Ann Thorac Surg 2000;70:264-269
© 2000 The Society of Thoracic Surgeons
Original articles: General thoracic
Effects of storage and reperfusion oxygen content on substrate metabolism in the isolated rat lung
Paul E. Meyer, MDa,b,c,d,
Michael E. Jessen, MDa,b,c,d,
Jayendra B. Patel, BSa,b,c,d,
Robert Y. Chao, MDa,b,c,d,
Craig R. Malloy, MDa,b,c,d,
Dan M. Meyer, MDa,b,c,d
a Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA
c Division of Cardiology, Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA
b Department of Radiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA
d Dallas Veterans Affairs Medical Center, Dallas, Texas, USA
Address reprint requests to Dr Dan Meyer, Division of Thoracic and Cardiovascular Surgery, Department of Surgery, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235
e-mail: dan.meyer{at}email.swmed.edu
Presented in part at the Forty-sixth Annual Meeting of the Southern Thoracic Surgical Association, San Juan, Puerto Rico, Nov 4–6, 1999.
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Abstract
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Background. Lung transplantation requires a period of storage and ischemia; we examined the largely unknown effects of that period on intermediary metabolism.
Methods. Two groups of isolated rat lung blocks (n = 16 each) were flushed with Euro-Collins solution and harvested. The lung blocks were immediately ventilated and either perfused for 30 minutes with an erythrocyte-based solution containing carbon 13 labeled substrates (group 1) or stored for 6 hours at 1°C and then reperfused (group 2). Half of each group was reperfused at a physiologic PO2 the other half at high PO2. Analysis of carbon 13 isotopomers was performed to determine substrate utilization through aerobic pathways in lung tissue.
Results. Lungs from both groups oxidized all major substrates. The contribution of fatty acids to acetyl-coenzyme acid oxidized in the citric acid cycle was significantly higher in group 2 than in group 1 (31.3% ± 2.2% versus 22.0% ± 2.1%, p < 0.05). Perfusate PO2 did not affect substrate preference. Gas exchange was worse in stored lungs.
Conclusions. After a period of hypothermic ischemia and storage, substrate preference in lung tissue exhibits a switch towards fatty acids. As fatty acid oxidation occurring after ischemia is deleterious in other organs, strategies to inhibit this process in stored lungs may warrant further investigation.
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Introduction
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Lung transplantation has become a viable treatment option for many patients with end-stage pulmonary diseases. The long-term results, however, have not compared favorably to those of other solid organ transplants. Early graft dysfunction complicates a significant percentage of lung transplants and may influence the development of bronchiolitis obliterans, a chronic and often terminal complication of lung transplantation. Although the causes of both early and late graft dysfunction are multifactorial, adequate lung preservation may be important in limiting these processes.
Current preservation solutions were developed for the protection of solid organs with minimal, if any, adaptation of these solutions for the metabolic requirements of the lung. In part this has been related to our incomplete understanding of intermediary metabolism of the lung, because measurement of aerobic metabolism in that organ is difficult. In this study, a novel technology (carbon 13 [13C] nuclear magnetic resonance [NMR] spectroscopy) was used to simultaneously evaluate the utilization of multiple substrates in an isolated lung model. We examined the effects on intermediary metabolism of two variables (1): an ischemic storage period and (2) reperfusion PO2 levels, simulating transplant conditions encountered clinically.
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Material and methods
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Materials
Adult male Sprague-Dawley rats weighing 280 to 300 g were given food and water ad libitum and used in an institutionally-approved research protocol. All animals were cared for in compliance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources (National Academy of Sciences) and published by the National Institutes of Health (NIH publication 86–23, revised 1985).
13C-enriched compounds and perfusate composition
We obtained [3-13C]-sodium L-lactate, [3-13C]-sodium pyruvate, and [1,3-13C]-ethyl-acetoacetate and [U-13C]-fatty acids from either Cambridge Isotope Labs (Andover, MA) or Martek (Columbia, MD). Unenriched glucose and other biochemicals were obtained from Sigma (St. Louis, MO). These substrates were incorporated into a modified Krebs-Henseleit solution containing physiologic concentrations of electrolytes (118 mmol NaCl, 4.7 mmol KCl, 1.2 mmol MgSO4, 1.2 mmol NaH2PO4, 25 mmol NaHCO3, and 1.2 mmol CaCl2) and substrates (5.5 mmol glucose, 1.2 mmol lactate, 0.12 mmol pyruvate, 0.17 mmol acetoacetate, and 0.35 mmol fatty acids). These concentrations are considered normal in the fed, rested rat [1]. This solution was combined with isolated porcine red blood cells (RBCs) to yield a hematocrit of 20%.
The porcine RBCs were isolated from fresh whole blood and stored in acid citrate dextrose solution. The whole blood was centrifuged at 3,000 revolutions per minute for 15 minutes and plasma was removed. The remaining RBCs were combined with phosphate buffer solution in a 1-to-1 ratio and centrifuged again to wash the RBCs and again isolate the RBC fraction. This washing process was repeated three times. The prepared RBCs were then stored overnight in a 1-to-1 ratio with a solution containing phosphate buffer solution and glucose. All red cells were used within 48 hours of overnight storage to avoid increased red cell fragility and resulting hemolysis.
Experimental protocol and groups
Animals were anesthetized with 4% chloral hydrate (1 mL/100 g administered intraperitoneally) and heparinized (1,000 U/100 g). After tracheostomy, rats were ventilated with a Harvard rodent ventilator (Harvard Apparatus, Holliston, MA) using room air at 50 breaths/min, a tidal volume of 10 mL/kg, and positive end-expiratory pressure of 5 cm H2O. A double lumen catheter was inserted into the main pulmonary artery through the right ventricular outflow tract to allow infusion of pulmonoplegic solution (Euro-Collins solution, Baxter Healthcare, Irvine, CA). The left atrium was incised to allow unrestricted effluent drainage. Euro-Collins solution (60 mL/kg) was delivered to the lung at a pressure of 25 cm H2O, and ice slush was placed in the pleural cavity. After pulmonoplegic solution administration, the heart-lung block was excised in the semiinflated state (using room air) and randomly assigned to one of two groups. Group 1 (n = 16) was reperfused immediately with the labeled RBC solution without a storage interval. Group 2 (n = 16) was placed in Euro-Collins solution and stored at 1°C for 6 hours, after which the lung blocks were reperfused with the identical labeled perfusate. Each group was further divided into two subgroups (n = 8 each) based on the level of oxygenation of the reperfusing solution. One half of group 1 (group 1-low) was reperfused with RBC solution oxygenated to a PO2 of 35 to 45 mm Hg, modeling normal pulmonary artery oxygen tension. The other half (group 1-high) was perfused with the same solution gassed with 95% O2/5% CO2 to yield a supraphysiologic PO2 (> 550 mm Hg). Group 2 was divided into identical subsets (groups 2-low and 2-high) based on inflow oxygenation. Perfusate that passed through the pulmonary circulation was not recirculated.
The perfusate solution was assayed for substrate concentrations using commercially available kits. During reperfusion, lung function was evaluated by measurements of pulmonary artery pressure, airway pressure, and inflow and outflow blood gas levels taken at 0-, 10-, 20-, and 30-minute timepoints. After 30 minutes of reperfusion, the lung tissue was freeze-clamped, stored in liquid nitrogen, and later extracted with cold perchloric acid, neutralized with KOH, freeze-dried, and dissolved in 0.5 mL of D2O for 13C NMR analysis.
Ex vivo perfusion apparatus
The perfusion apparatus was primed with the RBC solution with labeled substrates (described above) that had been circulated through a membrane oxygenator (Maxima-Plus, Medtronic, Minneapolis, MN) by a centrifugal pump (Medtronic-BioMedicus, Eden Prairie, MN) (Fig 1). A blender was used to maintain perfusate oxygenation at the desired level, providing either 95% O2/5% CO2 to achieve a supraphysiologic oxygen level or 95% O2/5% CO2 and 95% N2/5% CO2 blended to yield a physiologic oxygen tension (35 to 45 mm Hg). Perfusate was drawn from a reservoir and delivered to the lung block through the pulmonary artery catheter at 20 mL/min using a calibrated roller pump. The lungs were ventilated at the same tidal volume, respiratory rate, and positive end-expiratory pressure as had been present during procurement, at a FIOC2 of 0.40.
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Fig 1. Perfusion apparatus. A reservoir supplied the perfusate, which was circulated and oxygenated with an oxygenator and blender. Perfusate was drawn from this circuit and delivered to the lung block at a rate of 20 mL/min. The lungs were ventilated with a rodent ventilator during reperfusion.
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13C NMR spectroscopy
Tissue extracts were placed in 5-mm tubes. Proton-decoupled 13C spectra were obtained at 100.3 MHz on a 9.4 T Bruker Omega spectrometer (Billerica, MA). Spectra were acquired using a 45-degree carbon pulse, collecting 15000 data points at 22,000 to 26,000 Hz with a 1.0-second delay between pulses. Relative multiplet areas in the glutamate carbon resonances were determined with the use of commercial software (NMR–1, Tripos, St. Louis, MO).
By examining the enrichment pattern of the 4 and 5 carbons of glutamate, 13C NMR spectroscopy determines the contributions of acetoacetate, lactate plus pyruvate, fatty acids, and unlabeled sources to acetyl-coenzyme A (acetyl-CoA) oxidized in the tricarboxylic acid (Krebs) cycle. Acetyl CoA will be labeled in one of four possible patterns: enriched in carbon 1 (derived only from acetoacetate), enriched in carbon 2 (derived only from pyruvate or lactate in the perfusate), enriched in both carbons 1 and 2 (derived only from fatty acids in the perfusate), or unenriched (derived from glucose in the perfusate or endogenous stores). Data obtained from these spectra were then analyzed using the non–steady-state technique as described previously [2] to quantify the relative contribution of each substrate to the total acetyl-CoA pool oxidized within the lung tissue. This analysis does not require conditions of metabolic or isotopic steady state.
Statistical analysis
Data are reported as mean ± SEM. The perfusate assay values and pulmonary substrate utilization data were compared by one-way analysis of variance. Lung performance data were compared by repeated measures analysis of variance using commercially available software (SAS Institute, Cary, NC).
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Results
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Perfusate composition
Analysis of perfusate composition revealed no significant differences in substrate concentration among groups. The PO2 of the inflow perfusate was higher (by design) in the high-oxygenation subgroups than in the physiologic oxygenation groups (Table 1).
Lung performance
Rat lungs perfused with a solution oxygenated at physiologic levels (groups 1-low and 2-low) increased the oxygen level of the pulmonary venous effluent. However, lungs stored for 6 hours (group 2-low) exhibited a marked deterioration in their ability to oxygenate over the 30 minute timecourse of the experiment, ending with significantly lower PO2 in the pulmonary venous effluent than did the nonischemic lungs (group 1-low). Lungs in all groups exhibited evidence of pulmonary edema over the reperfusion interval.
Lungs perfused with highly oxygenated solution were found to release oxygen as the perfusate passed through the pulmonary bed. Again, this release of oxygen declined over time in the stored lungs (group 2-high) but remained relatively constant in the nonischemic group (group 1-high), as shown in Table 2. These patterns of gas exchange differed significantly between groups.
No significant differences in mean pulmonary artery pressure (p = 0.43) or peak airway pressure (p = 0.18) were observed over time among groups and their subsets (Fig 2).
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Fig 2. (A) Plot of mean pulmonary artery pressure over time for all subgroups. There were no significant differences in mean pulmonary artery pressure among groups (n = 16 each) or subsets (n = 8 each). (B) Plot of peak airway pressure over time for all subgroups. There were no significant differences in peak airway pressure among groups (n = 16 each) or subsets (n = 8 each). In both figures, results are expressed as mean ± standard error of the mean and compared by repeated-measures analysis of variance.
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Substrate utilization
Substrate utilization patterns in the two groups are displayed in Table 3. Isotopomer analysis revealed that a 6-hour period of ischemia significantly altered the substrate utilization of the lung. Compared with nonischemic controls, ischemic rat lungs preferentially oxidized fatty acids at the expense of unlabeled substrates. Subset analysis revealed that the level of perfusate oxygenation during reperfusion had no effect on substrate preference in either stored or immediately perfused lungs. The raw data for all four subsets are shown in Table 4.
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Comment
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The principal function of the lung is to oxygenate blood for use by the tissues. Because of this unique role, the oxygen content of blood exiting the organ exceeds that entering it and measurement of oxygen consumption by the lung tissue is not directly possible. This factor also complicates the study of oxidative metabolism of the lung, with the result that information available about intermediary metabolism in lung tissue is limited compared with information available about most other organs.
Prior studies of lung tissue have addressed the consumption of many substrates in a limited fashion. Glucose, which has been the most extensively studied substrate, has been shown to be taken up by the lung in a concentration-dependent fashion [3]. The fate of glucose cleared from the pulmonary circulation is under debate, with evidence for lactate production by anaerobic metabolism [3–6], complete oxidation to CO2 through aerobic pathways within the citric acid cycle [7, 8], and diversion of glucose carbons into biosynthetic pathways for phospholipid synthesis and nicotinamide adenine dinucleotide phosphate production [9]. Lactate is cleared from solution by the lung as its concentration increases relative to glucose [7, 8], emphasizing the importance of studying substrate utilization at physiologic concentrations. Lactate taken up by the lung can undergo oxidation to CO2 [7] or may be used in phospholipid production, particularly in the perinatal period [10]. Other investigators have demonstrated clearance of nonesterified fatty acids and 3-hydroxybuturyate (a ketone body) by the lung [3, 9], and oxidation of fatty acids has been described [8]. Thus the lung appears to be capable of uptake and oxidation of most major substrate classes.
As noted above, studies that quantify uptake do not necessarily imply that the substrates entering the pulmonocyte will be oxidized completely. Furthermore, carbon 14 (14C) tracer studies that define oxidation of the labeled substrate (by 14CO2 release) are limited to the measurement of only a single substrate for any given experimental conditions. On the other hand, 13C NMR isotopomer analysis measures only the substrates that are metabolized within the citric acid cycle and thus contribute most to energy production in the cell. Substrates taken up but diverted to biosynthetic pathways are not included in this analysis. Furthermore, this technique allows measurements of multiple substrates in a single experiment.
The hallmark of aerobic metabolism is the complete oxidation of carbon-containing compounds with the generation of reducing equivalents. These reducing equivalents are reoxidized through the electron transport chain with molecular oxygen as the terminal electron acceptor. We were concerned that the low oxygen tension normally present in the pulmonary artery blood would significantly alter aerobic metabolism by limiting oxygen availability. No bronchial circulation is provided in this model or in clinical lung transplantation. For this reason we studied lungs reperfused with a physiologic PO2 (35 to 45 mm Hg) and also with the maximal obtainable PO2 ( > 550 mm Hg). It is of interest that substrate oxidation profiles were not affected by oxygenation at these two extremes. Although absolute flux through the citric acid cycle was not measured, no changes in substrate selection were seen between the low and high subsets of either group.
The ability of stored lungs to oxygenate perfusate was significantly inferior to that seen in immediately perfused lung blocks. The degree of impairment was substantially inferior to that seen in clinical lung transplantation after a similar storage interval, suggesting that the model is more sensitive to ischemic damage than are other transplant models. Our results occurred despite the absence of any rejection phenomenon and the elimination of leukocytes and other potential mediators of reperfusion injury in this model. Of interest, no significant difference in airway or pulmonary artery pressures was found between stored and nonstored groups. This contrasts with previous studies, which have suggested that high potassium solutions such as Euro-Collins can cause vasoconstriction, V/Q mismatch, and increased pressures in reperfused lungs [11].
The principal finding in this study is that lung ischemia and hypothermic storage in a model of transplant preservation were accompanied by a relative increase in fatty acid utilization by the lung tissue. The metabolic mechanism behind this observation is not defined, but studies in other organs have found qualitatively similar shifts in substrate selection after ischemia, either as a result of inhibition of pyruvate dehydrogenase [12], or through decrease in malonyl-coenzyme A levels which normally serve to inhibit fatty acid metabolism at the level of carnitine-palmitoyl transferase [13]. This increase in fatty acid use was accompanied by a significant decline in the utilization of unlabeled sources of acetyl-CoA. Under the conditions studied, these unlabeled sources could represent exogenous glucose in the perfusate or endogenous glycogen or triglycerides. In general, significant quantities of endogenous stores have not been described in pulmonocytes, and studies done with a substrate combination identical to that used in this study but with labels in the glucose component found exogenous glucose to contribute less than 4% of the acetyl-CoA oxidized within the citric acid cycle [14].
This study has several noteworthy limitations. The use of an isolated lung does not completely model conditions encountered in clinical transplantation, because no effects of the immune response or immunosuppressive drugs are present. The perfusate includes erythrocytes but no leukocytes, which may substantially alter reperfusion injury. Substrates were provided in carefully controlled concentrations that mimic those observed in the fed rat. Surgery, anesthesia, and presence of heparin and other medications may, however, alter the concentrations seen clinically. The model does not include any neurohumoral mediators, such as insulin or epinephrine, which may further modify metabolism. Finally, isotopomer analysis determines the relative contributions of each available substrate to acetyl-CoA oxidized within the lung. We do not obtain absolute flux measurements; those may be more relevant under conditions in which the overall metabolic rate may be altered.
In this experiment, there was an association between decreased function after ischemia and an increased utilization of fatty acids as a substrate for oxidative metabolism. Fatty acids have been shown to be detrimental to postischemic myocardium [15], and it is possible that a similar relationship may exist between fatty acid utilization and lung function. Strategies designed to suppress fatty acid utilization in cardiac tissue after ischemia have been shown to improve cardiac performance during reperfusion [16, 17]. Further studies evaluating inhibitors of fatty acid catabolism during storage or reperfusion of lung tissue may be warranted.
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Acknowledgments
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This study was supported by grants from the American Lung Association of Texas and the National Institutes of Health (P41-RR02584), a Merit Review and Clinical Investigator Award of the Department of Veterans Affairs, and the Sarah M. and Charles E. Seay Distinguished Chair in Thoracic Surgery.
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Discussion
Ann. Thorac. Surg. 2000 70: 269.
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Abstract
Introduction
