Beneficial Effects of Methionine on Myocardial Hemodynamic and Cellular Functions in Hemorrhagic Shock Rakesh Kapoor, M. Pharm*
+ Jawahar Kalra, M.D., Ph.D., F.A.C.A., F.I.C.A. and Kailash Prasad, M.D., Ph.D., F.A.C.A., F.I.C.A.* SASKATOON, CANADA
Abstract
Hypochlorous acid (HOCI), produced by activated polymorphonuclear (PMN) leukocytes, has been reported to depress cardiac function and contractility. Various mechanisms exist for activation of PMN leukocytes during hemorrhagic shock and reperfusion. In order to determine the role of HOCI in hemorrhagic shock and reinfusion, the authors studied the effects of shock and reinfusion on the cardiac function, contractility, blood lactate, blood gases, and creatine kinase (CK) and MB fraction of CK (MBCK) with and without methionine (quencher of HOCI) in anesthetized dogs. Dogs were divided into two groups: Group I, hemorrhagic shock (two hours) and reinfusion (two hours): Group II, hemorrhagic shock and reinfusion with methionine treatment. Cardiac index, mean arterial pressure, and index of cardiac contractility were similar in the two groups during shock. Postinfusion recovery of cardiac function and contractility was better in group II. Increases in blood lactate were similar in the two groups during shock. The rate of return of blood lactate to preshock values after reinfusion was greater in group II. The increases in serum CK and MBCK of the two groups during shock were similar but not significant. Following reinfusion the levels of these enzymes increased significantly, but the increases in group II were less. These results suggest that HOCI produced by activated PMN leukocytes may play a role in cardiac damage during hemorrhagic shock and reinfusion. Methionine may have beneficial effects in the hemodynamic and metabolic recovery and may reduce cellular damage during hemorrhage shock and reinfusion.
Departments of *Physiology and Pathology, College of Medicine and Royal University Hospital, University of Saskatch+ Saskatoon, Saskatchewan, Canada This work was supported by a grant from Heart and Stroke Foundation of Saskatchewan, Canada, and forms a part of the thesis for Ph.D. degree of Mr. Rakesh Kapoor. Presented at the 32nd Annual Meeting of the International College of Angiology, Toronto, Canada, June, 1990 From the
ewan,
294
295 Introduction various possible mechanisms may lead to an increase in the levels of oxygen free radicals (OFR). Ischemia increases the level of xanthine and xanthine oxidase activity. 1,2 Increased levels of xanthine and xanthine oxidase activity in the presence of tissue oxygen favor the production of OFR [superoxide anion (02), hydrogen peroxide (H202) and hydroxyl radical (·OH) ].3 During hemorrhage there is an increase in the blood levels of catecholamines,4 angiotensin 11,5 and prostaglandins6 catecholamines through autoxidation would produce OFR.’ Angiotensin has been shown to directly stimulate the synthesis and release of prostaglandins.’ During a prostaglandin synthesis there is production of OFR.9 Leukotrienes have been implicated as a mediator of hemorrhagic shock.’° Leukocytes metabolize arachidonic acid, giving rise to leukotrienes, including leukotriene B4 (LTB4) . &dquo; During ischemia and reperfusion, there is an increase in the release of platelet-activating factor (PAF).’2 Hydrogen peroxide has been shown to release PAF from endothelial15 cells.’3 Polymorphonuclear (PMN) leukocytes are known to be activated by LTB4 14 and PAF to produce OFR. Hydrogen peroxide reacts with leukocyte myelopoxidase to form an enzyme-substrate complex that oxidizes various halides, in particular Cl-, to highly reactive toxic products such as hypochlorous acid (HOCI).&dquo; Highly reactive oxygen metabolites act on unsaturated fatty acids to produce lipid peroxides that increase membrane fluidity and permeability with loss of membrane integrity.&dquo; OFR generated by xanthine and xanthine oxidase and by activated PMN leukocytes have been shown to depress the cardiac function and contractility. &dquo;’19 Also a quencher of hypochlorous acid 20 has been shown to have a protective effect against activated-PMN-leukocyte-induced depression in cardiac function and contractility.’9 It is possible that during hemorrhagic shock there is an increase in the level of HOCI in addition to an increase in OFR. This increase in HOCI might have deleterious effects on all organ systems in the body, including the cardiovascular system. The cellular damage would release creatine kinase (CK) and MB fraction of CK (MBCK). It was proposed, therefore, to measure the cardiac function and contractility, blood lactate, CK, and MBCK during hemorrhagic shock and reperfusion. We also studied the effects of HOCI quencher (methionine) during hemorrhagic shock and reperfusion to determine whether HOCI is involved.
During hemorrhagic shock,
Materials and Methods Adult mongrel dogs of either sex weighing between 15 and 25 kg were anesthetized with pentobarbital sodium (30 mg/kg) intravenously and intubated with a closed cuff endotrachial tube. The lungs were ventilated with room air by means of a respiratory pump with a volume of 20 mL/kg and a respiratory rate of 20/min. Additional doses (5 mg/kg) of pentobarbital sodium were given intravenously as and when needed to maintain anesthesia.
296
Hemodynamics Hemodynamic measurements were made by the previously described method.’9 A 7 french (F) gauge catheter was positioned in the left ventricle to record left ventricular pressure. The first derivative of the left ventricular pressure (dp/dt) was recorded with a differentiating device coupled to left ventricular pressure at a frequency response of 100 Hz. A 7F cournand catheter was positioned at the aortic arch through the carotid artery to record the aortic pressure. The right femoral vein was used to pass a 7F triplelumen catheter equipped with thermistor tip into the pulmonary artery to measure cardiac output by the thermodilution method.2’ A catheter was placed in the right atrium through the external jugular vein to measure right atrial pressure and to collect blood samples for biochemical measurement. The right femoral artery was used to bleed the animal. The right femoral vein was used to transfuse the shed blood. Lead II ECG was monitored throughout the experiment. All pressure recordings were made by use of pressure transducers on a recorder. Cardiac output was measured in triplicate with a cardiac output computer. Cardiac index (CI) was calculated as previously described.19,22 The ratio of dp/dt at common peak isovolumic pressure (CPIP) to pulmonary arterial wedge pressure (PAW) was used as one index of myocardial contractility because it is not affected by preload or afterload.z3 In this study PAW pressure has been used instead of left ventricular end-diastolic pressure as a representative of preload. In addition, the V max was used as another index of myocardial contractility. Cardiac effort (pressure and heart rate product), an index of myocardial oxygen consumption, was calculated as previously described.’9 Blood Gases and Lactate Lactate levels in mixed venous blood were measured by means of a lactic dehydrogenase system on an analyzer. 24 Partial pressure of oxygen (P02), carbon dioxide pressure (PC02), bicarbonate (HC03), and pH of arterial blood were measured with an automatic pH/blood gas
system. CK and MBCK Measurements The method of measurement of CK and MBCK was that described earlier by Prasad and Bharadwaj25 and Prasad et a1.26 Creatine kinase-isoenzymes were separated by use of the column chromatography method of Mercer . 27 The CK and MBCK measurements were made by means of a creatine kinase reagent test kit. Protocol of Studies The dogs were divided into two groups. Dogs in group I (n 7) were subjected to hemorrhagic shock and reperfusion. The dogs were bled through the arterial line into standard blood bank bags containing 63 mL of Citrate phosphate dextrose adenine (CPDA) solution per 450 mL of blood over a period of five to ten minutes to bring the mean arterial pressure to 50 ± 5 mm Hg. The collected blood was maintained at ambient temperature until it was reperfused. The mean arterial pressure (MAP) of 50 ± 5 mm Hg was maintained for two hours by withdrawal or reinfusion of blood. At the end of two hours of shock period, the shed blood was transfused through the right femoral vein over a period =
297 of twenty to thirty minutes and the dogs were observed for a further period of two hours, at the end of which they were sacrificed. Group II (n 6) were subjected to hemorrhagic shock and reperfusuion similar to group I. However, the dogs in this group received methionine 30 mg/kg IV before bleeding, before reinfusion of shed blood, and after one hour of reinfusion. An equivalent volume of normal saline was given intravenously in group I. The hemodynamic measurements and collection of mixed venous blood for lactate and CK and MBCK and of arterial blood for blood gases were made before hemorrhage, at minutes 30, 60, and 120 of induction of shock, and at minutes 15, 30, 60, 90, and 120 after completion of reinfusion of shed blood. The words reinfusion and reperfusion are used interchangeably. =
-
Statistical Analysis The data were analyzed by two-way analysis of variance using repeated measure followed by least significant difference (LSD) test28 for comparison within and between the groups. The difference was considered significant if P was less than 0.05. Results
Hemodynamics The changes in the hemodynamic parameters with hemorrhagic shock and reinfusion in the presence and absence of methionine are summarized in Figures 1 and 2 and Table I. The mAP declined to 50 ± 5 mm Hg within five to ten minutes and was maintained at that level for two hours in both the shock with and shock without methionine treat-
(Fig. 1). This fall in blood pressure amounted to an approximate decline of 55.9~10 and 67.4°~0 of the preshock values in group I and group II respectively. Cardiac index during shock decreased to approximately 25.6070 and 18.6070 of preshock values in group I and group II respectively. Index of myocardial contractility (dp/dt at CPIP:PAW) declined to the same level in both the groups during the early part of shock (Fig. 2). However, the decline was less, although not significantly, in the methionine-treated group. In both groups the Vmax, another index of myocardial contractility, did not change significantly during shock. Heart rate increased while the mean right atrial pressure (mRAP) and cardiac effort (CE) decreased during shock in both the groups. Reinfusion of shed blood improved the hemodynamic parameters. The mean arterial pressure (mAP) tended to return to preshock values in both groups; however, it never achieved preshock level in the untreated group. After an initial rise, it started to fall gradually and it reached significantly lower than preshock values by minute 120 postreinfusion. Post-reinfusion mAP was higher in the methionine-treated group. Postreinfusion recovery of CI was significantly higher in the methionine-treated group. CI returned to preshock values in the treated group but fell below the preshock value by minute 120 postreinfusion. In the untreated group, CI never reached the preshock level after reinfusion. The index of myocardial contractility (dp/dt at CPIP:PAW) recovered completely after reinfusion of shed blood in the treated group but decreased again at the minute 120 period of postreinfusion. The recovery of this index of myocardial contractility to the preshock level in the untreated group was transient and gradually declined significantly thereafter.
ment
298
FIG. 1. Mean arterial pressure
(mAP) and cardiac index (CI) during shock and reperfusion without and with methionine (Meth) treatment. Each point represents the mean t SE. In the mAP tracing the preshock values are indicated at the five-to tenminute level. The changes in the mAP at zero minute indicate the pressure after hemorrhage to shock level. Cardiac index and other parameters were measured at thirty minutes after initiation of shock. *P < 0.05, comparison of the value at different time courses with respect to preshock values in the respective group. tip < 0.05, intergroup
comparison.
did not change significantly from the preshock values in either group and remained in preshock levels. However, by minute 120 of postreinfusion V max had decreased significantly from the preshock values in the untreated group. There was a decrease in the heart rate and an increase in the CE after reinfusion of shed blood in both groups. The mRAP after reinfusion of shed blood increased, however, only in the methionine-treated group. The postreinfusion values of mRAP was higher in the methionine-treated group. These results indicate that the hemodynamic parameters were similar in the two groups during the shock phase. The postinfusion recovery of cardiac function and contractility was, however, significantly better in the group with methionine treatment.
Vmax
Blood Lactate and Gases Blood lactate levels in the two groups before and during hemorrhagic shock and
postin-
299
FIG. 2. Changes in Vna, and at CPIP/PAW in the two groups. Points on the tracings represent mean ± SE. *P
+ Jawahar Kalra, M.D., Ph.D., F.A.C.A., F.I.C.A. and Kailash Prasad, M.D., Ph.D., F.A.C.A., F.I.C.A.* SASKATOON, CANADA
Abstract
Hypochlorous acid (HOCI), produced by activated polymorphonuclear (PMN) leukocytes, has been reported to depress cardiac function and contractility. Various mechanisms exist for activation of PMN leukocytes during hemorrhagic shock and reperfusion. In order to determine the role of HOCI in hemorrhagic shock and reinfusion, the authors studied the effects of shock and reinfusion on the cardiac function, contractility, blood lactate, blood gases, and creatine kinase (CK) and MB fraction of CK (MBCK) with and without methionine (quencher of HOCI) in anesthetized dogs. Dogs were divided into two groups: Group I, hemorrhagic shock (two hours) and reinfusion (two hours): Group II, hemorrhagic shock and reinfusion with methionine treatment. Cardiac index, mean arterial pressure, and index of cardiac contractility were similar in the two groups during shock. Postinfusion recovery of cardiac function and contractility was better in group II. Increases in blood lactate were similar in the two groups during shock. The rate of return of blood lactate to preshock values after reinfusion was greater in group II. The increases in serum CK and MBCK of the two groups during shock were similar but not significant. Following reinfusion the levels of these enzymes increased significantly, but the increases in group II were less. These results suggest that HOCI produced by activated PMN leukocytes may play a role in cardiac damage during hemorrhagic shock and reinfusion. Methionine may have beneficial effects in the hemodynamic and metabolic recovery and may reduce cellular damage during hemorrhage shock and reinfusion.
Departments of *Physiology and Pathology, College of Medicine and Royal University Hospital, University of Saskatch+ Saskatoon, Saskatchewan, Canada This work was supported by a grant from Heart and Stroke Foundation of Saskatchewan, Canada, and forms a part of the thesis for Ph.D. degree of Mr. Rakesh Kapoor. Presented at the 32nd Annual Meeting of the International College of Angiology, Toronto, Canada, June, 1990 From the
ewan,
294
295 Introduction various possible mechanisms may lead to an increase in the levels of oxygen free radicals (OFR). Ischemia increases the level of xanthine and xanthine oxidase activity. 1,2 Increased levels of xanthine and xanthine oxidase activity in the presence of tissue oxygen favor the production of OFR [superoxide anion (02), hydrogen peroxide (H202) and hydroxyl radical (·OH) ].3 During hemorrhage there is an increase in the blood levels of catecholamines,4 angiotensin 11,5 and prostaglandins6 catecholamines through autoxidation would produce OFR.’ Angiotensin has been shown to directly stimulate the synthesis and release of prostaglandins.’ During a prostaglandin synthesis there is production of OFR.9 Leukotrienes have been implicated as a mediator of hemorrhagic shock.’° Leukocytes metabolize arachidonic acid, giving rise to leukotrienes, including leukotriene B4 (LTB4) . &dquo; During ischemia and reperfusion, there is an increase in the release of platelet-activating factor (PAF).’2 Hydrogen peroxide has been shown to release PAF from endothelial15 cells.’3 Polymorphonuclear (PMN) leukocytes are known to be activated by LTB4 14 and PAF to produce OFR. Hydrogen peroxide reacts with leukocyte myelopoxidase to form an enzyme-substrate complex that oxidizes various halides, in particular Cl-, to highly reactive toxic products such as hypochlorous acid (HOCI).&dquo; Highly reactive oxygen metabolites act on unsaturated fatty acids to produce lipid peroxides that increase membrane fluidity and permeability with loss of membrane integrity.&dquo; OFR generated by xanthine and xanthine oxidase and by activated PMN leukocytes have been shown to depress the cardiac function and contractility. &dquo;’19 Also a quencher of hypochlorous acid 20 has been shown to have a protective effect against activated-PMN-leukocyte-induced depression in cardiac function and contractility.’9 It is possible that during hemorrhagic shock there is an increase in the level of HOCI in addition to an increase in OFR. This increase in HOCI might have deleterious effects on all organ systems in the body, including the cardiovascular system. The cellular damage would release creatine kinase (CK) and MB fraction of CK (MBCK). It was proposed, therefore, to measure the cardiac function and contractility, blood lactate, CK, and MBCK during hemorrhagic shock and reperfusion. We also studied the effects of HOCI quencher (methionine) during hemorrhagic shock and reperfusion to determine whether HOCI is involved.
During hemorrhagic shock,
Materials and Methods Adult mongrel dogs of either sex weighing between 15 and 25 kg were anesthetized with pentobarbital sodium (30 mg/kg) intravenously and intubated with a closed cuff endotrachial tube. The lungs were ventilated with room air by means of a respiratory pump with a volume of 20 mL/kg and a respiratory rate of 20/min. Additional doses (5 mg/kg) of pentobarbital sodium were given intravenously as and when needed to maintain anesthesia.
296
Hemodynamics Hemodynamic measurements were made by the previously described method.’9 A 7 french (F) gauge catheter was positioned in the left ventricle to record left ventricular pressure. The first derivative of the left ventricular pressure (dp/dt) was recorded with a differentiating device coupled to left ventricular pressure at a frequency response of 100 Hz. A 7F cournand catheter was positioned at the aortic arch through the carotid artery to record the aortic pressure. The right femoral vein was used to pass a 7F triplelumen catheter equipped with thermistor tip into the pulmonary artery to measure cardiac output by the thermodilution method.2’ A catheter was placed in the right atrium through the external jugular vein to measure right atrial pressure and to collect blood samples for biochemical measurement. The right femoral artery was used to bleed the animal. The right femoral vein was used to transfuse the shed blood. Lead II ECG was monitored throughout the experiment. All pressure recordings were made by use of pressure transducers on a recorder. Cardiac output was measured in triplicate with a cardiac output computer. Cardiac index (CI) was calculated as previously described.19,22 The ratio of dp/dt at common peak isovolumic pressure (CPIP) to pulmonary arterial wedge pressure (PAW) was used as one index of myocardial contractility because it is not affected by preload or afterload.z3 In this study PAW pressure has been used instead of left ventricular end-diastolic pressure as a representative of preload. In addition, the V max was used as another index of myocardial contractility. Cardiac effort (pressure and heart rate product), an index of myocardial oxygen consumption, was calculated as previously described.’9 Blood Gases and Lactate Lactate levels in mixed venous blood were measured by means of a lactic dehydrogenase system on an analyzer. 24 Partial pressure of oxygen (P02), carbon dioxide pressure (PC02), bicarbonate (HC03), and pH of arterial blood were measured with an automatic pH/blood gas
system. CK and MBCK Measurements The method of measurement of CK and MBCK was that described earlier by Prasad and Bharadwaj25 and Prasad et a1.26 Creatine kinase-isoenzymes were separated by use of the column chromatography method of Mercer . 27 The CK and MBCK measurements were made by means of a creatine kinase reagent test kit. Protocol of Studies The dogs were divided into two groups. Dogs in group I (n 7) were subjected to hemorrhagic shock and reperfusion. The dogs were bled through the arterial line into standard blood bank bags containing 63 mL of Citrate phosphate dextrose adenine (CPDA) solution per 450 mL of blood over a period of five to ten minutes to bring the mean arterial pressure to 50 ± 5 mm Hg. The collected blood was maintained at ambient temperature until it was reperfused. The mean arterial pressure (MAP) of 50 ± 5 mm Hg was maintained for two hours by withdrawal or reinfusion of blood. At the end of two hours of shock period, the shed blood was transfused through the right femoral vein over a period =
297 of twenty to thirty minutes and the dogs were observed for a further period of two hours, at the end of which they were sacrificed. Group II (n 6) were subjected to hemorrhagic shock and reperfusuion similar to group I. However, the dogs in this group received methionine 30 mg/kg IV before bleeding, before reinfusion of shed blood, and after one hour of reinfusion. An equivalent volume of normal saline was given intravenously in group I. The hemodynamic measurements and collection of mixed venous blood for lactate and CK and MBCK and of arterial blood for blood gases were made before hemorrhage, at minutes 30, 60, and 120 of induction of shock, and at minutes 15, 30, 60, 90, and 120 after completion of reinfusion of shed blood. The words reinfusion and reperfusion are used interchangeably. =
-
Statistical Analysis The data were analyzed by two-way analysis of variance using repeated measure followed by least significant difference (LSD) test28 for comparison within and between the groups. The difference was considered significant if P was less than 0.05. Results
Hemodynamics The changes in the hemodynamic parameters with hemorrhagic shock and reinfusion in the presence and absence of methionine are summarized in Figures 1 and 2 and Table I. The mAP declined to 50 ± 5 mm Hg within five to ten minutes and was maintained at that level for two hours in both the shock with and shock without methionine treat-
(Fig. 1). This fall in blood pressure amounted to an approximate decline of 55.9~10 and 67.4°~0 of the preshock values in group I and group II respectively. Cardiac index during shock decreased to approximately 25.6070 and 18.6070 of preshock values in group I and group II respectively. Index of myocardial contractility (dp/dt at CPIP:PAW) declined to the same level in both the groups during the early part of shock (Fig. 2). However, the decline was less, although not significantly, in the methionine-treated group. In both groups the Vmax, another index of myocardial contractility, did not change significantly during shock. Heart rate increased while the mean right atrial pressure (mRAP) and cardiac effort (CE) decreased during shock in both the groups. Reinfusion of shed blood improved the hemodynamic parameters. The mean arterial pressure (mAP) tended to return to preshock values in both groups; however, it never achieved preshock level in the untreated group. After an initial rise, it started to fall gradually and it reached significantly lower than preshock values by minute 120 postreinfusion. Post-reinfusion mAP was higher in the methionine-treated group. Postreinfusion recovery of CI was significantly higher in the methionine-treated group. CI returned to preshock values in the treated group but fell below the preshock value by minute 120 postreinfusion. In the untreated group, CI never reached the preshock level after reinfusion. The index of myocardial contractility (dp/dt at CPIP:PAW) recovered completely after reinfusion of shed blood in the treated group but decreased again at the minute 120 period of postreinfusion. The recovery of this index of myocardial contractility to the preshock level in the untreated group was transient and gradually declined significantly thereafter.
ment
298
FIG. 1. Mean arterial pressure
(mAP) and cardiac index (CI) during shock and reperfusion without and with methionine (Meth) treatment. Each point represents the mean t SE. In the mAP tracing the preshock values are indicated at the five-to tenminute level. The changes in the mAP at zero minute indicate the pressure after hemorrhage to shock level. Cardiac index and other parameters were measured at thirty minutes after initiation of shock. *P < 0.05, comparison of the value at different time courses with respect to preshock values in the respective group. tip < 0.05, intergroup
comparison.
did not change significantly from the preshock values in either group and remained in preshock levels. However, by minute 120 of postreinfusion V max had decreased significantly from the preshock values in the untreated group. There was a decrease in the heart rate and an increase in the CE after reinfusion of shed blood in both groups. The mRAP after reinfusion of shed blood increased, however, only in the methionine-treated group. The postreinfusion values of mRAP was higher in the methionine-treated group. These results indicate that the hemodynamic parameters were similar in the two groups during the shock phase. The postinfusion recovery of cardiac function and contractility was, however, significantly better in the group with methionine treatment.
Vmax
Blood Lactate and Gases Blood lactate levels in the two groups before and during hemorrhagic shock and
postin-
299
FIG. 2. Changes in Vna, and at CPIP/PAW in the two groups. Points on the tracings represent mean ± SE. *P
