostaglandins in severe congestive heart failure. Relation to activation of the renin-angiotensin system and hyponatremia. N Engl J Med 1984;310:347-52. 14. Jennings RB, Reimer KA. Lethal myocardial ischemia injury. Am J Path01 1982:102:241-55.
American
June 1989 Heart Journal
15. Chambers DE, Parks D.4, Patterson G, Roy R, McCord JM, Yoshida S, Parmley LF, Downey JM. Xanthine oxidase as a source of free radical damage in myocardial ischemia, ,J Mol Co11 Cardiol 1985;17:145-52. 16. Guarnieri C, Flamigni F, Caldarera CM. Role of oxygen in the cellular damage induced by re-oxygenation of hypoxic heart. J Mol Cell Cardiol 1980;12:797-808. 17. Freeman BA, Crapo JD. Biology of disease. Free radicals and tissue injury. Lab Invest 1982;47:412-26. 18. Meerson FZ, Kagon VE, Kozlov YP, Belkina LM, Arkkhipenko YV. The role of lipid peroxidation in pathogenesis of ischemic damage and the antioxidant protection of the heart. Basic Res Cardiol 1982;77:465-85. 19. Halliwell B, Gutteridge MC. Oxygen radicals and tissue damage. In Caldarera CM, Harris P, eds. Advances in studies on heart metabolism. Bologna: Cooperativa Libraria Universitavia Editrice Bologna 1982:403-11. 20. Siegel JH, Sonnenblick EH, Judge RD, Wilson WS. The quantification of myocardial contractility in dog and man. Cardiologia 1964;45:189-221. 21. Yang SS, Bentivoglio LG. Maranhao V, Goldberg H. From cardiac catheterization data to hemodynamic parameters. Philadelphia: FA Davis Company, 1972;157. 22. Prasad K, Khatter JC, Bharadwaj B. Intra- and extracellular electrolytes and sarcolemmal ATPase in the failing heart due to pressure overload in dogs. Cardiovasc Res 1979:13:95104. 23. Prasad K, Khatter JC, Bharadwaj B. Cardiovascular function in experimental mitral insufficiency in dogs. Jpn Heart J 1977;18:823-40. 24. Prasad K, O’Neil CL, Bharadwaj B. Effects of chronic digoxin treatment on cardiac function, electrolytes, and sarcolemmal ATPase in the canine failing heart due to chronic mitral regurgitation. Am J Cardiol 1984;108:1487-94. 25. Ettinger JS: Suter PF. Canine cardiology. Philadelphia: WB Saunders Company, 1970:209-11. 26. Westgard JO, Lahmever BL, Birnbaum ML. Use of the Du Pont “Automatic Clinical Analyzer” in direct determination of lactic acid in plasma stabilized with sodium fluoride. Clin Chem 1972;18:1334-8. 27. Prasad K, Kim RD, Debnath D. Effect of hydrocortisone on the hemodynamics and serum CK and MBCK enzymes in acute myocardial infarct in dogs. Can J Cardiol 1986;2:3441. 28. Burton KP, McCord JM, Ghai G. Myocardial alterations due to free radical generation. Am J Physiol 1984;246:H776-83. 29. Roy RS, McCord JM. Superoxide and ischemia: conversion of xanthine dehydrogenase to xanthine oxidase. In: Greenwald RA, Cohen G, eds. Oxy radicals and their scavenger systems. Cellular and medical asnects. vol. II. New York: Elsevier Science, 1983:145. I 30. McCord JM, Day ED. Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett 1978;86:139-42. 31 Fee JA, Valentine JS. Chemical and physical properties of superoxide. In: Michelson AM, McCord JM, Fridovich I, eds. Superoxide and superoxide dismutases. New York: Academic Press, 1976:239-77. 32. Werns SW, Shea MJ, Driscoll EM, Cohen C, Abrams GD, Pitts B, Lucchesi BR. The independent effects of oxygen radical scavengers on canine infarct size. Reduction by superoxide dismutase but not catalase. Circ Res 1985;56:895-8. 33. Shlafer M, Kane PF, Kirsh MM. Superoxide dismutase plus catalase enhance the efficacy of hypothermic cardioplegia to protect the globally ischemic, reperfused heart. J Thorac Cardiovasc Surg 1982;83:830-9. 34. Myers CL, Weiss SJ, Kirsh MM, Shepard BM, Shlafer M. Effect of supplementing hypothermic crystalloid cardioplegia solution with catalase, superoxide dismutase, allopurinol, or deferoxamine on functional recovery of globally ischemic and reperfused isolated hearts. ,J Thorac Cardiovasc Surg 1986:91:281-9X.
K. Prasad, MD, PhD, J. Kalra, MD, PhD, W. P. Chan, BSc, and A. K. Chaudhary, MPharm, PhD. Saskatoon, Saskatchewan, Canada Oxygen free radicals have been implicated as inducers of tissue injury in several pathologic conditions such as inflammation, irradiation, ischemia/reperfusion, circulatory shock injury, and respiratory disfree radicals tress syndrome.l-s Oxygen-derived include superoxide anion (O;), hydrogen peroxide (H,O,), and the hydroxyl radical (-OH). These radicals are generated by a number of cellular reactions.2,g 0; is catalyzed to H202 by superoxide dismutase (SOD) present in the biological system. H,O, is then catalytically reduced in the cell to H,O by catalase or glutathione peroxidase. There are various sources of oxygen free radicals including xanthine-xanthine oxidase,lO polymorphonuclear leukocytes,” auto-oxidation of catecholamines,12 and arachidonic acid metabolism.13 During tissue ischemia, it is not only that the production of xanthine and xanthine oxidase is increased,14z15 but that there
From the Departments of Physiology University of Saskatchewan. This
work
Received
was supported for publication
and Pathology,
by the Saskatchewan Sept. 2, 1988; accepted
Heart
College of Medicine, Foundation.
Jan. 6, 1989.
Reprint requests: K. Praaad, MD, Dept. of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N owo.
1196
is also a loss of both SOD and glutathione peroxidase,16 which would result in an increase in the level of oxygen free radicals. Oxygen free radicals have been suggested to exert their cytotoxic effect by causing peroxidation of membrane phospholipids, which can result in an increase in membrane fluidity, increasing permeability, and loss of membrane integrity.17-lg Also, oxygen free radicals have been reported to affect excitation-contraction coupling.18 The effect of oxygen free radicals on cardiovascular function and cardiac contractility is not clear. It is possible that such radicals might influence these properties. The present investigation therefore deals with the effects of exogenous oxygen free radicals on cardiac function, indices of cardiac contractility, serum creatine kinase, and blood lactate in anesthetized dogs. METHODS The experiments were carried out on mongrel dogs of either sex, weighing between 18 and 25 kg. The dogs were anesthetized with sodium pentobarbital(30 mg/kg) intravenously and intubated with a closed-cuff endotracheal tube. The lungs were ventilated with room air using a Harvard respiratory pump (Harvard Apparatus Co. Inc., S. Natick, Mass.) with a volume of 20 ml/kg and a respiratory rate of 20Jmin. Additional amounts of sodium
Volume 117 Number 6
pentobarbital (5 mg/kg) were administered intravenously when needed. Hemodynamic measurements. The femoral artery and vein were exposed. A 7F gauge Cournand (Cordis GF, Cordis Corp., Miami, Fla.) catheter was positioned at the aortic arch through the femoral artery to record aortic pressure. The same catheter was pushed into the left ventricle to record left ventricular pressure. Another 7F gauge Cournand (Cordis GF) catheter was positioned in the right atrium through the femoral vein to record right atria1 pressure and to collect blood samples for biochemical measurements. A Swan-Ganz triple lumen catheter (7F gauge) equipped with a thermistor tip (Baxter Healthcare Corp., Edwards Division, Santa Ana, Calif.) was passed into the pulmonary artery through the external jugular vein to measure pulmonary arterial pressure, pulmonary capillary wedge pressure, and cardiac output. Cardiac output was measured in triplicate with Edwards cardiac output computer (COM-1, Baxter Healthcare Corp., Scientific Division, McGaw Park, Ill.). Lead II of the electrocardiogram (ECG) was monitored throughout the experiment. The first derivative of left ventricular pressure (dp/dt) was recorded with a differentiating device coupled to left ventricular pressure at a frequency response of 100 Hz. All pressures were recorded with a Gould pressure transducer (Spectramed Inc., Cardiovascular Products Division, Oxnard, Calif.) and a Beckman R611 dynograph recorder (Beckman Instruments Inc., Brea, Calif.). The ratio of (dp/dt)/IIP was used as one index of myocardial contractility because it is not affected by preload or by a small change in ,the heart rate.20p21The dp/dt is not only affected by contractility but also by preload, afterload, and heart rate.21 The integrated isovolumetric pressure (IIP) was calculated by the method described previously by Prasad et a1.251 The other index of myocardial contractility that is not affected by preload and that is used in this experiment was (dp/dt)/PAW, where PAW is the pulmonary arterial wedge pressure. The left ventricular work index (LVWI), total systemic vascular resistance (TSVR), and cardiac index (CI) were calculated by previously described nnethods.23s24 The body surface area of the dogs was determined according to the method of Ettinger and Suter.25 Pulmonary vascular resistance (PVR) was calculated by the formula: PVR = PA&O, where PA,,, = mean pulmonary arterial pressure (in millimeters of mercury) and CO = cardiac output (in liters per minute). Biochemical measurements. Mixed venous blood was collected for the measurements of creatine kinase (CK) and lactate. Arterial blood was collected for measurement of PO, and pH. Blood lactate was measured using the lactic dehydrogenase system on an ACA Du Pont Discrete clinical analyzer (Du Pont Company, Wilmington, De1.)26 The method for measurement of CK was that described by Prasad et a1.27Two milliliters of blood was collected in a test tube containing 0.1 ml ethyleneglycol-bis @-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (20pM solution) and was centrifuged to collect plasma. N-acetylcysteine (1 mmol) was added at 0.1 ml/ml of plasma as a reducing agent. The CK determination was made using a
Oxygen
free radicals
and cardiovascular
function
1197
Fig. 1. A typical tracing of the effect of xanthine plus xanthine oxidase (X + X0) in the absence and in the presence of superoxide dismutase (SOD) or SOD plus catalase on the left ventricular pressure (LIP) and its first derivative (dp/dt). The control tracing before administration of any drug is designated as “0” minutes. The arrow marks the beginning of the bolus administration of xanthine plus xanthine oxidase and the subsequent strips of tracings are shown at intervals at the top of each strip of tracing. SOD or SOD plus catalase were administered continuously throughout the observation period. Top tracing shows the effect of X + X0. Middle tracing shows the effect of X + X0 in the presence of SOD. Bottom tracing shows the effect of X + X0 in the presence of SOD plus catalase. Vertical axis indicates the magnitude of various hemodynamic parameters. Zero point in the LVP and dpldt tracings is the base line. Positive dpldt is the rate of pressure development and the (-) dp/dt is the rate of pressure drop (rate of relaxation) of the ventricle. Note the marked decrease in the LVP and its dpldt with X + X0. Also note the ineffectiveness of X + X0 in decreasing the LVP and its dpldt in the presence of SOD or SOD + catalase.
creatine kinase reagent test kit (SVR CK test kit, Behrmg Diagnostics, Hoechst Canada Inc., Montreal, Quebec, Canada). PO, and pH were measured with Corning 175 automatic pH/blood gas system (Corning Medical, Medfield, Mass.).
1190
June 1999 American Heart Journal
Prasad et al.
_ ” ‘E 0 .
6000
r *
6000
5: ul* I-
LI 05
’ 15 TIME
1 30
l
gl E r
4000
z
2000
I 00
L
1 15
1 30
~~ II 05
(mln)
’
TIME
J 60
(min)
2. Effects of xanthine plus xanthine oxidase (X + X0) in the absence and presence of superoxide dismutase (SOD) or SOD plus catalase on the cardiac index, pulmonary vascular resistance (PVR), mean pulmonary arterial pressure (mPAP), and total systemic vascular resistance (TSR). The results are expressed as mean + SE. *p < 0.05, comparison of the values at different time courses with respect to the values before any treatment (0 minutes) in the respective groups (intragroup).
Fig.
Xanthine plus xanthine oxidase were used to generate oxygen free radicals, These substances have been used by various investigators in the past to generate oxygen free radica1s.10~28 SOD and catalase were used as scavengers of oxygen free radicals. The dogs were divided into three groups. Group I (n = 5) dogs received xanthine (0.14 mg/kg) plus xanthine oxidase (0.14 U/mg protein, Sigma Chemical Company, St. Louis, MO.) in the dose of 2 U/kg intravenously after initial hemodynamic and biochemical measurements. The effects of xanthine plus xanthine oxidase on the various hemodynamic and biochemical parameters were observed for a period of 1 hour. Group II (n = 5) dogs received SOD (3050 U/mg protein, Sigma Chemical Company) 5 minutes prior to the administration of xanthine plus xanthine oxidase in doses similar to dogs in group I. SOD in a dose of 1.4 mg/kg was administered intravenously in the bolus form followed immediately by a continuous infusion in a dose of 5 mg/kg/hr for the duration of study. Group III (n = 5) received SOD plus catalase (11000 U/mg, Sigma Chemical Company) 5 minutes prior to xanthine plus xanthine oxidase. Catalase and SOD each in the dose of 1.4 mg/kg intravenously were administered in a bolus form followed by a continuous infusion at a dose of 5 mg/kg/hr each for the entire duration of the study. The hemodynamic measurements and collections of blood samples for the measurement of lactate, CK, PO,, and pH were made before administration of any drug and after 5, 15, 30, and 60 minutes of xanthine plus xanthine oxidase administration in all three groups. Data analysis. The statistical analysis was made using paired t test for comparison in the same group and unpaired t test for comparison between two groups. Statistical significance was considered as p < 0.05. Protocol
of studies.
RESULTS Hemodynamics. A typical tracing of the effect of xanthine plus xanthine oxidase in the presence and absence of SOD and of SOD plus catalase on the left ventricular pressure and its dp/dt is shown in Fig. 1. Xanthine plus xanthine oxidase produced a marked decrease in the left ventricular pressure (LVP) and its dp/dt within 1 minute, reaching its lowest value within 5 minutes. The LVP and dp/dt then started to increase at about 10 to 15 minutes till the observation period (60 minutes). However, the recovery was not complete at the end of the observation period. The decreases in the LVP and its dp/dt with xanthine plus xanthine oxidase were less marked in the presence of SOD or SOD plus catalase. The changes in the hemodynamic parameters with exogenous oxygen free radicals (xanthine plus xanthine oxidase) in the absence and in the presence of SOD, and SOD plus catalase are summarized in Fig. 2 and 3 and Tables I and II. Xanthine plus xanthine oxidase. Oxygen free radicals produced a marked decrease in the mean arterial pressure (MAP), mean pulmonary arterial pressure (MPAP), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), left ventricular (+) dp/dt and (-) dp/dt, (dp/dt)/IIP, (dp/dt)/PAW, LVWI, CI, heart rate, and cardiac effort within 5 minutes. PVR and systemic TSVR increased with oxygen free radicals. No change was observed in mean right atria1 pressure (MRAP) and PAW. Most of the hemodynamics
Volume 117 Number
Oxygen free radicals and cardiovascular function
6
o-
150
F-;
140
4 ‘a5
130
I-
a 2 =-
2 m z
I
x-x0;
1199
700 600 0. -A 500 -.I 5: E
l - x-x0
+ SOD;
-
n -
x-x0
+ SOD + CATALASE
400
120 110
-
100
-
*
Ik
200
*
90’ -
300
,“’
100
L’
’
I
I
05
15
30
60
TIME
05
15 TIME
(min)
60
30 (min)
3. EBects of xanthine plus xanthine oxidase (X + X0) in the absence and presence of superoxide dismutase (SOD) or SOD plus catalase on the mean right atrial pressure (MRAP), heart rate, (dp/dt)/IIP, and cardiac effort. The results are expressed as mean + SE. *p < 0.05, comparison of the values at various time courses with respect to the values before any drug administration (0 minutes) in the respective groups.
Fig.
I. Changes in hemodynamics with various treatments
Table
Time (min) 0
__ Z 19.2
15 30 60
MAP (mm Hd
LVWZ (kg . MJminlM2)
PAW (mm Z-W
Group
Group
Group
Group
zz
zzz
Z
zz
zzz
Z
zz
zzz
Z
zz
zzz
4.1 +0.6
7.6 + 0.7
10.2 k 1.2
9.7 +0.7
10.5 It 1.1
12.5
25.6
156.0
119.2
151.3
7.5
7.0 ?c 1.2
zi 3.8 9.8
k4.0 10.0
k 11.2 67.5*
k6.8 81.0
k8.0 92.5
+1.6 0.9*
+ 1.9
k 1.8
+ 10.1
+8.1
+ 5.3
kO.3
kO.5
+0.5
AZ1.5
kO.3
+ 0.4
11.7 k4.4
8.8 + 0.7
12.5 +1.8
98.3 k 1.7
98.8 +6.2
2.5* kO.2
2.7 kO.5
3.3 kO.8
10.9 + 0.7
9.8 + 0.8
8.2 +0.6
3.3
2.9 20.3
4.5 r 0.8
9.2 kO.1
10.9 iz 1.5
8.5 kO.7
k9.2 5
L VEDP (mm fig)
96.7* k1.7
11.7
6.7
6.8
110.0
106.7
112.5
* 1.7
* 1.7 10.0
+ 1.2 10.0
+8.7 108.3
+ 1.7 100.0
zk 1.8 80.5
20.5 3.3
k1.4
k2.5
f 13.0
+ 2.9
27.4
f 0.8
8.3 ir0.8
1.7*
2.9 LO.4
2.3
2.2 +0.1
9.0
9.4
6.5
9.1
9.8
9.0
k 1.2
f 1.4
r 1.4
Results are expressetl BSmean+ SE. LVEDP, Left ventricular end-diastolic pressure; MAP, mean aortic pressure; LVWI, left ventricular work index; PAW, pulmonary arterial wedge pressure. Group I, Xanthine + xanthine oxidase; Group II, xanthine + xanthine oxidase in the presence of superoxide dismutase (SOD); Group III, xanthine + xanthine oxidase in the presence of SOD and catalase. *p < 0.05, comparison of the values at different time intervals with respect to the values before any treatment (0 minutes) in the respective groups.
changes tended to return toward control values at the end of the l-hour period of observation. However, the MAP, heart rate, LVEDP, LVWI, CI, cardiac effort, (-) dp/dt, and LVSP were still depressed at the end of the l-hour observation period. Xanthine plus xanthine oxidase and SOD. Left ventricular work index, (+) dp/dt, (-) dp/dt, (dp/ dt)lPAW, and cardiac effort decreased significantly.
TSVR increased significantly. No changes were observed in the PVR, MPAP, LVEDP, MAP, PAW, LVSP, heart rate, and MRAP. Xanthine plus xanthine oxidase in the presence of SOD produced changes in the hemodynamic parameters similar to that of xanthine plus xanthine oxidase. Although these changes with xanthine plus xanthine oxidase in the presence of SOD were less marked than in its
1200
Table
Time (min) 0 5 15 30 60
Prasad
et al.
American
June 1989 Heart Journal
II. Changes in hemodynamics with various treatments 1, VSP (mm Hd
i+)dp/dt (mm Hglsec)
Group
Group
__---.--. ___--
__ I
II
190.0 + 18.0 85.8* 19.1 132.5 k3.8 147.2 224.9 137.5 + 8.0
157.5 k 2.9 111.3 + 10.8 136.7 i4.4 145.0 i 2.9 137.5 t3.8
III 197.5 f 1.8 115.0 27.1 143.8 k 13.3 157.5 k5.3 125.0 27.1
__---
i-ldpldt (mm Hglsec) ---~__ --.-~-
I
II
III
2166.7 i 272.8 1100.0 Il15.5 1866.7 t 120.2 2300.0 + 173.2 2266.7 i 266.7
3133.3t i 176.4 1580.0* + 334.9 2333.3 + 185.6 2800.0 + 200.0 2433.3 + 338.3
2080.0 + 510.6 1600.0 k 141.8 2650.0 -t 390.0 3250.0 t 177.3 2200.0 + 141.8
I 2733.3 + 371.2 800.0* * 115.5 1833.3 k 152.8 1866.7 k 176.4 1533.3 k 352.8
+(dpldt)lPAW (Set- ‘)
___.
Group -____
.____-__
II
III
I
2233.3 k 166.7 930.0* + 155.9 1466.7t 266.7 1733.3 i: 266.7 1266.7 + 176.4
3000.0 ?I 709.2 900.0* * 70.9 1400.0 + 424.5 1750.0 z!I 319.0 1070.0 * 191.5
221.9 rf: 48.7 115.8 k4.3 174.6 k8.7 255.4 -+ 34.8 262.5 t 55.1
Group II 329.3 + 37.9 164.8’ IL 29.6 241.5 k31.6 263.6 -t 35.5 257.7 k41.4
III 192.2 i29.1 250.0 i 35.5 319.7 k25.5 352.9 + 28.5 262.3 + 57.1
Results are expressed as mean k SE. LVSP, Left ventricular systolic pressure; (+) dp/dt, rate of pressure development; (-) dp/dt, rate of relaxation; PAW, pulmonary arterial wedgle pressure. Notations of the groups are same as described in Table I. *p < 0.05, comparison of the values at different time intervals with respect to the values before any treatment (0 minutes) in the respective groups. tb < 0.05, group I versus group II or group III.
absence, they were not significant. Decreases in the heart rate and cardiac effort were greater in the presence of SOD than in its absence.
changes in the PO, and pH with xanthine plus xanthine oxidase in the presence and in the absence of SOD or SOD plus catalase were not significant.
Xanthine plus xanthine oxidase and SOD plus catalase. Xanthine plus xanthine oxidase in the
DISCUSSION
presence of SOD and catalase produced very little change in the measured hemodynamic parameters. Catalase and SOD protected deleterious effects of exogenous oxygen free radicals produced by xanthine plus xanthine oxidase on the hemodynamic parameters. Lactate and creatine kinase. The summary of the results of blood lactate and CK is given in Fig. 4. Blood lactate before the administration of drugs (0 minutes) was 1.33 -t 0.56 mmol/L. Although there was a tendency for an increase in the blood lactate after administration of xanthine plus xanthine oxidase, this increase was not significant. The changes in the blood lactate with xanthine plus xanthine oxidase in the presence of SOD or SOD and catalase were not significantly different from those with xanthine plus xanthine oxidase alone. The value of blood CK before drug treatment was 137 f 60 IU/L. There was a tendency for an increase in the serum CK with xanthine plus xanthine oxidase treatment. However, these changes were not significant. Serum CK did not change with xanthine plus xanthine oxidase treatment in the presence of SOD or SOD plus catalase. The serum CK was significantly lower in the group of dogs treated with SOD or SOD plus catalase. PO* and pH. The changes in the PO, and pH with various interventions are summarized in Fig. 4. The
In the present study we used xanthine plus xanthine oxidase for generation of oxygen free radicals. Xanthine plus xanthine oxidase have been used by various investigators to generate oxygen free radica1s.‘“~2sO;, however, has also been generated by purine and xanthine oxidase.28 Oxygen free radicals generated by xanthine plus xanthine oxidase produced a decrease in the cardiac function and indices of myocardial contractility. This decrease in the myocardial contractility might be due to the depressant effect of oxygen free radicals on the excitation contraction coupling mechanism in the cardiac muscle. As such, it has been reported by Hess et al.‘O that oxygen free radicals generated by xanthine plus xanthine oxidase depressed the Ca++ accumulation by sarcoplasmic reticulum (SR) and Ca++ ATPase (adenosine triphosphatase) of SR. These effects of oxygen free radicals on SR would produce a decrease in the cardiac contractility and rate of relaxation of cardiac muscle. Our results showed that there was a decrease in the index of cardiac contractility [(-t) dp/dt, (dp/dt)/IIP, (dp/dt)/PAW] and rate of cardiac relaxation [(-) dp/dt]. The other mechanism for a decrease in the myocardial contractility might be the cell damage produced by oxygen free radicals. Xanthine plus xanthine oxidase has been shown to produce O;.2*g, 2g 0; is catalyzed to H,O, by SOD present in the
Volume 117 Number
Oxygen free radicals and cardiovascular function
6
l
- X-X0
o-
x-x0
.-
x-x0
0
+ SOD + SOD
30 TIME
+ CATALASE
60 (min)
m
- X-XO+SOD+
l-l
-
x-x0
m
-
X-X0
1201
CATALASE + SOD
-I 0 min
60
min
Fig. 4. Effects of xanthine plus xanthine oxidase (X + X0) in the absence or in the presence of superoxide dismutase (SOD) or SOD plus catalase on the blood lactate, serum creatine kinase (CK), PO,, and nH. The results are exnressed as mean rt SE. tr, < 0.05, X + X0 versus X + X0 in the absence and in the presence of SOD or SbD plus catalase. -
biologic system. H202 is then catalytically reduced to H,O by catalase or glutathione peroxidase. The major danger of H,O, accumulation is the production of . OH by Haber-Weiss reactio+ or the Fenton reaction.31 *OH is more toxic than 0; and H,0,.2,g These radicals produce lipid peroxidation of cell membrane, resulting in the loss of membrane integrity.2,gs1g This loss of cell integrity and cell damage would then affect the contractility. The fall in the blood pressure observed with oxygen free radicals might be due to a decrease in the CI. The TSVR and PVR increased with the administration of xanthine plus xanthine oxidase. It appears that oxygen free radicals depress the myocardial contractility and increase the tone in the peripheral vasculature. Decrease in the CI appears to be due mainly to a decrease in the cardiac contractility and to an increase in TSVR. The hemodynamic parameters were better preserved in SOD or SOD plus catalase treated dogs than in the dogs without these treatments. However, the protective effect of SOD or SOD plus catalase against the ca:rdiac depressant effect of oxygen free radicals was not complete. The protective effect of SOD plus catalase is probably due to their oxygen free radical sc:avenging property.2~5~g~32The protective effect of SOD and catalase against the injurious effect of oxygen free radicals on the myocardium is well established.5* 33,34Although the protective effect of SOD plus catalase was slightly better than that of SOD alone, the results were not significantly differ-
ent from each other. The decrease in the cardiac contractility and cardiac function might be due to 0, and aOH. There was a tendency for an increase in the blood lactate, but this increase was not significant. The increase in the blood lactate might be due to a decrease in the capillary perfusion. This decrease in the capillary perfusion might be due to an increase in the TSVR and PVR that were evident in this study. The increase in blood lactate might also be due to a decrease in the capillary perfusion as a result of vascular damage produced by oxygen free radicals. As such, exogenous oxygen free radicals have been reported to produce structural alterations in the vascular endothelium including vacuoles, blebs, and edema.2* It is surprising that SOD and catalase were unable to affect this slight but not significant increase in the oxygen free radicalsinduced blood lactate. The serum CK increased markedly in the dogs with exogenous oxygen free radicals. This increase in serum CK suggests the tissue damage. As has been discussed earlier, oxygen free radicals are known to damage the biologic tissue through lipid peroxidation. The damage would produce release of CK in the blood through various tissues. The CK level in the blood did not rise in the groups of dogs treated with SOD or SOD plus catalase. This is to be expected, because SOD and cat&se are scavengers of oxygen free radicals. There was no change in the arterial PO, and pH of the arterial blood.
1202
Prasad
et al.
These results show that oxygen free radicals depressed the myocardial contractility and cardiac function and increased the serum CK. SOD and catalase, the scavengers of oxygen free radicals, attenuated the effects of oxygen free radicals on the hemodynamic parameters and biochemical changes. The cardiac depression with oxygen free radicals might be due to the myocardial cell damage and/or its depressing effect on the Ca++ uptake by SR and Cat+ ATPase of SR. These results suggest that oxygen free radicals have a cardiac depressant effect and this effect can be prevented by the administration of scavengers of oxygen free radicals. This work forms a part of the thesis for the MSc degree of Mr. W.P. Chan. The authors acknowledge the technical assistance of Mr. P.K. Chattopadhyay and Mr. Dennis Duncan. REFERENCES
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