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Effect of increasing volume of cardioplegic solution on postischemic myocardial recovery
J
THORAC CARDIOVASC SURG
1987;94:234-40
Effect of increasing volume of cardioplegic solution on postischemic myocardial recovery Multidose cardioplegia has been reported to be superior to single-dose cardioplegia in protecting the heart during ischemia. However, large volumes of cardioplegic solution may be detrimental because of washout of adenine nucleotide degradation products that accumulate during ischemia, which limits recovery of adenosine triphosphate. We designed an experiment to test the effects of increasing the volume of cardioplegic solution on postischemic myocardial recovery. Four groups were studied: Group 1, initial 2 minute single dose of cardioplegic solution; Group 2, infusion of cardioplegic solution every 30 minutes for 1 minute; Group 3, infusion of cardioplegic solution every 20 minutes for 1 minute; and Group 4, infusion of cardioplegic solution every 20 minutes for 2 minutes. All groups were ischemic for 2 hours at 20° C. Although washout of nucleotide degradation products during the ischemic interval increased with higher volumes of cardioplegic infusion, the total washout (infusion plus initial 5 minutes of reperfusion) was not different among all groups. The multidose groups recovered function better and had significantly higher levels of total tissue purines after 30 minutes of reperfusion. There was no difference in adenosine triphosphate levels among all groups after reperfusion. We conclude that increasing the volume of cardioplegic solution, within a clinically relevant range is not associated with increasing loss of adenine nucleotides from the ceU or with impaired functional recovery of the heart.
Razi Saydjari, M.D., Gregory Asimakis, Ph.D., and Vincent R. Conti, M.D., Galveston, Texas
h e technique of multi-dose cold cardioplegia (CP) has extended the safe period of ischemic arrest for cardiac operations because it substantially slows myocardial metabolism and decreases myocardial energy use during arrest. However, adenine mucleotide degradation still does occur during ischemia with this technique.l' Consequently, the degradation products are lost from the myocardium during infusion of CP solution and upon reperfusion. Several investigators have suggested that the loss of these degradation products, which are needed as precursors for purine nucleotide resynthesis, may limit functional recovery." Continuous infusion of large volumes of CP solution during arrest has been shown in the isolated heart model to be detrimental because of washout of adenine nucleotide degradation products during the ischemic arrest period, which in From the Division of Cardiovascular and Thoracic Surgery, Department of Surgery, John Sealy Hospital, University of Texas Medical Branch, Galveston, Texas Received for publication July 14, 1986. Accepted for publication Sept. 9, 1986. Address for reprints: Vincent R. Conti, M.D., Division of Cardiothoracic and Thoracic Surgery, University of Texas Medical Branch, Galveston, Texas 77550.
234
tum limits the availability of precursors of the salvage pathway for purine synthesis.' However, the significance of washout of nucleotide degradation products during multidose CP as it is commonly used clinically remains unanswered. The present study was designed to test the effects of increasing the volume of CP solution infused during arrest on postischemic myocardial recovery. The infusion times and durations during arrest were chosen to closely reflect the range used under clinical conditions with the technique of multidose CPo Methods
Care of the animals in this study was in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Experimental design. An isolated, nonworking rat heart model was used in this study. Hearts were divided into four groups that varied regarding the duration of CP infusion during arrest. Each group received an initial 2 minute dose of CP solution. Group I received only the initial dose of CP solution, whereas Group II subse-
Volume 94 Number 2 August 1987
Postischemic myocardial recovery 2 3 5
quently received a 1 minute dose of CP solution every 30 minutes, Group III a 1 minute dose every 20 minutes, and Group IV a 2 minute dose every 20 minutes. The total time of CP infusion for Groups I to IV was 2, 5, 7, and 12 minutes, respectively. All hearts were ischemic for 2 hours and were maintained at 20 C during ischemia and during infusions. Hearts in each group were then reperfused at 37 C for 30 minutes. Perfusion techniques. The nonworking heart perfusion apparatus described by Apstein, Mueller, and Hood' was used for this study. Hearts were removed from heparinized (sodium heparin, 1 mg) and anesthetized (sodium pentobarbitol, 50 mg) male SpragueDawley rats (200 to 250 gm) and placed immediately into a beaker of iced Krebs-Henseleit bicarbonate buffer (KHB). After aortic cannulation, a 10 minute period of preliminary retrograde (Langendorff) perfusion at a perfusion pressure of 100 em H 20 was begun with oxygenated KHB (37 C). The CP solution used clinically at our institution was also used for this experiment. It is a modification of the CP solution originally described by Tyers and associates? and contains the following constituents in milliequivalents per liter: sodium, 139; potassium, 20; magnesium, 3; chloride, 101; calcium, 0.9; bicarbonate, 20; acetate, 27; and gluconate, 23. It also contained glucose 500 mg/dl, heparin 2.5 U /ml, and had an osmolarity of approximately 320 mOsm. Hearts were reperfused with KHB (37 C) for a 30 minute recovery period after ischemia. Hemodynamic measurements. Hearts studied for determination of functional recovery underwent placement of a balloon within the left ventricle during the first 5 minutes of initial perfusion. For placement of the balloon, the left atrium was opened and the cannula and balloon were passed through the atrium into the left ventricle as described by Igo and co-workers." The balloon was inflated with water at 10 #1 increments to a maximum volume of 60 #1 and the maximum developed pressure was recorded. The balloon was then deflated and remained deflated until measurements were once again recorded after approximately 25 minutes of reperfusion. Recovery was expressed as a percentage of prearrest maximum developed pressure. Maximum developed pressure was monitored with a Watanabe twelve-channel Mark IV Linearcorder recorder interfaced to Statham type P231D pressure transducers. Washout of degradation products. For the determination of baseline efflux of nucleotides from hearts before ischemia, coronary effluents were collected over a 30 second time period at the 9 minute point of initial perfusion. The entire coronary effluent was then collect0
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ed with each infusion of CP solution for all groups. The coronary effluent was continuously collected for the first 5 minutes of reperfusion; separate aliquots were collected 30 seconds after this to determine whether or not washout returned to the preischemic rate. The effluents were frozen until analyzed. Myocardial metabolite analysis. Hearts were freeze-clamped with Wollenberger tongs cooled to the temperature of liquid nitrogen. For the determination of initial metabolite levels, hearts were freezed-clamped after 10 minutes of initial perfusion. Hearts in each group were freezed-clamped immediately before reperfusion to determine metabolite levels at the end of ischemia. To determine metabolite levels after reperfusion, those hearts that were used to determine functional recovery were freezed-clamped after 30 minutes of reperfusion (hemodynamic measurements were made 25 minutes after reperfusion). The frozen heart wafers were ground to a fine powder in liquid nitrogen with a mortar and pestle, extracted in 1N perchloric acid and neutralized with potassium hydroxide for later analysis of nucleotides and nucleotide degradation products. The protein pellets were saved for analysis. Analysis of extract and coronary effluents. Adenosine triphosphate (ATP) and creatine phosphate levels were determined by standard enzymatic techniques." Adenosine monophosphate and diphosphate were separated and quantitated by reverse-phase high-performance liquid chromotography (HPLC). Samples (20 #1) were injected on an Altex Ultrasphere ODS column
The JOurnal of Thoracic and Cardiovascular Surgery
2 3 6 Saydjari, Asimakis, Conti
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(5 f.L particle-size, 25 cm by 4.6 mm). The samples were eluted with a buffer consisting of monobasic potassium phosphate 30 mmol/L, tetrabutylammonium sulfate 15 mmol/L, and 19% acetonitrile, pH 6.0. For the determination of nucleosides (adenosine, hypoxanthine, and inosine), the samples were eluted with buffer consisting of monobasic potassium phosphate 10 mmol/L and 2% acetonitrile, pH 4.0. In each case, the flow rate was maintained at 1.5 ml/min with a Beckman Model 11OA pump. The effluent was monitored with a Beckman 160 absorbance detector (254 nm) interfaced to a chart recorder. A comparison was made between elution profiles of standards and elution profiles of the samples.
Internal standards were also run to verify peak identification. The HPLC buffers and all samples to be applied to the column were filtered through 0.45 /lm cellulose nitrate filters. Protein determination. After extraction in perchloric acid, 4 ml of 6N potassium hydroxide was added to each protein pellet that remained when the neutralized supernatant was removed. The samples were incubated at 30° C for 6 hours. The dissolved protein samples were diluted 1:60 with water. The samples were then assayed for protein content by the method of Lowry and associates." Analysis of results. Results are expressed as
Volume 94 Number 2
Postischemic myocardial recovery
Auqust 1987
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Results Purine washout with each infusion of CP solution. The total infusion times of CP solutions for Groups I, II, III, and IV were 2, 5, 7, and 12 minutes, respectively; the total volumes of coronary effluent during CP were 20.6 ± 1.1,46.1 ± 3.6,66.2 ± 4.9, and 92.7 ± 4.5 mi/ gm wet weight, respectively. Fig. 1 shows that in the multidose groups the amount of washout increased with each CP infusion until a plateau was reached at about 60 minutes of ischemia. A comparison of Groups III and IV shows that most of the washout of degradation products occurs during the first minute of CP infusion and is not substantially increased by doubling the infusion duration. Cumulative purine washout during CPo Although increasing the time of CP infusion from 1 to 2 minutes did not significantly increase the amount of washout from any single infusion (Fig. 1), the cumulative effect of all the CP infusions was that washout of adenine nucleotides increased significantly with increasing volumes of CP (Fig. 2). The washout observed in Group I hearts was low because there was only a single infusion at the onset of ischemia.
Purine washout during reperfusion. The volumes of coronary effluent during the first 5 minutes of reperfusion were not significantly different among the groups. The washout during the first 5 minutes of reperfusion was two to three times greater for the single-dose group than for the groups that had multidose CP (Fig. 2). Hearts that had larger volumes of CP solution during arrest had less purine washout during reperfusion. Total purine washout (infusion plus 5 minute reperfusion). Total measured washout among all groups was not statistically different (Fig. 2). Therefore, the increased washout resulting from higher CP volume infusion was nearly equally counterbalanced by a decreased measured washout during the first 5 minutes of reperfusion. Thus the total measured washout was not statistically different among groups. In addition to collecting the first 5 minutes of effluent during reperfusion, we also collected individual 30 second samples of effluent immediately after the 5 minute point of reperfusion. The amount of purines washed out during this period was used as an indication of the magnitude of continued washout during reperfusion. Comparison was made to the control washout rate (l0.8 nmol/gm wet weight/min) determined by collecting a 30 second sample after 9 minutes of initial perfusion before ischemia. The rates of efflux just after the 5 minute point of reperfusion for Groups I, II, III, and IV were 105.1 ± 11.5,26.1 ± 4.8, 18.8 ± 3.4, and 14.8 ± 1.1 nmol/gm wet weight/min, respectively.
The Journal of
2 3 8 Saydjari, Asimakis, Conti
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Thoracic and Cardiovascular Surgery
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Although the rates of washout in the two higher-dose multidose groups returned to near control values after 5 minutes of reperfusion, the rate of washout was approximately ten times the control value in the single-dose group (p < 0.001). The rate in Group II was also significantly higher than control, although barely so (p < 0.05). Total tissue purines at the end of ischemia. Control hearts perfused for an initial 10 minute period had a total tissue purine content of 52.4 ± 2.1 nmol/rng protein (Fig. 3). Total tissue purines after 120 minutes of ischemia for Groups I, II, III, and IV were 50.2 ± 4.6, 45.0 ± 1.5, 43.6 ± 3.3, and 40.6 ± 3.3 nmol/mg protein, respectively (Fig. 3). These values were not statistically different although they tended to be lower with increasing CP infusion. This is consistent with the observation that increasing the CP perfusion volume resulted in greater washout during the period of ischemia (Fig. 2). Total tissue purines after reperfusion. Total tissue purines in nanomoles per milligram of protein for Groups I, II, III, and IV after 30 minutes of reperfusion were as follows: 26.2 ± 2.0,31.6 ± 1.7,31.7 ± 1.3, and 35.5 ± 2.3, respectively (Fig. 3). Groups III and IV had levels that were significantly higher than Group I levels (p < 0.05). The results suggest that total purine washout, which includes the entire 30 minutes of reperfusion, is decreased when increasing volume of CP infusion is given during arrest. Because the total purine content of
the effluents collected from the CP infusions and the first 5 minutes of reperfusion were not different between the groups, the difference in the tissue purine content at the end of reperfusion must reflect ongoing washout rate observed in the single-dose groups in the 30 second sample taken after the 5 minute reperfusion collection. Tissue ATP and creatine phosphate. At the end of ischemia, each of the multidose groups had significantly higher levels of ATP than the single-dose group hearts, in which ATP levels decreased to approximately 20% of control values (Fig. 4). There was no statistically significant difference among the multidose groups at the end of ischemia. Reperfusion resulted in a significant increase in the ATP levels in the Group I hearts, from approximately 20% to 50% of control values. The multidose groups showed only a slight enhancement of ATP levels after reperfusion, and no significant difference was found among all groups after reperfusion. Tissue levels of creatine phosphate at the end of ischemia decreased to 15% to 20% of the control value in each of the experimental groups (Fig. 5) and recovered to levels that were not statistically different among the groups. Functional recovery after reperfusion. The average recovery of maximum developed pressure of Group I hearts was 56% of the preischemic control value (Fig. 6), and although this value was lower than the recovery of the multidose hearts, the differences between the single-dose group and each of the multidose groups were not statistically significant. Discussion A number of recent studies have suggested that depletion of adenine nucleotides from the myocardium may be an important mechanism that limits the metabolic and functional recovery after ischemic arrest."? Despite present methods of myocardial preservation, myocardial energy consumption proceeds to some degree during ischemic arrest and ATP is dephosphory1ated, which permits adenosine and its metabolites, inosine and hypoxanthine, to be washed from the myocardium. Regeneration can occur by one of two pathways: de novo synthesis, which is a relatively slow metabolic pathway, and the salvage pathway, which must use the precursors adenosine or inosine. 13. 14 Depletion of these precursors may inhibit regeneration of sufficient adenine nucleotides to provide for the energyrequiring functions of the myocardium." These metabolic mechanisms have recently been reviewed' and interventions to counteract loss of these intermediates . from the myocardium have been suggested in experimental studies.t'
Volume 94
Postischemic myocardial recovery
Number 2 August 1987
One of these studies, by De Witt, Jochim, and Behrendt,' showed that delivering increasing volumes of CP solution to the myocardium during arrest was associated with increased washout of adenine nucleosides from the myocardium and impaired functional recovery. They concluded that the most important period of washout occurred during CP infusion, and they recommended limiting the volume of CP solution delivered during arrest to prevent this detrimental effect. However, in their study, significant injury to the myocardium occurred when CP solution was given as a continuous single infusion of 10, 30, and 60 minutes. Other experimental studies have suggested that delivering increasing amounts of CP solution during arrest with a multidose technique may be beneficial and improve myocardial protection. IS, 16 Our own clinical practice has called for delivering larger volumes of CP solution when needed to cool the myocardium adequately and maintain arrest, especially when longer ischemic times become necessary and when severe coronary artery disease is present; this technique has consistently provided excellent myocardial protection. Clinical practice 'might be affected by recommendations based on the study of De Witt, Jochim, and Behrendt,' who used prolonged continuous anoxic CP infusion, a practice not used clinically. Therefore, we studied the effect of increased volumes of crystalloid CP solution administered with the same intermittent multidose techique as is used clinically with the volume, intervals, and durations of infusion reflecting the range commonly used clinically. In our study, a 2 hour ischemic period of 20° C was used in our groups that varied by the duration and volume of CP solution administered, with the highest dose group receiving 2 minute infusions every 20 minutes (total 12 minutes of CP infusion). We found that increasing volume of CP solution did result in more washout of adenine nucleosides in the coronary effluent during arrest but that correspondingly less was subsequently washed out during the first 5 minutes of reperfusion after arrest. Although tissue levels of total tissue purines were not different at the end of arrest, after the 30 minute recovery period there were significantly lower cellular levels of total tissue purines in the hearts receiving only the single dose of CP solution compared with the two higher-dose groups. This is consistent with the increased ongoing washout rate in the single-dose group that was observed in the sample taken after the initial 5 minute reperfusion period during which the entire coronary effluent was collected. Despite these metabolic differences, functional recovery as measured by recovery of developed pressure was not statis-
239
tically different between groups. Functional recovery, though not significantly improved with multidose CP, did not deteriorate with increasing volume of CP infusion during arrest. This study used the isolated rat heart model with functional recovery measured with an intraventricular balloon. This model has the advantage of allowing nearly total control of experimental conditons and accurate measurement of metabolic and functional consequences of individual interventions. However, only crystalloid perfusates are used in this model and it does not reproduce other conditions present during cardiopulmonary bypass. This study was done well within the period of stable function of this model, and the absence of sanguineous perfusate greatly simplified measurement of adenine nucleotide metabolite levels in the coronary effluent. Recent data has cast doubt on the importance of ATP levels as an indicator of the metabolic status and functional capacity of the myocardium. 17, 18 Although the rate of turnover and efficiency of ATP use to generate energy and mechanical work may be more important than static levels of ATP at any time, the studies that have correlated adenine nucleotide loss with decreased myocardial function strongly suggest that methods of myocardial preservation should be structured to minimize the detrimental effects that may be due to this mechanism.t- 19 Our data indicate that when smaller doses of CP solution are used, washout of adenine nucleosides occurs to the same degree as or a greater degree than with multidose techniques, similar to those used clinically, but occurs proportionately more during the reperfusion period rather than during the CP reinfusions. -Because the degree of loss of adenine nucleotides during arrest and reperfusion seems to be related to the magnitude of the ischemic insult rather than the volume of CP solution infused, the objective of any method of myocardial preservation should be to use the volume of CP solution necessary to maintain profound hypothermia and complete arrest. REFERENCES 1. Wright RN, Levitsky S, Holland C, Feinberg H. Benefi-
cial effects of potassium cardioplegia during intermittent aortic cross-clamping and reperfusion. J Surg Res 1978; 24:201-9,
2. Pasque MK, Wechsler AS. Metabolic intervention to affect myocardial recovery following ischemia. Ann Surg 1984;200: 1-12.
3. DeWitt OF, Jochim KE, Behrendt OM. Nucleotide degradation and functional impairment during cardioplegia: amelioration by inosine. Circulation 1984;67:171-8. 4. Ely SW, Mentzer RM, Lasley RO, Lee BK, Berne RM.
The Journal of
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5.
6.
7.
8.
9.
10.
11.
Functional and metabolic evidence of enhanced myocardial tolerance to ischemia and reperfusion with adenosine. J THORAC CARDIOVASC SURG 1985;90:549-56. Foker JE, Einzig S, Wang T. Adenosine metabolism and myocardial preservation. J THORAC CARDIOVASC SURG 1980;80:506-16. Pasque MK, Spray TL, Pellom GL, et al. Riboseenhanced myocardial recovery following ischemia in the isolated working rat heart. J THORAC CARDIOVASC SURG 1982;83:390-8. Silverman NA, Kohler J, Feinberg H, Levitsky S. Beneficial metabolic effect of nucleoside augmentation on reperfusion injury following cardioplegic arrest. Chest 1983;83:787-92. Apstein CS, Mueller M, Hood Jr WB. Ventricular contraction and compliance with global ischemia and reperfusion: their effect on coronary resistance in the rat. Circ Res 1977;41:206-17. Tyers GFO, Manley NJ, Williams EH, Shaffer CW, Williams DR, Kurusz M. Preliminary clinical experience with isotonic hypothermic postassium-induced arrest. J THORAC CARDIOVASC SURG 1977;74:674-81. Igo SR, Kyger ER, Lande AJ, Dudrick SJ. Improved intraventricular balloons for isolated rat hearts. Proceedings of the thirty-second annual conference on Engineering in Medicine and Biology, Bethesda, Maryland: LC 61-24788, 1979. Lamprecht W, Trautschold I. Adenosine-5'-phosphate: determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis, vol 4, English 2nd ed. New York: Academic Press, 1974:2101-10.
Thoracic and Cardiovascular Surgery
12. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with the folin phenol reagent. J BioI Chern 1951;193:265-75. 13. Manfredi JP, Holmes EW. Purine salvage pathways in myocardium. Ann Rev Physiol 1985;47:691-705. 14. Zimmer HG, Trendelenberg C, Kammermeier H, Gerlach E. De novo synthesis of myocardial adenine nucleotides in the rat. Circ Res 1973;32:635-42. 15. Engelman RM, Rousou JH, Lemeshow S. High-volume crystalloid cardioplegia: an improved method of myocardial preservation. J THORAC CARDIOVASC SURG 1983; 86:87-96. 16. Conti VR, Kao RL. Metabolic and functional effects of carbohydrate substrate with single-dose and multiple-dose potassium cardioplegia. Ann Thorac Surg 1983;36:3207. 17. Taegtmeyer H, Roberts AFC, Raine AEG. Energy metabolism in reperfused heart muscle: metabolic correlates to return of function. J Am ColI CardioI1985;6:86470. 18. Rosenkranz ER, Vinten-Johansen J, Buckberg GD, Okamoto F, Edwards H, Bugyi H. Benefits of normothermic induction of blood cardioplegia in energy-depleted hearts, with maintenance of arrest by multidose cold blood cardioplegic infusions. J THORAC CARDIOVASC SURG 1982; 84:667-77. 19. House DJ, Stewart DA, Chain EB. Recovery from cardiac bypass and elective cardiac arrest: the metabolic consequences of various cardioplegic procedures in the isolated rat heart. Circ Res 1974;35:448-57.
THORAC CARDIOVASC SURG
1987;94:234-40
Effect of increasing volume of cardioplegic solution on postischemic myocardial recovery Multidose cardioplegia has been reported to be superior to single-dose cardioplegia in protecting the heart during ischemia. However, large volumes of cardioplegic solution may be detrimental because of washout of adenine nucleotide degradation products that accumulate during ischemia, which limits recovery of adenosine triphosphate. We designed an experiment to test the effects of increasing the volume of cardioplegic solution on postischemic myocardial recovery. Four groups were studied: Group 1, initial 2 minute single dose of cardioplegic solution; Group 2, infusion of cardioplegic solution every 30 minutes for 1 minute; Group 3, infusion of cardioplegic solution every 20 minutes for 1 minute; and Group 4, infusion of cardioplegic solution every 20 minutes for 2 minutes. All groups were ischemic for 2 hours at 20° C. Although washout of nucleotide degradation products during the ischemic interval increased with higher volumes of cardioplegic infusion, the total washout (infusion plus initial 5 minutes of reperfusion) was not different among all groups. The multidose groups recovered function better and had significantly higher levels of total tissue purines after 30 minutes of reperfusion. There was no difference in adenosine triphosphate levels among all groups after reperfusion. We conclude that increasing the volume of cardioplegic solution, within a clinically relevant range is not associated with increasing loss of adenine nucleotides from the ceU or with impaired functional recovery of the heart.
Razi Saydjari, M.D., Gregory Asimakis, Ph.D., and Vincent R. Conti, M.D., Galveston, Texas
h e technique of multi-dose cold cardioplegia (CP) has extended the safe period of ischemic arrest for cardiac operations because it substantially slows myocardial metabolism and decreases myocardial energy use during arrest. However, adenine mucleotide degradation still does occur during ischemia with this technique.l' Consequently, the degradation products are lost from the myocardium during infusion of CP solution and upon reperfusion. Several investigators have suggested that the loss of these degradation products, which are needed as precursors for purine nucleotide resynthesis, may limit functional recovery." Continuous infusion of large volumes of CP solution during arrest has been shown in the isolated heart model to be detrimental because of washout of adenine nucleotide degradation products during the ischemic arrest period, which in From the Division of Cardiovascular and Thoracic Surgery, Department of Surgery, John Sealy Hospital, University of Texas Medical Branch, Galveston, Texas Received for publication July 14, 1986. Accepted for publication Sept. 9, 1986. Address for reprints: Vincent R. Conti, M.D., Division of Cardiothoracic and Thoracic Surgery, University of Texas Medical Branch, Galveston, Texas 77550.
234
tum limits the availability of precursors of the salvage pathway for purine synthesis.' However, the significance of washout of nucleotide degradation products during multidose CP as it is commonly used clinically remains unanswered. The present study was designed to test the effects of increasing the volume of CP solution infused during arrest on postischemic myocardial recovery. The infusion times and durations during arrest were chosen to closely reflect the range used under clinical conditions with the technique of multidose CPo Methods
Care of the animals in this study was in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Experimental design. An isolated, nonworking rat heart model was used in this study. Hearts were divided into four groups that varied regarding the duration of CP infusion during arrest. Each group received an initial 2 minute dose of CP solution. Group I received only the initial dose of CP solution, whereas Group II subse-
Volume 94 Number 2 August 1987
Postischemic myocardial recovery 2 3 5
quently received a 1 minute dose of CP solution every 30 minutes, Group III a 1 minute dose every 20 minutes, and Group IV a 2 minute dose every 20 minutes. The total time of CP infusion for Groups I to IV was 2, 5, 7, and 12 minutes, respectively. All hearts were ischemic for 2 hours and were maintained at 20 C during ischemia and during infusions. Hearts in each group were then reperfused at 37 C for 30 minutes. Perfusion techniques. The nonworking heart perfusion apparatus described by Apstein, Mueller, and Hood' was used for this study. Hearts were removed from heparinized (sodium heparin, 1 mg) and anesthetized (sodium pentobarbitol, 50 mg) male SpragueDawley rats (200 to 250 gm) and placed immediately into a beaker of iced Krebs-Henseleit bicarbonate buffer (KHB). After aortic cannulation, a 10 minute period of preliminary retrograde (Langendorff) perfusion at a perfusion pressure of 100 em H 20 was begun with oxygenated KHB (37 C). The CP solution used clinically at our institution was also used for this experiment. It is a modification of the CP solution originally described by Tyers and associates? and contains the following constituents in milliequivalents per liter: sodium, 139; potassium, 20; magnesium, 3; chloride, 101; calcium, 0.9; bicarbonate, 20; acetate, 27; and gluconate, 23. It also contained glucose 500 mg/dl, heparin 2.5 U /ml, and had an osmolarity of approximately 320 mOsm. Hearts were reperfused with KHB (37 C) for a 30 minute recovery period after ischemia. Hemodynamic measurements. Hearts studied for determination of functional recovery underwent placement of a balloon within the left ventricle during the first 5 minutes of initial perfusion. For placement of the balloon, the left atrium was opened and the cannula and balloon were passed through the atrium into the left ventricle as described by Igo and co-workers." The balloon was inflated with water at 10 #1 increments to a maximum volume of 60 #1 and the maximum developed pressure was recorded. The balloon was then deflated and remained deflated until measurements were once again recorded after approximately 25 minutes of reperfusion. Recovery was expressed as a percentage of prearrest maximum developed pressure. Maximum developed pressure was monitored with a Watanabe twelve-channel Mark IV Linearcorder recorder interfaced to Statham type P231D pressure transducers. Washout of degradation products. For the determination of baseline efflux of nucleotides from hearts before ischemia, coronary effluents were collected over a 30 second time period at the 9 minute point of initial perfusion. The entire coronary effluent was then collect0
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ed with each infusion of CP solution for all groups. The coronary effluent was continuously collected for the first 5 minutes of reperfusion; separate aliquots were collected 30 seconds after this to determine whether or not washout returned to the preischemic rate. The effluents were frozen until analyzed. Myocardial metabolite analysis. Hearts were freeze-clamped with Wollenberger tongs cooled to the temperature of liquid nitrogen. For the determination of initial metabolite levels, hearts were freezed-clamped after 10 minutes of initial perfusion. Hearts in each group were freezed-clamped immediately before reperfusion to determine metabolite levels at the end of ischemia. To determine metabolite levels after reperfusion, those hearts that were used to determine functional recovery were freezed-clamped after 30 minutes of reperfusion (hemodynamic measurements were made 25 minutes after reperfusion). The frozen heart wafers were ground to a fine powder in liquid nitrogen with a mortar and pestle, extracted in 1N perchloric acid and neutralized with potassium hydroxide for later analysis of nucleotides and nucleotide degradation products. The protein pellets were saved for analysis. Analysis of extract and coronary effluents. Adenosine triphosphate (ATP) and creatine phosphate levels were determined by standard enzymatic techniques." Adenosine monophosphate and diphosphate were separated and quantitated by reverse-phase high-performance liquid chromotography (HPLC). Samples (20 #1) were injected on an Altex Ultrasphere ODS column
The JOurnal of Thoracic and Cardiovascular Surgery
2 3 6 Saydjari, Asimakis, Conti
-
CJCP infusion washout
800
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(5 f.L particle-size, 25 cm by 4.6 mm). The samples were eluted with a buffer consisting of monobasic potassium phosphate 30 mmol/L, tetrabutylammonium sulfate 15 mmol/L, and 19% acetonitrile, pH 6.0. For the determination of nucleosides (adenosine, hypoxanthine, and inosine), the samples were eluted with buffer consisting of monobasic potassium phosphate 10 mmol/L and 2% acetonitrile, pH 4.0. In each case, the flow rate was maintained at 1.5 ml/min with a Beckman Model 11OA pump. The effluent was monitored with a Beckman 160 absorbance detector (254 nm) interfaced to a chart recorder. A comparison was made between elution profiles of standards and elution profiles of the samples.
Internal standards were also run to verify peak identification. The HPLC buffers and all samples to be applied to the column were filtered through 0.45 /lm cellulose nitrate filters. Protein determination. After extraction in perchloric acid, 4 ml of 6N potassium hydroxide was added to each protein pellet that remained when the neutralized supernatant was removed. The samples were incubated at 30° C for 6 hours. The dissolved protein samples were diluted 1:60 with water. The samples were then assayed for protein content by the method of Lowry and associates." Analysis of results. Results are expressed as
Volume 94 Number 2
Postischemic myocardial recovery
Auqust 1987
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Fig. 4. Tissue ATP levels. See text.
means ± standard error of the mean. A total 13 hearts were used for each observation. analysis was determined by Student's t test. less than 0.05 was considered to indicate a difference.
o
Fig. 5. Tissue creatine phosphate levels. See text. of eight to Statistical A p value significant
Results Purine washout with each infusion of CP solution. The total infusion times of CP solutions for Groups I, II, III, and IV were 2, 5, 7, and 12 minutes, respectively; the total volumes of coronary effluent during CP were 20.6 ± 1.1,46.1 ± 3.6,66.2 ± 4.9, and 92.7 ± 4.5 mi/ gm wet weight, respectively. Fig. 1 shows that in the multidose groups the amount of washout increased with each CP infusion until a plateau was reached at about 60 minutes of ischemia. A comparison of Groups III and IV shows that most of the washout of degradation products occurs during the first minute of CP infusion and is not substantially increased by doubling the infusion duration. Cumulative purine washout during CPo Although increasing the time of CP infusion from 1 to 2 minutes did not significantly increase the amount of washout from any single infusion (Fig. 1), the cumulative effect of all the CP infusions was that washout of adenine nucleotides increased significantly with increasing volumes of CP (Fig. 2). The washout observed in Group I hearts was low because there was only a single infusion at the onset of ischemia.
Purine washout during reperfusion. The volumes of coronary effluent during the first 5 minutes of reperfusion were not significantly different among the groups. The washout during the first 5 minutes of reperfusion was two to three times greater for the single-dose group than for the groups that had multidose CP (Fig. 2). Hearts that had larger volumes of CP solution during arrest had less purine washout during reperfusion. Total purine washout (infusion plus 5 minute reperfusion). Total measured washout among all groups was not statistically different (Fig. 2). Therefore, the increased washout resulting from higher CP volume infusion was nearly equally counterbalanced by a decreased measured washout during the first 5 minutes of reperfusion. Thus the total measured washout was not statistically different among groups. In addition to collecting the first 5 minutes of effluent during reperfusion, we also collected individual 30 second samples of effluent immediately after the 5 minute point of reperfusion. The amount of purines washed out during this period was used as an indication of the magnitude of continued washout during reperfusion. Comparison was made to the control washout rate (l0.8 nmol/gm wet weight/min) determined by collecting a 30 second sample after 9 minutes of initial perfusion before ischemia. The rates of efflux just after the 5 minute point of reperfusion for Groups I, II, III, and IV were 105.1 ± 11.5,26.1 ± 4.8, 18.8 ± 3.4, and 14.8 ± 1.1 nmol/gm wet weight/min, respectively.
The Journal of
2 3 8 Saydjari, Asimakis, Conti
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Thoracic and Cardiovascular Surgery
t
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Experimental group Fig. 6. Functional recovery defined as percent recovery of maximal developed pressure after 25 minutes of reperfusion.
Although the rates of washout in the two higher-dose multidose groups returned to near control values after 5 minutes of reperfusion, the rate of washout was approximately ten times the control value in the single-dose group (p < 0.001). The rate in Group II was also significantly higher than control, although barely so (p < 0.05). Total tissue purines at the end of ischemia. Control hearts perfused for an initial 10 minute period had a total tissue purine content of 52.4 ± 2.1 nmol/rng protein (Fig. 3). Total tissue purines after 120 minutes of ischemia for Groups I, II, III, and IV were 50.2 ± 4.6, 45.0 ± 1.5, 43.6 ± 3.3, and 40.6 ± 3.3 nmol/mg protein, respectively (Fig. 3). These values were not statistically different although they tended to be lower with increasing CP infusion. This is consistent with the observation that increasing the CP perfusion volume resulted in greater washout during the period of ischemia (Fig. 2). Total tissue purines after reperfusion. Total tissue purines in nanomoles per milligram of protein for Groups I, II, III, and IV after 30 minutes of reperfusion were as follows: 26.2 ± 2.0,31.6 ± 1.7,31.7 ± 1.3, and 35.5 ± 2.3, respectively (Fig. 3). Groups III and IV had levels that were significantly higher than Group I levels (p < 0.05). The results suggest that total purine washout, which includes the entire 30 minutes of reperfusion, is decreased when increasing volume of CP infusion is given during arrest. Because the total purine content of
the effluents collected from the CP infusions and the first 5 minutes of reperfusion were not different between the groups, the difference in the tissue purine content at the end of reperfusion must reflect ongoing washout rate observed in the single-dose groups in the 30 second sample taken after the 5 minute reperfusion collection. Tissue ATP and creatine phosphate. At the end of ischemia, each of the multidose groups had significantly higher levels of ATP than the single-dose group hearts, in which ATP levels decreased to approximately 20% of control values (Fig. 4). There was no statistically significant difference among the multidose groups at the end of ischemia. Reperfusion resulted in a significant increase in the ATP levels in the Group I hearts, from approximately 20% to 50% of control values. The multidose groups showed only a slight enhancement of ATP levels after reperfusion, and no significant difference was found among all groups after reperfusion. Tissue levels of creatine phosphate at the end of ischemia decreased to 15% to 20% of the control value in each of the experimental groups (Fig. 5) and recovered to levels that were not statistically different among the groups. Functional recovery after reperfusion. The average recovery of maximum developed pressure of Group I hearts was 56% of the preischemic control value (Fig. 6), and although this value was lower than the recovery of the multidose hearts, the differences between the single-dose group and each of the multidose groups were not statistically significant. Discussion A number of recent studies have suggested that depletion of adenine nucleotides from the myocardium may be an important mechanism that limits the metabolic and functional recovery after ischemic arrest."? Despite present methods of myocardial preservation, myocardial energy consumption proceeds to some degree during ischemic arrest and ATP is dephosphory1ated, which permits adenosine and its metabolites, inosine and hypoxanthine, to be washed from the myocardium. Regeneration can occur by one of two pathways: de novo synthesis, which is a relatively slow metabolic pathway, and the salvage pathway, which must use the precursors adenosine or inosine. 13. 14 Depletion of these precursors may inhibit regeneration of sufficient adenine nucleotides to provide for the energyrequiring functions of the myocardium." These metabolic mechanisms have recently been reviewed' and interventions to counteract loss of these intermediates . from the myocardium have been suggested in experimental studies.t'
Volume 94
Postischemic myocardial recovery
Number 2 August 1987
One of these studies, by De Witt, Jochim, and Behrendt,' showed that delivering increasing volumes of CP solution to the myocardium during arrest was associated with increased washout of adenine nucleosides from the myocardium and impaired functional recovery. They concluded that the most important period of washout occurred during CP infusion, and they recommended limiting the volume of CP solution delivered during arrest to prevent this detrimental effect. However, in their study, significant injury to the myocardium occurred when CP solution was given as a continuous single infusion of 10, 30, and 60 minutes. Other experimental studies have suggested that delivering increasing amounts of CP solution during arrest with a multidose technique may be beneficial and improve myocardial protection. IS, 16 Our own clinical practice has called for delivering larger volumes of CP solution when needed to cool the myocardium adequately and maintain arrest, especially when longer ischemic times become necessary and when severe coronary artery disease is present; this technique has consistently provided excellent myocardial protection. Clinical practice 'might be affected by recommendations based on the study of De Witt, Jochim, and Behrendt,' who used prolonged continuous anoxic CP infusion, a practice not used clinically. Therefore, we studied the effect of increased volumes of crystalloid CP solution administered with the same intermittent multidose techique as is used clinically with the volume, intervals, and durations of infusion reflecting the range commonly used clinically. In our study, a 2 hour ischemic period of 20° C was used in our groups that varied by the duration and volume of CP solution administered, with the highest dose group receiving 2 minute infusions every 20 minutes (total 12 minutes of CP infusion). We found that increasing volume of CP solution did result in more washout of adenine nucleosides in the coronary effluent during arrest but that correspondingly less was subsequently washed out during the first 5 minutes of reperfusion after arrest. Although tissue levels of total tissue purines were not different at the end of arrest, after the 30 minute recovery period there were significantly lower cellular levels of total tissue purines in the hearts receiving only the single dose of CP solution compared with the two higher-dose groups. This is consistent with the increased ongoing washout rate in the single-dose group that was observed in the sample taken after the initial 5 minute reperfusion period during which the entire coronary effluent was collected. Despite these metabolic differences, functional recovery as measured by recovery of developed pressure was not statis-
239
tically different between groups. Functional recovery, though not significantly improved with multidose CP, did not deteriorate with increasing volume of CP infusion during arrest. This study used the isolated rat heart model with functional recovery measured with an intraventricular balloon. This model has the advantage of allowing nearly total control of experimental conditons and accurate measurement of metabolic and functional consequences of individual interventions. However, only crystalloid perfusates are used in this model and it does not reproduce other conditions present during cardiopulmonary bypass. This study was done well within the period of stable function of this model, and the absence of sanguineous perfusate greatly simplified measurement of adenine nucleotide metabolite levels in the coronary effluent. Recent data has cast doubt on the importance of ATP levels as an indicator of the metabolic status and functional capacity of the myocardium. 17, 18 Although the rate of turnover and efficiency of ATP use to generate energy and mechanical work may be more important than static levels of ATP at any time, the studies that have correlated adenine nucleotide loss with decreased myocardial function strongly suggest that methods of myocardial preservation should be structured to minimize the detrimental effects that may be due to this mechanism.t- 19 Our data indicate that when smaller doses of CP solution are used, washout of adenine nucleosides occurs to the same degree as or a greater degree than with multidose techniques, similar to those used clinically, but occurs proportionately more during the reperfusion period rather than during the CP reinfusions. -Because the degree of loss of adenine nucleotides during arrest and reperfusion seems to be related to the magnitude of the ischemic insult rather than the volume of CP solution infused, the objective of any method of myocardial preservation should be to use the volume of CP solution necessary to maintain profound hypothermia and complete arrest. REFERENCES 1. Wright RN, Levitsky S, Holland C, Feinberg H. Benefi-
cial effects of potassium cardioplegia during intermittent aortic cross-clamping and reperfusion. J Surg Res 1978; 24:201-9,
2. Pasque MK, Wechsler AS. Metabolic intervention to affect myocardial recovery following ischemia. Ann Surg 1984;200: 1-12.
3. DeWitt OF, Jochim KE, Behrendt OM. Nucleotide degradation and functional impairment during cardioplegia: amelioration by inosine. Circulation 1984;67:171-8. 4. Ely SW, Mentzer RM, Lasley RO, Lee BK, Berne RM.
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Functional and metabolic evidence of enhanced myocardial tolerance to ischemia and reperfusion with adenosine. J THORAC CARDIOVASC SURG 1985;90:549-56. Foker JE, Einzig S, Wang T. Adenosine metabolism and myocardial preservation. J THORAC CARDIOVASC SURG 1980;80:506-16. Pasque MK, Spray TL, Pellom GL, et al. Riboseenhanced myocardial recovery following ischemia in the isolated working rat heart. J THORAC CARDIOVASC SURG 1982;83:390-8. Silverman NA, Kohler J, Feinberg H, Levitsky S. Beneficial metabolic effect of nucleoside augmentation on reperfusion injury following cardioplegic arrest. Chest 1983;83:787-92. Apstein CS, Mueller M, Hood Jr WB. Ventricular contraction and compliance with global ischemia and reperfusion: their effect on coronary resistance in the rat. Circ Res 1977;41:206-17. Tyers GFO, Manley NJ, Williams EH, Shaffer CW, Williams DR, Kurusz M. Preliminary clinical experience with isotonic hypothermic postassium-induced arrest. J THORAC CARDIOVASC SURG 1977;74:674-81. Igo SR, Kyger ER, Lande AJ, Dudrick SJ. Improved intraventricular balloons for isolated rat hearts. Proceedings of the thirty-second annual conference on Engineering in Medicine and Biology, Bethesda, Maryland: LC 61-24788, 1979. Lamprecht W, Trautschold I. Adenosine-5'-phosphate: determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, ed. Methods of enzymatic analysis, vol 4, English 2nd ed. New York: Academic Press, 1974:2101-10.
Thoracic and Cardiovascular Surgery
12. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with the folin phenol reagent. J BioI Chern 1951;193:265-75. 13. Manfredi JP, Holmes EW. Purine salvage pathways in myocardium. Ann Rev Physiol 1985;47:691-705. 14. Zimmer HG, Trendelenberg C, Kammermeier H, Gerlach E. De novo synthesis of myocardial adenine nucleotides in the rat. Circ Res 1973;32:635-42. 15. Engelman RM, Rousou JH, Lemeshow S. High-volume crystalloid cardioplegia: an improved method of myocardial preservation. J THORAC CARDIOVASC SURG 1983; 86:87-96. 16. Conti VR, Kao RL. Metabolic and functional effects of carbohydrate substrate with single-dose and multiple-dose potassium cardioplegia. Ann Thorac Surg 1983;36:3207. 17. Taegtmeyer H, Roberts AFC, Raine AEG. Energy metabolism in reperfused heart muscle: metabolic correlates to return of function. J Am ColI CardioI1985;6:86470. 18. Rosenkranz ER, Vinten-Johansen J, Buckberg GD, Okamoto F, Edwards H, Bugyi H. Benefits of normothermic induction of blood cardioplegia in energy-depleted hearts, with maintenance of arrest by multidose cold blood cardioplegic infusions. J THORAC CARDIOVASC SURG 1982; 84:667-77. 19. House DJ, Stewart DA, Chain EB. Recovery from cardiac bypass and elective cardiac arrest: the metabolic consequences of various cardioplegic procedures in the isolated rat heart. Circ Res 1974;35:448-57.