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Ann Thorac Surg 2000;69:1799-1805
© 2000 The Society of Thoracic Surgeons
a Department of Thoracic and Cardiovascular Surgery, Medical School, University of Tromsø, Tromsø, Norway
b Department of Medical Physiology, Medical School, University of Tromsø, Tromsø, Norway
Address reprint requests to Dr Elvenes, Department of Thoracic and Cardiovascular Surgery, Institute of Clinical Medicine, University of Tromsø, N-9038 Tromsø, Norway
e-mail: oddpe{at}fagmed.uit.no
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Abstract |
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Methods. Substrate oxidation, blood flow, and myocardial function were measured before, during, and after 3 hours of WCBCP in 7 pigs.
Results. Free fatty acid and glucose oxidation decreased by 60% ± 3.8% and 94% ± 1.2%, respectively, during cardioplegia (both p < 0.05) and increased to 62% ± 28% and 122% ± 62% of baseline during the early recovery phase (p < 0.05 for glucose). One hour after WCBCP oxidation rates were similar to baseline. The transient postcardioplegic increase in substrate oxidation was associated with a 43% ± 23% elevation of oxygen consumption (MVO2) compared with baseline and a 62% ± 18% increase in myocardial blood flow. Cardiac output and mean arterial pressure did not change significantly after WCBCP, although myocardial function (stroke work, left ventricular end-systolic pressure, end-diastolic pressure, contractility, and efficiency) was depressed (p < 0.05). End-diastolic pressure and contractility improved from early to late phase of recovery, whereas the other indicators of ventricular function remained depressed.
Conclusions. Myocardial substrate oxidation was preserved after 3 hours of WCBCP, although ventricular function was moderately impaired. Thus, WCBCP with a seemingly normal substrate and oxygen supply was associated with a reduced cardiac efficiency.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Theoretically, myocardial substrate utilization is determined by the availability of the substrate, ie, the plasma concentration [2]. However, studies have shown almost no free fatty acid (FFA) oxidation in the early reperfusion phase after coronary operation using cold crystalloid cardioplegia and only minimal FFA oxidation after cold blood cardioplegia [3, 4]. It also has been suggested that the systemic neuroendocrine response with high arterial concentrations of stress hormones antagonizes insulin action and stimulates lipolysis and gluconeogenesis [5]. Postoperatively, uptake of glucose is reduced, although some uptake of lactate persists [3, 6]. Svennson and coworkers [4] concluded that oxidation of exogenous FFA and carbohydrates cannot account for the myocardial demand of substrates after cardiac operation, and accordingly that utilization of endogenous substrates may account for a major part of myocardial energy production in the early postoperative phase.
The suggested dependence on endogenous substrates after operation [4] could be questioned given the fact that the amounts of endogenous glycogen and lipids are limited [7]. Moreover, a reduced uptake of substrates postoperatively would indicate a profound defect in metabolic regulation, at odds with what should be expected after good preservation during cross clamping.
In a previous study we have demonstrated an overreliance on fatty acids as a source of energy for the heart during WCBCP in pigs [8], indicating that this procedure alters the regulation of myocardial metabolism.
The aim of the present study was to assess whether substrate metabolism was preserved in the recovery phase after 3 hours of WCBCP in the pig model. Also, the preservation of myocardial function during this nonischemic arrest period was assessed to address the effectiveness of this cardioprotective regimen.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Anesthesia
Seven locally bred domestic pigs of either sex weighing 41 to 52 kg (mean weight, 44.3 kg) were included in the study. All animals were premedicated with intramuscular injections of 1,000 mg ketamine (Ketalar, Parke-Davis, Scandinavia AB, Solna, Sweden) and 2 mg atropine (Atropine, Hydro Pharma, Oslo, Norway) in the animal department. The pigs were then transported to the operating room and given an intravenous bolus infusion of 10 mg/kg pentobarbital (Pentobarbital, Nycomed Pharma, Oslo, Norway) and 0.020 mg/kg fentanyl (Leptanal, Janssen-Cilag, Beerse, Belgium) and 0.3 mg/kg midazolam (Dormicum, Roche, Basel, Switzerland). The animals were tracheostomized and mechanically ventilated (Servo 900, Elema-Schønander, Stockholm, Sweden) with 0.5 FiO2 and respiratory rate was set to 20 breaths per minute. Anesthesia was maintained by continuous infusion of 4 mg · kg-1 · h-1 pentobarbital, 0.020 mg · kg-1 · h-1 fentanyl, and 0.3 mg · kg-1 · h-1 midazolam into the external jugular vein using a venous catheter (Secalon Seldy, Ohmeda, Denmark). Tidal volume was adjusted by means of repeated arterial blood gas analyses (BGM, Allied Instrumentation Laboratory, Milan, Italy) to achieve pCO2 and pH within normal ranges (3.5 to 5.7 mm Hg and 7.34 to 7.47, respectively). Sodium chloride (0.9%) enriched with glucose (1.25 g glucose/1,000 ml sodium chloride) was given for basal fluid replacement (10 mg · kg-1 · h-1). Anesthesia depth was checked regularly by testing the ciliary reflex and reaction to pain in the nasal cartilage.
Experimental setup
The experimental protocol is outlined in Figure 1. We principally used the same experimental setup as described in detail in previous studies from our group including a separate extracorporeal circuit for the heart [8, 9]. The blood was oxygenated using two separate membrane oxygenators (Monolyth, Sorin Biomedica, Saluggia, Italy) along with two separate reservoirs: one baby-sized set for the heart and one adult-sized set for the rest of the body. Cardiac output (CO) and the myocardial blood flow (MBF) were measured with ultrasonic transit-time probes (Cardio-Med, Medistim, Oslo, Norway) on the pulmonary artery and the left and the right main stem of the coronary arteries.
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The extracorporeal circuits were primed with homologous fresh blood from a crossmatched donor pig. The cardioplegic blood solution was prepared by mixing 200 mL St Thomas’ solution No. 2 (Plegisol, Abbot Laboratories, Chicago, IL) with 800 mL blood. The activating clotting time (ACT; Hemochrom 400, Techidyne-Corp, Edison, NJ) was kept above 500 seconds at all times. The temperature in the blood cardioplegia circuit was 37°C during the cardiac arrest period, whereas temperature was lowered to 26°C in the systemic line. Systemic temperature was gradually elevated to 37°C during the last half hour before declamping of the aorta and the pulmonary artery.
Mean arterial pressure (MAP), central venous pressure, and pressure in the cardioplegic line were measured in the femoral artery, external jugular vein, and root of the aorta, respectively, with calibrated transducers (Transpac 3, Abbot Critical Care Systems, Chicago, IL) connected to an amplifier (Gould ES 2000, Valley View, OH), digitized (LABview, National Instruments, Austin, TX), and stored (Macintosh Quadra 950, Apple Computer Inc, Cupertino, CA). Heart rate was obtained from the pulsatile flow signal by means of a pressure processor (Gould ES 2000) and then digitized. The automatized sampling rate for all channels was 0.25 Hz.
A 6F, 12-electrode, dual-field, pigtail combined microtip and conductance catheter for continuous measurements of left ventricular (LV) pressures and volumes (Millar Instruments Inc, Houston, TX) was introduced into the LV through the left carotid artery. The catheter was positioned along the long axis of the LV and connected to a conductance conditioner (Leycom Sigma-5, Cardiodynamics BV, Leiden, The Netherlands). Pressure calibration was performed before insertion. Positioning of the catheter was evaluated by palpation of the apex. The catheter was reinserted after weaning from CPB.
Blood samples were taken from the aortic root and coronary sinus at the time points indicated in Figure 1. These samples were drawn simultaneously from all cites into preheparinized plastic syringes and transferred to plastic tubes that were immediately cooled on ice and centrifuged at 4°C with 14,000 rpm. The plasma was stored at -70°C for later determination of plasma metabolite levels.
Intraventricular measurements
The conductance-catheter method is based on measuring time-varying electrical conductances of five segments of blood in the LV [10, 11]. Time-varying conductance is converted to volume by the formula:
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is a gain factor, relating conductance volume to an independent method, eg, ultrasonic flow probe; L is the interelectrode distance; 
is blood resistivity; G(t) is the summed segmental conductances; and Gp is the total offset conductance (related to right ventricle and myocardial wall) [11]. Calculation of conductance-derived measurements was performed using the Conduct-PC software (CPCW version V3.15, Cardiodynamics BV). The heart cycle was defined to start at the peak of the R wave in the QRS complex, corresponding to end diastole. End systole was calculated as proposed by Sagawa [12]. Conductance-derived pressures and volumes were assessed 60 minutes before CPB, and at 30 and 60 minutes during postcardioplegic recovery. Two sets of data were obtained throughout 10-second intervals: one set during control preload and one set during vena cava occlusion (VCO). VCO was performed by snaring the inferior vena cava.
Substrate oxidation
An ethanolic mixture of 3H-labeled oleic acid (NET-289 [9,10-3H(N)]-oleic acid) and 14C-labeled glucose (NEC-042X D[14C(U)]-glucose) was dried under a steam of N2 as described previously [8]. Both substances were purchased from NEN Life Science Products (Boston, MA). The dried substances were subsequently redissolved in 65 mL pig plasma to give a final radioactivity of 25 (oleate) and 7 (glucose) µCi/mL. The labeled substrates were administered to the systemic circuit through a central venous line using an infusion pump set to a speed of 30 mL/h for the first 15 minutes. Thereafter the pump speed was reduced to 7 mL/h, corresponding to 175 µCi/h of [3H]-oleic acid and 50 µCi/h of [14C]-glucose. The cardioplegic circuit was enriched with a bolus of labeled substrates, followed by a continuous infusion to maintain adequate radioactive substrate concentrations.
Arterial and coronary sinus samples were drawn simultaneously for determination of 3H2O [13] and 14CO2 [13, 14]. Parallel samples were taken for determination of the chemical and radioactive substrate concentrations.
Chemical analyses
Plasma concentrations of glucose, lactate, and FFA were determined enzymatically using a semiautomatic analyzer (Cobas, Fara II, Roche, Basel, Switzerland). The standard reagents for glucose and lactate analyses were purchased from Boehringer Mannheim (Mannheim, Germany) and for FFA analyses from Wako Chemicals (Neuss, Germany). Oxygen saturation was determined in blood samples from all the sample lines (ABL3, Acid Base Laboratory/Hemoxymeter, Radiometer, Copenhagen, Denmark).
Calculations
Myocardial oxidation of FFA and glucose was calculated based on the production (arterial-coronary sinus difference) of 3H2O and 14CO2, respectively, as well as the plasma-specific activity of the substrates. Determination of radioactivity was carried out on a beta-scintillation counter (Packard 1900 TR Liquid Scintillation Analyzer, Packard Instruments, Groningen, The Netherlands).
Calculation of MVO2 (mLO2 · min-1 · heart-1) and substrate uptake (nmol · min-1 · heart-1) was based on the differences in oxygen content and substrate concentrations between arterial and coronary sinus samples multiplied by the coronary flow [8].
Stroke work (SW; mm Hg/mL), which corresponds to the area of the pressure-volume loop, was calculated as an integral of all sampled (200 Hz) pressures and volumes between end diastole and end systole, and converted to joules (1 mm Hg/mL = 1.33 · 10-4 J). Left ventricular contractility was assessed from the slope (PRSWI; mm Hg) of the linear SW to end-diastolic volume (Ved) relationship (preload recruitable stroke work) on a beat-beat basis during VCO [15]. The PRSWI is a load-independent index of LV contractility as long as an intervention-related shift in its volume-axis intercept (Vw) does not occur [16]. LV external efficiency (%) was calculated from the ratio between SW and MVO2, after conversion of MVO2 to J · beat-1 · heart-1 (1 mLO2 = 20.2 J).
Because of the weight range of the pigs, weight-specific values of the cardiac output index (CI; mL · min-1 · kg-1) are given, whereas MBF is given as the actually measured mean for the hearts (mL · min-1 · heart-1).
Statistics
All data are presented as mean ± standard error of the mean (SEM). Calculation and data analysis were performed using a spreadsheet (Microsoft Excel 5.0, Microsoft Corporation, Redmond, WA) and statistical package (SPSS 8.0, SPSS Inc, Chicago, IL). After testing and finding a normal distribution of our data they were examined for changes over time using repeated measures analysis of variance [17]. Posthoc comparisons were performed when the F values indicated statistical differences, using the two-sided Dunnett’s test against baseline conditions as control. Differences were considered to be statistically significant if p was less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Metabolic indicators
The ratio between oxidized substrates did not change throughout the experiment. Baseline arterial FFA, glucose, and lactate concentrations were 320 ± 23 µmol/L, 6.0 ± 0.5 mmol/L, and 1.1 ± 0.1 mmol/L, respectively. During cardiac arrest when the heart was supplied with cardioplegic blood from a separate oxygenator, arterial FFA values were approximately 60% (130 ± 11 µmol/L) below baseline, whereas glucose concentrations remained relatively unchanged. The concentration of lactate in the perfusion line increased slowly during the period of cardiac arrest so that the value measured at 180 minutes (3.6 ± 0.4 mmol/L) was significantly higher (p = 0.03) than the value measured at 30 minutes of cardioplegia (2.3 ± 0.2 mmol/L). The hematocrit values were 23.9 ± 1.03, 18.5 ± 0.96, 18.4 ± 1.11, 25.1 ± 1.36, and 25.2 ± 1.44 before, during (early and late), and after (early and late) WCBCP, respectively.
Table 1 shows net uptake of the main myocardial energy substrates throughout the experiment. FFA uptake declined to values 75% to 90% (p = 0.019, Table 1) below baseline during cardioplegia and recovered to values not different from baseline during postcardioplegic recovery. Glucose uptake showed no significant changes during the experimental protocol. Myocardial lactate uptake was reduced markedly (p < 0.05) during cardioplegia, but increased to values almost twofold those of baseline (not significant [NS]) during postcardioplegic recovery.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Our model with a closed circuit for the heart is a prerequisite to avoid systemic hyperkalemia without a hemofiltration setup. At the same time the heart is protected from the systemic stress-induced inflammatory reaction. This model is therefore well suited for studies of the quiescent, blood perfused oxygenated heart. One objection to this model is that the heart is excluded from pulmonary filtration, liver metabolism, and renal excretion during standstill, but we did not observe any indications of accumulated toxic metabolites in the closed heart circuit [9].
We have demonstrated previously a predominant oxidation of FFA during WCBCP [8]. The present results confirm this finding and show in addition that FFA is the predominant energy substrate also in the recovery phase after normothermic continuous blood-based cardioplegic arrest. We calculated that the contribution of FFA to the total ATP production amounted to 77%, 94%, and 70% before, during, and after cardioplegia, respectively. These values are probably slightly overestimated, because the contribution of lactate to the overall oxidative energy production could not be assessed in this study. Other models have provided variable results. Steigen and coworkers [18] reported that fatty acid oxidation was reduced by 56% after hypothermia and rewarming in dogs. Teoh and coworkers [3] reported a considerably reduced palmitate oxidation after cold blood or crystalloid cardioplegia in humans. These authors [19] also demonstrated that lactate was quantitatively more important than glucose and fatty acids for oxidative metabolism after cold blood cardioplegia using isotope techniques. The discrepancy between our results and those by Teoh and colleagues [19] with respect to myocardial substrate preference could be related to the difference in myocardial temperature during the cardiac arrest period. It is reasonable to suggest that normal temperature and oxygenation of the myocardium during the cardiac arrest period preserve the metabolic pathways better than procedures using low temperature in which ischemic episodes also may occur. Hence, the energy requirements for mechanical activity of the heart can be met adequately when the heart resumes beating after standstill.
In line with previous observations, we found that glucose uptake was essentially zero before, during, and after cardioplegia [8, 20, 21]. This probably could be explained by insulin resistance resulting from the surgical trauma [5]. Apparently, however, glucose uptake was sufficient to supply and label the myocardial glycogen pool, allowing measurement of glucose oxidation. A persisting flux through the glycolytic pathway also was indicated by the finding that the myocardium was taking up considerable proportions of the arterial supply of lactate which was fed into the tricarboxylic acid cycle for further breakdown. Lopaschuk and coworkers [22] have shown that fatty acid-induced inhibition of glucose oxidation contributes significantly to postischemic cardiac dysfunction in isolated rat hearts. In our model cardiac function was depressed despite a preserved glucose oxidation, excluding low glycolytic flux as a mechanism for the observed decline in cardiac function after WCBCP.
The long cardioplegic period may lead to perturbations of ion gradients both between the cytosol and the interstitial space, as well as between the cytosol and the sarcoplasmatic reticulum. It is obvious that extra energy is needed to restore these gradients in the postcardioplegic period, although the quantitative importance of this process is difficult to estimate. In addition, impaired interstitial fluid (lymph) drainage and edema resulting from standstill and clamps on the big vessels may lead to myocardial stiffness [23, 24] and reduced diastolic function. In accordance with this notion we observed an increased EDP in the postcardioplegic period. It should be noted, however, that both EDP and cardiac contractility (PRSWI, Fig 4) improved significantly from the early to late postcardioplegic recovery period, indicative of a reversible functional impairment.
The finding that MBF and MVO2 were increased in the early recovery period after cardiac arrest while at the same time LV performance was reduced shows that the rate of recovery of mitochondrial metabolism after WCBCP exceeds the rate of recovery of contractile function, resulting in a drop in cardiac efficiency (Fig 4). This suggests that a greater proportion of mitochondrial ATP production is used for noncontractile purposes or to overcome a possible decrease in heart chamber distensibility.
Finally, our results may indicate uncoupling of oxidative phosphorylation during cardioplegia and in the early recovery period. As shown in Figure 2, the relative changes in the calculated ATP production did not match the corresponding changes in MVO2. One plausible explanation for this observation could be that the theoretical values used to calculate energy (ATP) yield are too high, which would be the case if ATP production were partly uncoupled from oxidative metabolism. Normally, uncoupling of oxidative phosphorylation has been associated with high levels of FFA. In the present model the myocardium was not exposed to high plasma levels of FFA [25, 26], but the fact that more than 90% of the ATP production was derived from FFA oxidation during cardioplegia could argue in favor of uncoupling.
In conclusion, this study demonstrates a temporarily increased energy demand and a reduced cardiac efficiency in the early recovery phase after WCBCP without any significant shift in substrate preference. In our model, normal metabolic function was restored 60 minutes after cardiac arrest, but recovery of mechanical function was still lagging behind. Further studies are warranted to elucidate why a seemingly adequate perfusion of the nonbeating heart renders a somewhat depressed function.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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p < 0.05 (30 versus 60 minutes).