Sodium Pentobarbital Versus α-Chloralose Anesthesia
Experimental Production of Substantially Different Slopes in the Transmural CP/ATP Ratios Within the Left Ventricle of the Canine Myocardium
Background Transmural analyses of the creatine phosphate (CP)/ATP ratio in various lamina of the canine myocardium have previously revealed significant variations in the CP/ATP ratio, with the subendocardial layer displaying a decreased ratio relative to the subepicardial layer. Without exception, these results were obtained under sodium pentobarbital anesthesia. These findings have been interpreted to imply that the normal endocardium may be operating in the oxygen-limited domain or that there are transmurally varying set points for the regulation of oxidative phosphorylation.
Methods and Results In this work, we examine the effect of the anesthetic regimen on the transmural CP/ATP ratio within the left ventricular wall of the canine myocardium using spatially localized 31P-nuclear magnetic resonance (NMR) and an open-chest model. Two anesthetics were compared, α-chloralose and sodium pentobarbital. Under sodium pentobarbital, the CP/ATP ratio ranged from 1.92±0.06 to 2.51±0.08 from endocardium to epicardium, resulting in a transmural slope in the CP/ATP ratio of 0.149±0.047 (n=22). Under α-chloralose, CP/ATP ratios ranged from 2.18±0.05 to 2.32±0.06, with a transmural slope of 0.035±0.018 (n=38). Thus, the transmural slope in CP/ATP ratio was nearly four times greater with sodium pentobarbital than with α-chloralose, and the difference in these slopes was statistically significant (P=.029). No difference was observed in average CP/ATP obtained from the entire wall with either anesthetic.
Conclusions These results demonstrate that the transmural trend in CP/ATP ratio previously reported in the myocardium is likely to be a direct reflection of the sodium pentobarbital anesthetic regimen, not truly reflecting the trend in the normal unanesthetized animal. Moreover, since the transmural variation in CP/ATP ratio was greatly reduced with α-chloralose, it appears unlikely that the endocardium in the normal unanesthetized heart is operating in the oxygen-limited domain. These results also point to the importance of the anesthetic regimen in biochemical analysis, indicate the necessity of increased caution in directly translating results obtained under anesthesia, and demonstrate the unique power of in vivo NMR to extract such subtle biochemical information.
Previous transmural analyses of the creatine phosphate (CP)/ATP ratio in the canine myocardium have generally used biopsies followed by chemical analysis of the resultant extracts.1 2 3 4 While considerable scatter in the CP/ATP ratio is reported in these classic studies, they share the common finding that there is a slight drop in the CP/ATP ratio in the endocardium. These findings have recently been confirmed5 by localized transmural 31P-nuclear magnetic resonance (NMR).6 As a rule, in these studies, ATP levels were found to be constant across the heart wall. However, CP, and as a result the CP/ATP ratio, were significantly reduced in the subendocardium. Consequently, the calculated free ADP was found to be higher in the subendocardium versus the epicardium. Interestingly, these results were obtained, without exception, under sodium pentobarbital anesthesia.1 2 3 4 5 6 More importantly, the findings of reduced CP/ATP ratios in the endocardium have been interpreted to imply that the endocardium in the normal dog may be operating in the oxygen-limited domain or that there are transmurally varying set points for the regulation of oxidative phosphorylation.6 7 The oxygen-limited domain is a condition in which the ability of the endocardium to perform work and consume oxygen would be determined by oxygen availability.
However, it is extremely unlikely that the normal myocardium would evolve in such a way as to permit the normal disease-free endocardium to operate continuously in the oxygen-limited domain. Thus, we searched for possible experimental causes of these effects and monitored the transmural CP/ATP ratios in the in vivo canine myocardium while comparing two well-known anesthetics, sodium pentobarbital and α-chloralose. When the autonomic control of the cardiovascular system is considered, α-chloralose has been advanced as a more physiological anesthetic than sodium pentobarbital.8 9 10 11
All studies were conducted under institutional approval and conformed to the guiding principles of the American Physiological Society. Dogs weighing between 15 and 20 kg were preanesthetized with ketamine HCl (20 mg/kg). This was followed by an initial infusion of 2 to 3 g of α-chloralose (10 mg/mL in 0.9% NaCl) and maintained with repeated 1-g boluses as needed. Alternatively, sodium pentobarbital anesthesia was initiated with 30 mg/kg and was maintained with increments of 2.5 mg/kg as necessary to sustain surgical anesthesia as judged by palpebral reflex and loss of tone in masseter muscles. Dogs anesthetized with sodium pentobarbital were given fluids comparable in volume to those given the animals receiving α-chloralose. Arterial blood gases were monitored, and ventilation was adjusted to sustain a Paco2 of approximately 35 to 40 mm Hg.
A left thoracotomy was performed at the fifth intercostal space. The pericardial sac was then opened, and a 28-mm surface coil was sutured onto the myocardium over the region of the ventricle perfused by the left anterior descending coronary artery. A polyethylene catheter was inserted at the apex of the heart into the left ventricle and was used to monitor cardiac function while providing the input signal for cardiac gating and respiratory control.12 A venous catheter inserted into the cranial vena cava through the right external jugular vein was used for administration of fluids and drugs and for right atrial pressure measurements. Regional blood flows were determined by radiolabeled microspheres as previously described in detail.13 14 Radiolabeled microspheres were injected into the left atrium through a polystyrene catheter. Blood for a reference sample was withdrawn from a catheter placed through a femoral artery and into the descending thoracic aorta. A catheter was also inserted through the contralateral femoral artery to monitor mean arterial pressure. There were no variations between these two experimental groups other than the anesthetic regimen. There were also no variations in surgical timing, instrumentation, or NMR examination between the two groups.
Once surgery was completed, the animal was placed within the bore of a 4.7-T, 40-cm magnet for observation. The proton resonance was used to adjust field homogeneity, after which the 31P studies were initiated. All transmural 31P-NMR measurements were obtained with the FLAX-ISIS spatial localization sequence as reported previously,5 6 and the fully relaxed acquisition conditions15 obtained with 12-second repetition times were used. Normal saline boluses were given to maintain mean right atrial pressure. Hemodynamic parameters are summarized in Table 1⇓. Radiolabeled microspheres were injected before (141Ce) and after (51Cr) the acquisition of two FLAX-ISIS 31P-NMR data sets at basal workloads. After completion of the NMR studies, the myocardium beneath the surface coil was sectioned into epicardial, midmyocardial, and endocardial lamina. The kidneys were sectioned to validate blood flow measurements. Tissue counts were obtained with an autogamma counter (Packard 9042). After correction for spectral overlap and peak heights, tissue blood flows were calculated. A 10% difference in kidney blood flow or tissue microsphere numbers of <400 were criteria for exclusion. The presence of heartworms was also considered an immediate basis for exclusion. Under these criteria, no animals were excluded on the basis of blood flow, and five animals were excluded because of the presence of heartworms, resulting in a total of 22 sodium pentobarbital and 38 α-chloralose studies. Mean arterial pressures, cardiac indexes, and regional blood flow measurements were obtained only in a subset of animals (7 α-chloralose and 12 sodium pentobarbital). NMR spectra were integrated by use of standard GE omega software.
All data analysis was performed with the s-plus statistical analysis package (Statistical Sciences, Inc). Since we were most interested in intervoxel variations from epicardium to endocardium and not in absolute subject-to-subject differences, intraventricular wall differences in CP/ATP were analyzed in each animal. This was accomplished by fitting the data points obtained for all five voxels to the linear equation Yi=ΔmXi+Y0, where Xi represents a particular voxel of interest and Yi the experimental value of CP/ATP for that voxel. For this analysis, the voxels were assumed to be equally separated. The value of Xi increased from 1 to 5 in moving from the endocardium to the epicardium. All errors are reported as SD, with the exception of CP/ATP values, which are reported as SEM. From the signal-to-noise ratio of the spectra, the approximate error of integration for the CP/ATP ratio was assigned as ±15%. Statistical methods were then applied to the analysis of the calculated transmural slopes (Δm) for CP/ATP by use of a standard Student’s t test. Regional blood flow measurements were also compared against rate-pressure products and cardiac index by use of Pearson correlation analysis with Bonferroni corrections for multiple comparisons.
Differences in rate-pressure products, cardiac indexes, blood flows, blood flow ratios, mean arterial pressures, and total fluids for sodium pentobarbital and α-chloralose anesthetics are summarized in Table 1⇑. There were no differences between groups in experiment length or total intravenous fluids. In the subset of animals with blood flow measurements, lower myocardial blood flows were observed with α-chloralose. Similarly, although mean arterial pressures were lower under α-chloralose, there were no statistically significant differences in cardiac indexes between the two anesthetics. Importantly, the α-chloralose animals displayed endocardial/epicardial blood flow ratios of 1.20±0.17, suggesting adequate endocardial perfusion. Significant differences in rate-pressure products were observed between the two anesthetic regimens (P<.001). In addition, the ratio of global blood flow to average rate-pressure product was nearly identical with both anesthetics (see Table 1⇑). This suggests no differences in flow/perfusion mismatch between these two groups. Pearson correlation analysis on these data also revealed a strong relation between endocardial blood flows and cardiac index (P<.001, r=.82) or rate-pressure product (P=.005, r=.68) in the sodium pentobarbital animals. No such correlation for endocardial blood flows was observed in the α-chloralose group. Epicardial flows were not correlated to any of these parameters with either anesthetic.
Representative FLAX-ISIS spectra obtained under α-chloralose and sodium pentobarbital anesthesia are displayed in Figs 1⇓ and 2⇓, respectively. Subjective examination of these spectra quickly reveals (without integration) that the α-chloralose anesthetic regimen results in a nearly transmurally invariant CP/ATP ratio and that sodium pentobarbital results in a noticeable gradation in this ratio, with a decrease in the ratio toward the subendocardium. Transmural CP/ATP ratios observed under sodium pentobarbital and α-chloralose anesthesia at basal workloads are summarized in Fig 3A⇓ and 3B⇓, respectively, and a summary of transmural slopes can be found in Table 2⇓. The CP/ATP ratios under sodium pentobarbital anesthesia increased from 1.92±0.06 to 2.51±0.06 from endocardium to epicardium, resulting in a transmural slope of 0.149±0.047 (Fig 3A⇓). Interestingly, the CP/ATP ratio was essentially transmurally invariant under α-chloralose anesthesia, ranging from 2.18±0.05 in the endocardium to 2.32±0.06 in the epicardium, resulting in a transmural slope of 0.035±0.018 (Fig 3B⇓). The difference in these two slopes was statistically significant (P=.029). All NMR spectra were devoid of any signs of ischemia, namely increases in inorganic phosphate and decreases in intracellular pH values. The average myocardial CP/ATP ratio from all voxels was 2.23±0.06 under α-chloralose anesthesia and 2.17±0.07 under sodium pentobarbital. The presence of nearly identical global CP/ATP ratios under both anesthetic regimens strongly argues against perfusion/work mismatch in these animals.
The trend in CP/ATP transmural slopes was conserved in a smaller subset analysis of animals in which blood flows were also obtained (Table 2⇑). Thus, when only those animals with endocardial/epicardial blood flow ratios >1.0 were considered, the α-chloralose animals (n=6) had a transmural slope of 0.098±0.124 and the sodium pentobarbital animals (n=7) had an average transmural slope of 0.214±0.328.
Comparison of hemodynamic parameters in Table 1⇑ reveals that sodium pentobarbital animals had broader ranges in basal rate-pressure products and blood flows compared with the α-chloralose group. Indeed, α-chloralose more consistently provided low basal rate-pressure products and transmural blood flows. Nonetheless, although the sodium pentobarbital data were characterized by larger SDs for all measured parameters, statistically significant correlations were observed for endocardial blood flow and cardiac index or rate-pressure products with this anesthetic. Such correlations were not observed in the endocardium of α-chloralose animals. One should note in this regard, however, that although tighter standard deviations were obtained in the α-chloralose animals, fewer regional blood flow measurements were made with this anesthetic. Epicardial flows were not correlated to any of these parameters with either anesthetic.
It is interesting to note that the average CP/ATP ratio across all voxels did not differ significantly (2.23 versus 2.17) with either anesthetic regimen. This fact argues strongly against ischemia or work/perfusion mismatch in these two groups of animals. Nonetheless, changes in transmural slopes for CP/ATP were highly significant with sodium pentobarbital and α-chloralose (0.149 versus 0.035, respectively). Moreover, our results with sodium pentobarbital are in excellent agreement with previous studies, in which CP/ATP ratios (1.91, 1.89, 2.09, 2.35, 2.51) resulted in a 0.166 value for the transmural slope.6 Moreover, differences in transmural CP/ATP slopes between α-chloralose and sodium pentobarbital could not be explained by differences in rate-pressure products alone, since transmural CP/ATP ratios have previously been shown to be independent of changes in this variable.6
Importantly, analysis of a small subset of animals (6 α-chloralose, 7 sodium pentobarbital) from which blood flows were obtained and which had endocardial/epicardial blood flow ratios >1.0 (see Table 2⇑) also revealed a greater than twofold increase in the transmural slopes obtained under the sodium pentobarbital regimen. Although this data subset is too limited to help derive a mechanism responsible for changes in transmural CP/ATP slopes with these anesthetics, it is nonetheless encouraging that the trend in slopes between α-chloralose and sodium pentobarbital is preserved.
It has been reported previously16 that the variation in CP/ATP ratios across the myocardium was not observed in every animal studied with sodium pentobarbital anesthesia but rather only in those animals with endocardial blood flows <1 mL · min−1 · g−1. More importantly, transmural variations in CP/ATP ratios observed in these studies could be eliminated under hyperperfusion conditions induced with the intravenous infusion of carbochromene.16 These results could be interpreted as implying that the transmural variations in CP/ATP ratio under sodium pentobarbital anesthesia are the result of perfusion and work mismatch.
Although the magnitude and direction of transmural CP/ATP ratio gradients also varied from animal to animal in our studies, these variations could not be explained solely by the endocardial blood flows below 1 mL · min−1 · g−1. Indeed, several animals anesthetized with sodium pentobarbital in our study had significant transmural variations in CP/ATP ratios, with endocardial blood flows significantly above 1 mL · min−1 · g−1. While differences in endocardial blood flows have been reported in the dog between the awake state and under sodium pentobarbital anesthesia,17 flow changes are unlikely to be the only manifestation of this anesthetic. In this regard, it is interesting to note that average blood flows observed for the sodium pentobarbital group in this study were higher than flows observed with α-chloralose. Transmural trends observed in the sodium pentobarbital groups may be a reflection of the strong dependence of CP/ATP ratios on multivariable physiological determinants of work, vascular resistance, blood flow, etc. All of these parameters can be directly affected by the anesthetic regimen.
Our results also indicate that under α-chloralose anesthesia, the canine myocardium exhibits a greatly reduced transmural variation in its CP/ATP ratio. As a result, under α-chloralose anesthesia at basal workloads, the myocardium (and more specifically the endocardium) displays only the weakest signs of operating in the oxygen-limited domain. In this light, this result may reveal a slight effect of α-chloralose anesthesia in relation to the awake state. Nonetheless, these anesthetic effects make it unlikely that the normal unanesthetized myocardium is operating in the oxygen-limited domain. Furthermore, previously reported transmural variations in CP/ATP ratios in the normal myocardium are directly linked to the use of sodium pentobarbital, both in studies using NMR and in studies using biochemical assays.1 2 3 4 5 6 Although it is impossible to relate these findings to the CP/ATP ratio in the awake animal and although one cannot truly determine which anesthetic is providing the result that most closely mimics the awake state, this study nonetheless clearly points to the importance of anesthetic factors in the analysis of myocardial biochemistry.
It is interesting that important differences in myocardial biochemistry are detectable by NMR between these two anesthetic regimens. Cardiovascular function is known to be affected less by α-chloralose than by any other anesthetic regimen. However, to the best of our knowledge, cardiovascular parameters such as blood flow and cardiac index obtained with this anesthetic have not been compared directly with those obtained in animals in the awake state. Nonetheless, it is known that animals anesthetized with α-chloralose sustain heart rates and respiratory sinus arrhythmia nearly identical to those in the awake dog.8 9 10 Thus, investigators agree that α-chloralose permits more intact autonomic control of the cardiovascular system than any other anesthetic regimen.8 9 10 As previously reviewed by Booth,9 α-chloralose does not interfere with normal respiratory and cardioreflexes (eg, those initiated by baroreceptor and chemoreceptor activities). Conversely, it is well known that sodium pentobarbital is a ganglioplegic agent that blocks the parasympathetic system more than the sympathetic system, resulting in heart rate and cardiac output elevation.9 10 11 The cardiac outputs have been reported to rise by nearly 50% in a normal dog anesthetized with sodium pentobarbital versus the awake dog.9 As restated by Booth,9 the use of the dog anesthetized with sodium pentobarbital has long been criticized as a model of normal cardiovascular physiology.10
It is likely that the transmural trends observed in dogs are a reflection of the strong dependence of CP/ATP ratios on NADH availability and on the balance between myocardial oxygen consumption and myocardial oxygen demand. This balance depends on regional determinants of myocardial oxygen consumption: myocardial contractility and peak tension. Although heart rate is also a prime determinant of myocardial oxygen consumption, heart rate is constant among all voxels of the myocardium. Oxygen balance also depends on determinants of myocardial blood flow: pressure difference between the aorta and right atrium, regional vascular and microvascular resistances, including tissue forces that depend on heart rate, and peak myocardial tension. All of these parameters can in turn be directly affected by the anesthetic regimen.
In recent years, the desire to determine the CP/ATP ratio in the normal myocardium has led many investigators to view this ratio as a fixed parameter. This concept has been supported by the recent demonstration that the CP/ATP ratio in the anesthetized animal is independent of rate-pressure product.6 18 However, it has recently been established by NMR methods that the CP/ATP ratio in the normal myocardium falls at very high workloads.19 In addition, it has been well established in the isolated perfused heart that the level of Krebs cycle intermediates20 and the CP/ATP ratio21 depend on the nature of the oxidized substrate. For example, the CP/ATP ratio is lowest when pyruvate/glucose is oxidized and increases with glucose/insulin. This change in CP/ATP ratio is likely to be the result of changing NADH availability with these substrates. Substrates that enhance NADH availability under basal conditions would be expected to increase the CP/ATP ratio relative to substrates that decrease NADH availability. In addition, we have previously demonstrated that the in vivo canine myocardium, when presented with acetate, will oxidize this substrate at low rate-pressure products but changes to an endogenous substrate at high workloads.22 As a result, substrate selection is clearly a work-dependent phenomenon under in vivo conditions; subsequently, it may be more appropriate to view the CP/ATP ratio in the normal myocardium as a dynamic quantity whose value is dependent on (1) the nature of the oxidized substrate, (2) the global cardiac work, and (3) the transmural distribution of work, flow, and oxygen availability.
In summary, transmural variations in CP/ATP ratios previously reported in the normal myocardium are directly linked to the anesthetic regimen used. Under α-chloralose anesthesia, the canine myocardium exhibits only the slightest transmural variation in its CP/ATP ratio. Thus, it is highly unlikely that the in vivo myocardium in the unanesthetized animal (and more specifically the endocardium) is operating in the oxygen-limited domain.
This work was supported by Public Health Service grant RO-1-HL-45210 to Dr Robitaille. We would like to thank Y. Cheng and R. Bohr for assistance with surgical and manuscript preparation, respectively.
- Received July 26, 1994.
- Accepted August 19, 1994.
- Copyright © 1995 by American Heart Association
Boerth R, Covell JW, Seagren SC, Pool PE. High-energy phosphate concentrations in dog myocardium during stress. Am J Physiol. 1969;216:1103-1106.
Dunn RB, Griggs DM. Transmural gradients in ventricular tissue metabolites produced by stopping coronary blood flow in the dog. Circ Res. 1975;37:438-445.
Allison TB, Holsinger JW. Transmural metabolic gradients in the normal dog left ventricle: effect of right atrial pacing. Am J Physiol. 1977;233:H217-H221.
Dunn RB, McDonough KM, Griggs DM. High energy phosphate stores and lactate levels in different layers of the canine left ventricle during reactive hyperemia. Circ Res. 1979;44:788-795.
Robitaille PML, Merkle H, Sublett E, Hendrich K, Lew B, Path G, From AHL, Bache RJ, Garwood M, Ugurbil K. Spectroscopic imaging and spatial localization using adiabatic pulses and applications to detect transmural metabolite distribution in the canine heart. Magn Reson Med. 1989;10:14-37.
Robitaille PML, Merkle H, Lew B, Path G, Hendrich K, Lindstrom P, From AHL, Garwood M, Bache RJ, Ugurbil K. Transmural high energy phosphate distribution and response to altered workload in the normal canine myocardium as studied with spatially localized 31P NMR spectroscopy. Magn Reson Med. 1990;16:91-116.
Ugurbil K, From AHL. Nuclear magnetic resonance studies of kinetics and regulation of oxidative ATP synthesis in the myocardium. In: Schaefer S, Balaban RS, eds. Cardiovascular Magnetic Resonance Spectroscopy. Boston, Mass: Kluwer Academic Publishers; 1993:63-92.
Cox RH. Influence of chloralose anesthesia on cardiovascular function in trained dogs. Am J Physiol. 1972;223:660-667.
Booth NH. Intravenous and other parenteral anesthetics. In: Booth NH, McDonald LE, eds. Veterinary Pharmacology and Therapeutics. Ames, Iowa: Iowa State University Press; 1972:203-254.
Priano LL, Traber DL, Wilson RD. Barbiturate anesthesia: an abnormal physiological situation. J Pharmacol Exp Ther. 1969;165:126-135.
Manders WT, Vatner SF. Effects of sodium pentobarbital anesthesia on left ventricular function and distribution of cardiac output in dogs, with particular reference to the mechanism for tachycardia. Circ Res. 1976;39:512-517.
Zhang J, Yoshiyama M, Garwood M, From AHL, Bache RJ, Ugurbil L. Transmural high energy phosphate (HEP) distribution and response to coronary hyperperfusion. Soc Magn Reson Med Abs. 1990:862. Abstract.
Cobb FR, Bache RJ, Grenfield JC. Regional myocardial blood flow in awake dogs. J Clin Invest. 1974;53:1618-1625.
Balaban RS, Kantor HL, Katz LA, Briggs RW. Relation between work and phosphate metabolite in the in-vivo paced mammalian heart. Science. 1986;232:1121-1123.
Zhang J, Xu Y, Merkle H, Hendrich K, Dunker D, Path G, Bache R, From A, Ugurbil K. Metabolic responses of normal canine myocardium to very high workload: an in vivo 31P NMR study. Soc Magn Reson Med Abs. 1992:344. Abstract.
Neely JR, Denton RM, England PJ, Randle PJ. The effects of increased heart work on the tricarboxylate cycle and its interactions with glycolysis in the perfused rat heart. Biochem J. 1972;128:147-159.
Robitaille PML, Rath DP, Abduljalil AM, O’Donnell JM, Jiang Z, Zhang H, Hamlin RL. Dynamic 13C NMR analysis of oxidative metabolism in the in-vivo canine myocardium. J Biol Chem. 1993;268:26296-26301.