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Enhanced myocardial preservation by nicotinic acid, an antilipolytic compound
J
THORAC CARDIOVASC SURG
1988;96:81-7
Enhanced myocardial preservation by nicotinic acid, an antilipolytic compound Improved cardiac performance after hypothermic cardioplegic arrest The effect of nicotinic acid, an antilipolytic drug, on myocardial preservation was studied on the basis of cardiac performance after 2 hours of cardioplegic arrest. Isolated in situ pig hearts were subjected to 120 minutes of hypothermic potassium (35 mEq) crystalloid cardioplegic arrest followed by 60 minutes of reperfusion. The experimental group received nicotinic acid 0.08 mmoljL 15 minutes before cardioplegic arrest, whereas the control group received 15 minutes of unmodified perfusion. There was a marked decline in myocardial creatine phosphate levels during cardioplegic arrest in both groups that returned to the baseline level during reperfusion without a significant intergroup difference, and adenosine triphosphate levels remained stable throughout the experiment in both groups. Myocardial oxygen consumption during reperfusion was significantly higher in hearts treated with nicotinic acid, which was consistent with a significantly greater cardiac contractile force as evaluated by isovolumetric left ventricular pressure measurements. There appeared to be less cardiac membrane damage as measured by creatine kinase release during reperfusion, which was significantly inhibited by treatment with nicotinic acid. The present study supports the conclusion that nicotinic acid improves cardiac performance after hypothermic cardioplegic arrest.
Hajime Otani, MD, PhD, Richard M. Engelman, MD, Subhajit Datta, MD, Randall M. Jones, BS, Gerald A. Cordis, MS, John A. Rousou, MD, Robert H. Breyer, MD, and Dipak K. Das, PhD, Springfield. Mass.
Myocardial preservation induced by hypothermic cardioplegicarrest protects the ischemic heart primarily by reducing metabolic rate and energy demand. Intermittent infusion of either a crystalloid or blood cardioplegic solution, however, does not maintain optimal aerobic metabolism during hypothermic cardioplegic arrest. l This lack of adequate oxidative metabolism in the myocardium during hypothermic cardioplegic arrest
From the Department of Surgery, University of Connecticut School of Medicine, Farmington, Conn., and Baystate Medical Center, Springfield, Mass. Supported by National Institutes of Health Grants HL 22559-06, HL33889, and HL34360; American Heart Association Grant 11-202-856. Presented at the Thirty-sixth Annual Scientific Session, American College of Cardiology, New Orleans, La., March 1987. Received for publication April 3, 1987. Accepted for publication Nov. 2, 1987. Address for reprints: Richard M. Engelman, MD, Baystate Medical Center, 759 Chestnut St., Springfield, MA 01107.
was further suggested by our recent study- demonstrating an accumulation of reducing equivalents as evidenced by the rise in the reduced nicotinamide-adenine dinucleotide/nicotinamide-adenine dinucleotide ratio even after 40 minutes of arrest when no significant decline in the adenosine triphosphate (ATP) level was noted. The consequence of a reduced metabolic rate with oxygen deprivation is the inhibition of free fatty acid utilization, because the rate of free fatty acid oxidation is linearly related to the rate of oxidative phosphorylation.' The inhibition of ~-oxidation in conjunction with the stimulation of phospholipid degradation in ischemic and reperfused myocardium results in the intracellular accumulation of free fatty acids and their metabolites.t' These molecules have been shown by their detergent properties to disrupt membrane integrity, alter cellular structure, and inhibit activities of enzyme systems, which changes are potentially detrimental to both mechanical and electrophysiologic function."Although abnormal free fatty acid metabolism has been well characterized in normothermic global or 81
82
The Journal of Thoracic and Cardiovascular Surgery
Otani et al.
"'"',-y------------.,. 100
50
l IEQ
~; 3
otg
-ell>
is "0
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~~ o
15'
0'
0'
30'
Pre-Ischemia
15'
TIME (minutes)
30'
0
60'
45'
Reperfusion • (+) Nicotinic Acid
(-) Nicotinic Acid
Fig. 1. Effect of nicotinic acid on myocardial adenosine triphosphate and creatine phosphate as a function of time.
Table I. Effects of nicotinic acid on coronary flow and myocardial oxygen consumption Preischemia Time (min)
Pre-NA
Reperfusion Post-NA
15 min
30 min
45 min
60 min
6.95 ± 0.76 8.06 ± 0.83
5.86 ± 1.06 8.16 ± 1.64
5.20 ± 1.12* 7.53 ± 0.72
5.11±1.I1 7.86 ± 0.80
5.02 ± 1.25 8.31 ± l.01
181 ± 28 203 ± 22
132 ± 14 184 ± 20t
149 ± 16 209 ± 22t
132 ± 24 177 ± 33
126 ± 17* 182 ± 26
Coronary flow (ml/min/IOO gm) (-) NA (+)NA
6.96 ± 0.45 8.l3±0.46
Oxygen consumption (ILI/gm/min) (-) NA (+) NA
201 ± 43 209 ± 28
NA, Nicotinic acid. 'p < 0.05, reperfusion versus control. tp <0.05. (+) NA versus (-) NA.
regional ischemic heart models, no work has been done concerning free fatty acid metabolism and its relation to cardiac performance during hypothermic cardioplegic arrest and reperfusion. In view of the deleterious effects of increased free fatty acid levels in ischemic heart, we used nicotinic acid, an antilipolytic drug, in the isolated in situ pig heart, which was subjected to 120 minutes of hypothermic potassium crystalloid cardiopegic arrest followed by 60 minutes of reperfusion.
Materials and methods Animal preparation. Twenty-six Yorkshire pigs of either sex, weighing 15 to 23 kg, were tranquilized with ketamine (Ketaject, 50 mg/kg) and anesthetized with intravenous injection of pentobarbital (Nembutal, 25 rug/kg). Each animal was supported by controlled respiration with room air, and the chest was opened through a median sternotomy. Sodium heparin (500 units/kg) was administered, and the animal was
placed on cardiopulmonary bypass via the innominate artery and right atrial cannulation with a bubble oxygenator (Bentley BOS-5, American Bentley, Irvine, Calif). The isolated in situ pig heart preparation was originally described in 197Y and has been modified to fit our present needs. The method used is to initially place the pig on cardiopulmonary bypass and then perfuse the ascending aortic root proximal to an aortic cross-clamp in such a manner that only the coronary circulation is supplied with blood. The systemic circulation is discontinued and, after the systemic circulation has drained into the oxygenator, both the superior and inferior venae cavae are ligated. During the study, coronary perfusion pressure is maintained at 75 mm Hg, which is regulated by transducer cannulation of the aortic root. Myocardial perfusate temperature is maintained at 37° C unless hypothermia is used. The pericardial well is utilized as a constant temperature bath, and the fluid in which the heart is bathed is regulated at either normothermic or hypothermic levels. When appropriate, coronary effluent is collected through a cannula inserted into the right ventricular outflow
Volume 96 Number 1
Myocardial protection with nicotinic acid
July 1988
83
150 .,-------~m~%:mm_-------------T
1
i
100
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TIME (minutes)
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30'
45'
60'
Reperfusion
'p < .05
••• (+) Nicotinic Acid
"p < .005
Fig. 2. Effect of nicotinic acid on left ventricular developed pressure as a function of time.
25001---~~~~~----------1
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15'
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45'
60'
Reperfusion 'p < .05
"p < .005
Fig. 3. Effect of nicotinic acid on left ventricular maximum dpjdt as a function of time.
tract, and coronary flow is measured by timed collection from thiscannula. Blood samples are drawn from the arterial inflow line and, when appropriate, from the coronary sinus by direct cannulation via the right atrial wall. After the isolated heart model was stabilized for 15 minutes, experimental procedures were initiated. Thirteen hearts were treated with nicotinic acid 0.08 mmol/L (Sigma Diagnostics, St. Louis, Mo.) for 15 minutes before hypothermic cardioplegic arrest. The remaining 13 hearts received untreated perfusion for 15 minutes. Hypothermic cardioplegic arrest was induced
with 50 ml of chilled (4° C) hyperkalemic (35 mEqjL) crystalloid solution after the hearts were cooled to 10° C in a hypothermic topical solution in the pericardial well. Hypothermic cardioplegic arrest was maintained for 120 minutes with reinfusion of the same volume of cardioplegic solution at IS-minute intervals, and myocardial temperature (measured by temperature probe) was maintained at 8° to 10° C throughout the duration of arrest. Reperfusion was performed at normothermic temperature at a constant pressure (75 nun Hg) for 60 minutes with the heart in a normothermic bath. In
84
The Journal of Thoracic and Cardiovascular Surgery
Otani et al.
30r----~
Gi
..J
15'
30'
0'
15'
Pre-Ischemia
30'
45'
60'
Reperfusion
TIME (minutes) - -, (-) Nicotinic Acid
••
(+)
Nicotinic Acid
Fig. 4. Effect of nicotinic acid on left ventricular end-diastolic pressure as a function of time.
400
5l
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Qi
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0'
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TIME (minutes)
--, (-) Nicotinic Acid ••• (+) Nicotinic Acid
30'
45'
60'
Reperfusion 'p < .05
Fig. 5. Effect of nicotinic acid on creatine kinase release as a function of time.
the event of ventricular fibrillation, the heart was defibrillated by a direct shock of 20 watt/sec. Myocardial biopsy specimens were taken from six hearts in each group at indicated times with a dental drill and freeze-clamp techniques. Cardiac performance was evaluated in seven hearts in each group. Determination of high-energy phosphates. Myocardial tissue samles were homogenized with a 10 times volume of 6% perchloric acid in a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, N.Y.). After centrifugation of 10,000 gm, the supernatant fluid was neutralized and assayed for ATP and creatine phosphate by high-pressure liquid chromatography as recently described by our laboratory."
Measurements of cardiac performance. Myocardial oxygen consumption was measured according to the formula described previously." Left ventricular developed pressure (LVDP), maximum rate of rise of left ventricular pressure (LV max dp/dt), and left ventricular end-diastolic pressure (LVEDP) were determined under isovolumic conditions through a Millar Mikro-Tip catheter pressure transducer (Millar Instruments, Inc., Houston, Texas) within a 10 ml compliant balloon inserted into the left ventricle via the apex. Assay of creatine kinase. Creatine kinase (CK) was assayed in the perfusate plasma by the enzymatic assay
Volume 96 Number 1
Myocardial protection with nicotinic acid
July 1988
L-----
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15' 30' Pre-Ischemia
0'
15'
30' 45' Reperfusion
60'
TIME (minutes) -_. (-) Nicotinic Acid
•• (+) Nicotinic Acid
Fig. 6. Effect of nicotinic acid on plasma free fatty acid level as a function of time.
method" with a CK assay kit obtained from Sigma Diagnostics. The data were initially obtained 15 minutes after the preparation of the isolated in situ pig heart. Determination of plasma free fatty acids. Blood samples collected during nonbiopsy experiments were assayed for plasma free fatty acid levels. One milliliter of plasma was mixed with ice-cold chloroform-methanol solution (2: I, v Iv) containing 0.005% butylated hydroxy toluene. Twenty nanomoles of carbon : 17 was added as an internal standard to monitor the recoveries of free fatty acids during extraction procedures. The organic layer was aspirated and was taken into near dryness under a stream of nitrogen gas. A portion of the lipid extracts were used for determination of free fatty acids. The samples were added to a SEP-PAK silica cartridge (Waters, Milford, Mass.) for separation of the neutral lipids and free fatty acids from the phospholipids as described by Hamilton and Comai." Phenacyl esters of free fatty acids derived from the pooled eluents were analyzed by highpressure liquid chromatography according to the method of Wood and LeeY Statistical analysis. Both a paired t test and a two-sample Student's t test were used for statistical analysis. Results are expressed as mean ± standard error of the mean and were considered to be statistically significant when the p value was <0.05. Results
Effects of nicotinic acid on myocardial high-energy phosphate contents. Fig. 1 shows myocardial ATP and creatine phosphate contents as a function of time. There was no substantial change in the level of myocardial ATP during 120 minutes of hypothermic cardioplegic arrest and reperfusion in both animals treated with nicotinic acid and nontreated animals. Myocardial creatine phosphate contents, however, declined markedly during 120 minutes of arrest in both groups of animals
and then returned to the baseline value as early as 15 minutes after reperfusion in both groups of animals with no difference between the groups.
Effects of nicotinic acid on coronary ftow and myocardial oxygen consumption. Effects of nicotinic acid on coronary flow and myocardial oxygen consumption are shown in Table I. These measurements were not significantly altered during pretreatment with nicotinic acid before hypothermic cardioplegic arrest. Coronary flow decreased significantly during reperfusion in nontreated animals, whereas in animals treated with nicotinic acid flow was maintained near the baseline level. Oxygen consumption also decreased significantly during reperfusion in nontreated hearts. This decline in oxygen consumption was significantly inhibited in hearts treated with nicotinic acid.
Effects of nicotinic acid on cardiac contractile function. The effects of nicotinic acid on cardiac contractile function are shown in Figs. 2 through 4. There was a 30% decrease in LVDP and a 40% decrease in LV max dp / dt from baseline levels 15 minutes after reperfusion in nontreated hearts. Treatment with nicotinic acid prevented this decline in LVDP and LV max dp/dt significantly compared with the results in nontreated animals throughout the reperfusion period. Cardiac compliance as estimated by LVEDP was reduced only slightly during reperfusion in both groups of animals, and there was no significant difference between the groups. Effects of nicotinic acid on CK release. Because the release of CK is directly related to the integrity of the cellular membrane, we analyzed the effects of nicotinic
86
Otani et al.
acid on CK release (Fig. 5). Plasma CK levels were increased after reperfusion in nontreated and nicotinic acid-treated animals. There was a dramatic increase in CK release during the rest of the reperfusion period in nontreated animals, which was significantly inhibited in animals treated with nicotinic acid. Effects of nicotinic acid on plasma free fatty acid levels. Because nicotinic acid is known as a hypolipidemic drug, 13 we investigated the effect of nicotinic acid on plasma free fatty acid levels (Fig. 6). Plasma free fatty acid levels were essentially unchanged during these experiments, which indicated that treatment with nicotinic acid did not influence plasma free fatty acid levels in our experimental model. There was also no significant difference in the concentrations of the individual free fatty acid classes in the plasma fraction between nontreated animals and animals treated with nicotinic acid. Discussion The results of the present study demonstrate that pretreatment with nicotinic acid provides significant benefit as measured by the restoration of cardiac performance after 120 minutes of hypothermic cardioplegic arrest. Whereas nontreated animals showed a 30% to 40% reduction of cardiac contractility during reperfusion, treatment with nicotinic acid preserved myocardial mechanical function. A slight, not statistically significant, increase in LVEDP during reperfusion was not reversed by treatment with nicotinic acid. Improved cardiac contractility in the hearts treated with nicotinic acid was associated with higher coronary flow (which did not reach statistical significance) and myocardial oxygen consumption (p < 0.05) compared with nontreated hearts. An increase in CK release during reperfusion was also significantly inhibited in hearts treated with nicotinic acid, which suggested that the integrity of cellular membranes was improved. The results are consistent with the hypothesis that abnormal free fatty acid metabolism taking place in the ischemic heart is at least partly responsible for cardiac dysfunction after heart operations. The present study used 120 minutes of hypothermic cardioplegic arrest in the isolated in situ pig heart model without a previous ischemic insult. Only a small volume of cardioplegic solution was deliberately administered (50 m1 every 15 minutes) to permit other modalities of preservation to have potential for showing improvement. This experimental setting obviously produces less myocardial damage than one that uses the combination of normothermic regional ischemia and hypothermic cardioplegic arrest as previously done in this laboratory.": 15 Limitation of ischemic myocardial damage was
The Journal of Thoracic and Cardiovascular Surgery
reflected in the preservation of ATP during hypothermic cardioplegic arrest and reperfusion. Nevertheless, lowered creatine phosphate levels after 120 minutes of arrest indicated that metabolism was certainly inhibited in these hearts, which resulted in a marked imbalance between energy utilization and supply. Furthermore, cardiac performance was depressed in nontreated animals during reperfusion despite the fact that myocardial creatine phosphate content returned to a baseline level. Treatment with nicotinic acid improves energy utilization during reperfusion for restoration of normal cardiac function. In this regard, it has been shown that longchain fatty acid metabolites, which are known to accumulate in the ischemic heart as a result of the inhibition of l3-oxidation, are inhibitory to enzyme systems involved in energy-dependent ionic transport in th sarcolemmal membrane and the sarcoplasmic reticulum. 16• 17 Although the precise mechanism by which nicotinic acid improves ischemic myocardial function is not discussed here, this drug has been shown to decrease plasma free fatty acid levels when administered orally or intravenously," thereby reducing free fatty acid utilization by the ischemic myocardium. 18 This systemic hypolipidemic action is presumed to be due to inhibition of the lipolysis from adipose tissues. 19. 20 The evidence shown in the present study that nicotinic acid did not influence plasma free fatty acid levels in the isolated in situ pig heart model, in which there is no systemic circulation reaching the peripheral adipose tissues, supports this view. It must, therefore, be assumed that the beneficial effects of nicotinic acid on cardiac performance as seen in this study are primarily attributable to a direct interaction with myocardial free fatty acid metabolism. The present study for the first time provides conclusive evidence that antilipolytic therapy facilitates restoration of cardiac performance after hypothermic cardioplegic arrest by an intrinsic myocardial effect. REFERENCES 1. Kanter KR, Jaffin JH, Ehrlichman RJ, Flaherty JT, Gott VL, Gardner JT. Superiority of perfluorocarbon cardioplegia over blood or crystalloid cardioplegia. Circulation 1981;64(Pt 2):1175-80. 2. Engelman RM, Das KD, Otani H, Rousou lA, Breyer RH. Retrograde coronary sinus cardioplegia-influence on fatty acid metabolism during myocardial ischemia. In: Proceedings of the second international symposium on myocardial protection via the coronary sinus. New York: Springer-Verlag, 1986:215-20. 3. Oram JF, Bennetch SL, Neely JF. Regulation of fatty
Volume 96 Number 1 July 1988
acid utilization in isolated perfused rat hearts. J BioI Chern 1973;248:5299-309. 4. Neely JR, Rouetto MJ, Oram JF. Myocardial utilization of carbohydrate and lipids. Prog Cardiovasc Dis 1972; 15:289-329. 5. Das DK, Engelman RM, Rousou JA, Breyer RH, Otani H, Lemeshow S. Role of membrane phospholipids in myocardial injury induced by ischemia and reperfusion. Am J Physiol 1986;251:H71-9. 6. Corr PB, Gross RW, Sobel BE. Amphipathic metabolites and membrane dysfunction in ischemic myocardium. Circ Res 1984;55:135-54. 7. Engelman RM, Adler S, Gauge TH, Chandra R, Boyd AD. The effect of normothermic anoxic arrest and ventricular fibrillation on the coronary blood flow distribution of the pig. J THORAC CARDIOVASC SURG 1975; 69:858-69. 8. Cordis GA, Engelman RM, Das DK. Simultaneous quantification of myocardial adenine nuc1eotides and creatine phosphate by ion-pair reverse phase HPLC. J Chromatogr 1987;386:283-8. 9. Dobbs WA, Engelman RM, Rousou JH, Douglas DM, Lemeshow S, Avrunin JA. Performance of pig heart after 30 or 120 minutes' hypothermic arrest. J Surg Res 1983;35:132-41. 10. Oliver IT. A spectrophotometric method for the determination of creatine phosphokinase and myokinase. Biochern J 1955;61:116-22. II. Hamilton JG, Comai K. Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection. J Lipid Res 1984;25:1142-8.
Myocardial protection with nicotinic acid
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12. Wood R, Lee T. High-performance liquid chromatography of fatty acids: quantitative analysis of saturated monoenoic, polyenoic and geometrical isomers. J Chromatogr 1983;254:237-46. 13. Hotz W. Nicotinic acid and its derivatives. Adv Lipid Res 1983;20;195-217. 14. Otani H, Engelman RM, Rousou JA, Breyer RH, Lemeshow S, Das DK. Cardiac performance during reperfusion improved by pretreatment with oxygen freeradical scavengers. J THORAC CARDIOVASC SURG 1986; 91:290-5. 15. Otani H, Engelman RM, Breyer RH, Rousou lA, Lemeshow S, Das DK. Mepacrine, a phospholipase inhibitor. J THORAC CARDIOVASC SURG 1986;92:247-54. 16. Whitmer JT, Zdell-Wenger JA, Rouetto MJ, Neely JR. Control of fatty acid metabolism in ischemic and hypoxic hearts. J BioI Chern 1978;253:4305-9. 17. Adams RJ, Cohen OW, Gupte S, et al. In vitro effects of long-chain palmitylcarnitine on cardiac plasma membrane Na, K-ATPase, and sarcoplasmic reticulum Ca "ATPase and Ca transport. J Bioi Chern 1979;254:1240410. 18. Russell DC, Oliver MF. Effect of antilipolytic therapy on ST segment elevation during myocardial ischemia in man. Br Heart J 1978;40:117-23. 19. Carlson LA, Oro L. The effect of nicotinic acid on the plasma free fatty acids. Acta Med Scand 1962;172:6415. 20. Kudchodkar BJ, Sodhi HS, Horlick L, Mason DT. Mechanisms of hypolipidemic action of nicotinic acid. Clin Pharmacol Ther 1978;24:354-73.
THORAC CARDIOVASC SURG
1988;96:81-7
Enhanced myocardial preservation by nicotinic acid, an antilipolytic compound Improved cardiac performance after hypothermic cardioplegic arrest The effect of nicotinic acid, an antilipolytic drug, on myocardial preservation was studied on the basis of cardiac performance after 2 hours of cardioplegic arrest. Isolated in situ pig hearts were subjected to 120 minutes of hypothermic potassium (35 mEq) crystalloid cardioplegic arrest followed by 60 minutes of reperfusion. The experimental group received nicotinic acid 0.08 mmoljL 15 minutes before cardioplegic arrest, whereas the control group received 15 minutes of unmodified perfusion. There was a marked decline in myocardial creatine phosphate levels during cardioplegic arrest in both groups that returned to the baseline level during reperfusion without a significant intergroup difference, and adenosine triphosphate levels remained stable throughout the experiment in both groups. Myocardial oxygen consumption during reperfusion was significantly higher in hearts treated with nicotinic acid, which was consistent with a significantly greater cardiac contractile force as evaluated by isovolumetric left ventricular pressure measurements. There appeared to be less cardiac membrane damage as measured by creatine kinase release during reperfusion, which was significantly inhibited by treatment with nicotinic acid. The present study supports the conclusion that nicotinic acid improves cardiac performance after hypothermic cardioplegic arrest.
Hajime Otani, MD, PhD, Richard M. Engelman, MD, Subhajit Datta, MD, Randall M. Jones, BS, Gerald A. Cordis, MS, John A. Rousou, MD, Robert H. Breyer, MD, and Dipak K. Das, PhD, Springfield. Mass.
Myocardial preservation induced by hypothermic cardioplegicarrest protects the ischemic heart primarily by reducing metabolic rate and energy demand. Intermittent infusion of either a crystalloid or blood cardioplegic solution, however, does not maintain optimal aerobic metabolism during hypothermic cardioplegic arrest. l This lack of adequate oxidative metabolism in the myocardium during hypothermic cardioplegic arrest
From the Department of Surgery, University of Connecticut School of Medicine, Farmington, Conn., and Baystate Medical Center, Springfield, Mass. Supported by National Institutes of Health Grants HL 22559-06, HL33889, and HL34360; American Heart Association Grant 11-202-856. Presented at the Thirty-sixth Annual Scientific Session, American College of Cardiology, New Orleans, La., March 1987. Received for publication April 3, 1987. Accepted for publication Nov. 2, 1987. Address for reprints: Richard M. Engelman, MD, Baystate Medical Center, 759 Chestnut St., Springfield, MA 01107.
was further suggested by our recent study- demonstrating an accumulation of reducing equivalents as evidenced by the rise in the reduced nicotinamide-adenine dinucleotide/nicotinamide-adenine dinucleotide ratio even after 40 minutes of arrest when no significant decline in the adenosine triphosphate (ATP) level was noted. The consequence of a reduced metabolic rate with oxygen deprivation is the inhibition of free fatty acid utilization, because the rate of free fatty acid oxidation is linearly related to the rate of oxidative phosphorylation.' The inhibition of ~-oxidation in conjunction with the stimulation of phospholipid degradation in ischemic and reperfused myocardium results in the intracellular accumulation of free fatty acids and their metabolites.t' These molecules have been shown by their detergent properties to disrupt membrane integrity, alter cellular structure, and inhibit activities of enzyme systems, which changes are potentially detrimental to both mechanical and electrophysiologic function."Although abnormal free fatty acid metabolism has been well characterized in normothermic global or 81
82
The Journal of Thoracic and Cardiovascular Surgery
Otani et al.
"'"',-y------------.,. 100
50
l IEQ
~; 3
otg
-ell>
is "0
III ;:ltll
~~ o
15'
0'
0'
30'
Pre-Ischemia
15'
TIME (minutes)
30'
0
60'
45'
Reperfusion • (+) Nicotinic Acid
(-) Nicotinic Acid
Fig. 1. Effect of nicotinic acid on myocardial adenosine triphosphate and creatine phosphate as a function of time.
Table I. Effects of nicotinic acid on coronary flow and myocardial oxygen consumption Preischemia Time (min)
Pre-NA
Reperfusion Post-NA
15 min
30 min
45 min
60 min
6.95 ± 0.76 8.06 ± 0.83
5.86 ± 1.06 8.16 ± 1.64
5.20 ± 1.12* 7.53 ± 0.72
5.11±1.I1 7.86 ± 0.80
5.02 ± 1.25 8.31 ± l.01
181 ± 28 203 ± 22
132 ± 14 184 ± 20t
149 ± 16 209 ± 22t
132 ± 24 177 ± 33
126 ± 17* 182 ± 26
Coronary flow (ml/min/IOO gm) (-) NA (+)NA
6.96 ± 0.45 8.l3±0.46
Oxygen consumption (ILI/gm/min) (-) NA (+) NA
201 ± 43 209 ± 28
NA, Nicotinic acid. 'p < 0.05, reperfusion versus control. tp <0.05. (+) NA versus (-) NA.
regional ischemic heart models, no work has been done concerning free fatty acid metabolism and its relation to cardiac performance during hypothermic cardioplegic arrest and reperfusion. In view of the deleterious effects of increased free fatty acid levels in ischemic heart, we used nicotinic acid, an antilipolytic drug, in the isolated in situ pig heart, which was subjected to 120 minutes of hypothermic potassium crystalloid cardiopegic arrest followed by 60 minutes of reperfusion.
Materials and methods Animal preparation. Twenty-six Yorkshire pigs of either sex, weighing 15 to 23 kg, were tranquilized with ketamine (Ketaject, 50 mg/kg) and anesthetized with intravenous injection of pentobarbital (Nembutal, 25 rug/kg). Each animal was supported by controlled respiration with room air, and the chest was opened through a median sternotomy. Sodium heparin (500 units/kg) was administered, and the animal was
placed on cardiopulmonary bypass via the innominate artery and right atrial cannulation with a bubble oxygenator (Bentley BOS-5, American Bentley, Irvine, Calif). The isolated in situ pig heart preparation was originally described in 197Y and has been modified to fit our present needs. The method used is to initially place the pig on cardiopulmonary bypass and then perfuse the ascending aortic root proximal to an aortic cross-clamp in such a manner that only the coronary circulation is supplied with blood. The systemic circulation is discontinued and, after the systemic circulation has drained into the oxygenator, both the superior and inferior venae cavae are ligated. During the study, coronary perfusion pressure is maintained at 75 mm Hg, which is regulated by transducer cannulation of the aortic root. Myocardial perfusate temperature is maintained at 37° C unless hypothermia is used. The pericardial well is utilized as a constant temperature bath, and the fluid in which the heart is bathed is regulated at either normothermic or hypothermic levels. When appropriate, coronary effluent is collected through a cannula inserted into the right ventricular outflow
Volume 96 Number 1
Myocardial protection with nicotinic acid
July 1988
83
150 .,-------~m~%:mm_-------------T
1
i
100
..
'.,I'
1···· 1·····
t -----:r------r------r
NicotInIC
ACId
50
o
0'
15'
0'
30'
15'
TIME (minutes)
Pre-Ischemia --, (-) Nicotinic Acid
30'
45'
60'
Reperfusion
'p < .05
••• (+) Nicotinic Acid
"p < .005
Fig. 2. Effect of nicotinic acid on left ventricular developed pressure as a function of time.
25001---~~~~~----------1
2000 ~
......
}:::::I
C.
I.·•
T ••
1·······.r·
'0
E
1500
:::J
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'" ¥ '" 1000
~
.!!! E
i3 E
.~-
C Ql
>
500
15'
I
0'
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Pre-Ischemia - -, (-) Nicotinic Acid
15'
TIME (minutes) ••• (+) Nicotinic Acid
30'
45'
60'
Reperfusion 'p < .05
"p < .005
Fig. 3. Effect of nicotinic acid on left ventricular maximum dpjdt as a function of time.
tract, and coronary flow is measured by timed collection from thiscannula. Blood samples are drawn from the arterial inflow line and, when appropriate, from the coronary sinus by direct cannulation via the right atrial wall. After the isolated heart model was stabilized for 15 minutes, experimental procedures were initiated. Thirteen hearts were treated with nicotinic acid 0.08 mmol/L (Sigma Diagnostics, St. Louis, Mo.) for 15 minutes before hypothermic cardioplegic arrest. The remaining 13 hearts received untreated perfusion for 15 minutes. Hypothermic cardioplegic arrest was induced
with 50 ml of chilled (4° C) hyperkalemic (35 mEqjL) crystalloid solution after the hearts were cooled to 10° C in a hypothermic topical solution in the pericardial well. Hypothermic cardioplegic arrest was maintained for 120 minutes with reinfusion of the same volume of cardioplegic solution at IS-minute intervals, and myocardial temperature (measured by temperature probe) was maintained at 8° to 10° C throughout the duration of arrest. Reperfusion was performed at normothermic temperature at a constant pressure (75 nun Hg) for 60 minutes with the heart in a normothermic bath. In
84
The Journal of Thoracic and Cardiovascular Surgery
Otani et al.
30r----~
Gi
..J
15'
30'
0'
15'
Pre-Ischemia
30'
45'
60'
Reperfusion
TIME (minutes) - -, (-) Nicotinic Acid
••
(+)
Nicotinic Acid
Fig. 4. Effect of nicotinic acid on left ventricular end-diastolic pressure as a function of time.
400
5l
,------~
300
<':l Ql
Qi
a: Ql~
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<,
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200
Ql
100
15'
0'
30'
Pre-Ischemia
15'
TIME (minutes)
--, (-) Nicotinic Acid ••• (+) Nicotinic Acid
30'
45'
60'
Reperfusion 'p < .05
Fig. 5. Effect of nicotinic acid on creatine kinase release as a function of time.
the event of ventricular fibrillation, the heart was defibrillated by a direct shock of 20 watt/sec. Myocardial biopsy specimens were taken from six hearts in each group at indicated times with a dental drill and freeze-clamp techniques. Cardiac performance was evaluated in seven hearts in each group. Determination of high-energy phosphates. Myocardial tissue samles were homogenized with a 10 times volume of 6% perchloric acid in a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, N.Y.). After centrifugation of 10,000 gm, the supernatant fluid was neutralized and assayed for ATP and creatine phosphate by high-pressure liquid chromatography as recently described by our laboratory."
Measurements of cardiac performance. Myocardial oxygen consumption was measured according to the formula described previously." Left ventricular developed pressure (LVDP), maximum rate of rise of left ventricular pressure (LV max dp/dt), and left ventricular end-diastolic pressure (LVEDP) were determined under isovolumic conditions through a Millar Mikro-Tip catheter pressure transducer (Millar Instruments, Inc., Houston, Texas) within a 10 ml compliant balloon inserted into the left ventricle via the apex. Assay of creatine kinase. Creatine kinase (CK) was assayed in the perfusate plasma by the enzymatic assay
Volume 96 Number 1
Myocardial protection with nicotinic acid
July 1988
L-----
200
r
:g
~
n;:J u.,
:ll
g 100
85
J
----------1
NcoIr1C Acid
tt~
o+------,--T=
15' 30' Pre-Ischemia
0'
15'
30' 45' Reperfusion
60'
TIME (minutes) -_. (-) Nicotinic Acid
•• (+) Nicotinic Acid
Fig. 6. Effect of nicotinic acid on plasma free fatty acid level as a function of time.
method" with a CK assay kit obtained from Sigma Diagnostics. The data were initially obtained 15 minutes after the preparation of the isolated in situ pig heart. Determination of plasma free fatty acids. Blood samples collected during nonbiopsy experiments were assayed for plasma free fatty acid levels. One milliliter of plasma was mixed with ice-cold chloroform-methanol solution (2: I, v Iv) containing 0.005% butylated hydroxy toluene. Twenty nanomoles of carbon : 17 was added as an internal standard to monitor the recoveries of free fatty acids during extraction procedures. The organic layer was aspirated and was taken into near dryness under a stream of nitrogen gas. A portion of the lipid extracts were used for determination of free fatty acids. The samples were added to a SEP-PAK silica cartridge (Waters, Milford, Mass.) for separation of the neutral lipids and free fatty acids from the phospholipids as described by Hamilton and Comai." Phenacyl esters of free fatty acids derived from the pooled eluents were analyzed by highpressure liquid chromatography according to the method of Wood and LeeY Statistical analysis. Both a paired t test and a two-sample Student's t test were used for statistical analysis. Results are expressed as mean ± standard error of the mean and were considered to be statistically significant when the p value was <0.05. Results
Effects of nicotinic acid on myocardial high-energy phosphate contents. Fig. 1 shows myocardial ATP and creatine phosphate contents as a function of time. There was no substantial change in the level of myocardial ATP during 120 minutes of hypothermic cardioplegic arrest and reperfusion in both animals treated with nicotinic acid and nontreated animals. Myocardial creatine phosphate contents, however, declined markedly during 120 minutes of arrest in both groups of animals
and then returned to the baseline value as early as 15 minutes after reperfusion in both groups of animals with no difference between the groups.
Effects of nicotinic acid on coronary ftow and myocardial oxygen consumption. Effects of nicotinic acid on coronary flow and myocardial oxygen consumption are shown in Table I. These measurements were not significantly altered during pretreatment with nicotinic acid before hypothermic cardioplegic arrest. Coronary flow decreased significantly during reperfusion in nontreated animals, whereas in animals treated with nicotinic acid flow was maintained near the baseline level. Oxygen consumption also decreased significantly during reperfusion in nontreated hearts. This decline in oxygen consumption was significantly inhibited in hearts treated with nicotinic acid.
Effects of nicotinic acid on cardiac contractile function. The effects of nicotinic acid on cardiac contractile function are shown in Figs. 2 through 4. There was a 30% decrease in LVDP and a 40% decrease in LV max dp / dt from baseline levels 15 minutes after reperfusion in nontreated hearts. Treatment with nicotinic acid prevented this decline in LVDP and LV max dp/dt significantly compared with the results in nontreated animals throughout the reperfusion period. Cardiac compliance as estimated by LVEDP was reduced only slightly during reperfusion in both groups of animals, and there was no significant difference between the groups. Effects of nicotinic acid on CK release. Because the release of CK is directly related to the integrity of the cellular membrane, we analyzed the effects of nicotinic
86
Otani et al.
acid on CK release (Fig. 5). Plasma CK levels were increased after reperfusion in nontreated and nicotinic acid-treated animals. There was a dramatic increase in CK release during the rest of the reperfusion period in nontreated animals, which was significantly inhibited in animals treated with nicotinic acid. Effects of nicotinic acid on plasma free fatty acid levels. Because nicotinic acid is known as a hypolipidemic drug, 13 we investigated the effect of nicotinic acid on plasma free fatty acid levels (Fig. 6). Plasma free fatty acid levels were essentially unchanged during these experiments, which indicated that treatment with nicotinic acid did not influence plasma free fatty acid levels in our experimental model. There was also no significant difference in the concentrations of the individual free fatty acid classes in the plasma fraction between nontreated animals and animals treated with nicotinic acid. Discussion The results of the present study demonstrate that pretreatment with nicotinic acid provides significant benefit as measured by the restoration of cardiac performance after 120 minutes of hypothermic cardioplegic arrest. Whereas nontreated animals showed a 30% to 40% reduction of cardiac contractility during reperfusion, treatment with nicotinic acid preserved myocardial mechanical function. A slight, not statistically significant, increase in LVEDP during reperfusion was not reversed by treatment with nicotinic acid. Improved cardiac contractility in the hearts treated with nicotinic acid was associated with higher coronary flow (which did not reach statistical significance) and myocardial oxygen consumption (p < 0.05) compared with nontreated hearts. An increase in CK release during reperfusion was also significantly inhibited in hearts treated with nicotinic acid, which suggested that the integrity of cellular membranes was improved. The results are consistent with the hypothesis that abnormal free fatty acid metabolism taking place in the ischemic heart is at least partly responsible for cardiac dysfunction after heart operations. The present study used 120 minutes of hypothermic cardioplegic arrest in the isolated in situ pig heart model without a previous ischemic insult. Only a small volume of cardioplegic solution was deliberately administered (50 m1 every 15 minutes) to permit other modalities of preservation to have potential for showing improvement. This experimental setting obviously produces less myocardial damage than one that uses the combination of normothermic regional ischemia and hypothermic cardioplegic arrest as previously done in this laboratory.": 15 Limitation of ischemic myocardial damage was
The Journal of Thoracic and Cardiovascular Surgery
reflected in the preservation of ATP during hypothermic cardioplegic arrest and reperfusion. Nevertheless, lowered creatine phosphate levels after 120 minutes of arrest indicated that metabolism was certainly inhibited in these hearts, which resulted in a marked imbalance between energy utilization and supply. Furthermore, cardiac performance was depressed in nontreated animals during reperfusion despite the fact that myocardial creatine phosphate content returned to a baseline level. Treatment with nicotinic acid improves energy utilization during reperfusion for restoration of normal cardiac function. In this regard, it has been shown that longchain fatty acid metabolites, which are known to accumulate in the ischemic heart as a result of the inhibition of l3-oxidation, are inhibitory to enzyme systems involved in energy-dependent ionic transport in th sarcolemmal membrane and the sarcoplasmic reticulum. 16• 17 Although the precise mechanism by which nicotinic acid improves ischemic myocardial function is not discussed here, this drug has been shown to decrease plasma free fatty acid levels when administered orally or intravenously," thereby reducing free fatty acid utilization by the ischemic myocardium. 18 This systemic hypolipidemic action is presumed to be due to inhibition of the lipolysis from adipose tissues. 19. 20 The evidence shown in the present study that nicotinic acid did not influence plasma free fatty acid levels in the isolated in situ pig heart model, in which there is no systemic circulation reaching the peripheral adipose tissues, supports this view. It must, therefore, be assumed that the beneficial effects of nicotinic acid on cardiac performance as seen in this study are primarily attributable to a direct interaction with myocardial free fatty acid metabolism. The present study for the first time provides conclusive evidence that antilipolytic therapy facilitates restoration of cardiac performance after hypothermic cardioplegic arrest by an intrinsic myocardial effect. REFERENCES 1. Kanter KR, Jaffin JH, Ehrlichman RJ, Flaherty JT, Gott VL, Gardner JT. Superiority of perfluorocarbon cardioplegia over blood or crystalloid cardioplegia. Circulation 1981;64(Pt 2):1175-80. 2. Engelman RM, Das KD, Otani H, Rousou lA, Breyer RH. Retrograde coronary sinus cardioplegia-influence on fatty acid metabolism during myocardial ischemia. In: Proceedings of the second international symposium on myocardial protection via the coronary sinus. New York: Springer-Verlag, 1986:215-20. 3. Oram JF, Bennetch SL, Neely JF. Regulation of fatty
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acid utilization in isolated perfused rat hearts. J BioI Chern 1973;248:5299-309. 4. Neely JR, Rouetto MJ, Oram JF. Myocardial utilization of carbohydrate and lipids. Prog Cardiovasc Dis 1972; 15:289-329. 5. Das DK, Engelman RM, Rousou JA, Breyer RH, Otani H, Lemeshow S. Role of membrane phospholipids in myocardial injury induced by ischemia and reperfusion. Am J Physiol 1986;251:H71-9. 6. Corr PB, Gross RW, Sobel BE. Amphipathic metabolites and membrane dysfunction in ischemic myocardium. Circ Res 1984;55:135-54. 7. Engelman RM, Adler S, Gauge TH, Chandra R, Boyd AD. The effect of normothermic anoxic arrest and ventricular fibrillation on the coronary blood flow distribution of the pig. J THORAC CARDIOVASC SURG 1975; 69:858-69. 8. Cordis GA, Engelman RM, Das DK. Simultaneous quantification of myocardial adenine nuc1eotides and creatine phosphate by ion-pair reverse phase HPLC. J Chromatogr 1987;386:283-8. 9. Dobbs WA, Engelman RM, Rousou JH, Douglas DM, Lemeshow S, Avrunin JA. Performance of pig heart after 30 or 120 minutes' hypothermic arrest. J Surg Res 1983;35:132-41. 10. Oliver IT. A spectrophotometric method for the determination of creatine phosphokinase and myokinase. Biochern J 1955;61:116-22. II. Hamilton JG, Comai K. Separation of neutral lipids and free fatty acids by high-performance liquid chromatography using low wavelength ultraviolet detection. J Lipid Res 1984;25:1142-8.
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12. Wood R, Lee T. High-performance liquid chromatography of fatty acids: quantitative analysis of saturated monoenoic, polyenoic and geometrical isomers. J Chromatogr 1983;254:237-46. 13. Hotz W. Nicotinic acid and its derivatives. Adv Lipid Res 1983;20;195-217. 14. Otani H, Engelman RM, Rousou JA, Breyer RH, Lemeshow S, Das DK. Cardiac performance during reperfusion improved by pretreatment with oxygen freeradical scavengers. J THORAC CARDIOVASC SURG 1986; 91:290-5. 15. Otani H, Engelman RM, Breyer RH, Rousou lA, Lemeshow S, Das DK. Mepacrine, a phospholipase inhibitor. J THORAC CARDIOVASC SURG 1986;92:247-54. 16. Whitmer JT, Zdell-Wenger JA, Rouetto MJ, Neely JR. Control of fatty acid metabolism in ischemic and hypoxic hearts. J BioI Chern 1978;253:4305-9. 17. Adams RJ, Cohen OW, Gupte S, et al. In vitro effects of long-chain palmitylcarnitine on cardiac plasma membrane Na, K-ATPase, and sarcoplasmic reticulum Ca "ATPase and Ca transport. J Bioi Chern 1979;254:1240410. 18. Russell DC, Oliver MF. Effect of antilipolytic therapy on ST segment elevation during myocardial ischemia in man. Br Heart J 1978;40:117-23. 19. Carlson LA, Oro L. The effect of nicotinic acid on the plasma free fatty acids. Acta Med Scand 1962;172:6415. 20. Kudchodkar BJ, Sodhi HS, Horlick L, Mason DT. Mechanisms of hypolipidemic action of nicotinic acid. Clin Pharmacol Ther 1978;24:354-73.