JOURNAL
OF SURGICAL
RESEARCH
51,82-86
(1991)
Lung Glutamine Flux following Open Heart Surgery KENNETH
HERSKOWITZ, M.D., DONALD A. PLUMLEY, M.D., TOMAS D. MARTIN, M.D., R. DEAN HAUTAMAKI, EDWARD M. COPELAND III, M.D., AND WILEY W. SOUEIA, M.D., Sc.D.l Department
of Surgery, University
of Florida
College of Medicine,
Gainesville,
Florida
M.D.,
32601
Submitted for publication May 11, 1990
dant amino acid in the body and the amino acid most affected by these stress states [2,7,8]. Glutamine depletion in blood and skeletal muscle is commonly observed during critical illness [4,5,6,9]. This catabolic response contributes to a loss of lean body tissue and negative nitrogen balance. A major goal in the nutritional and metabolic care of such patients is the reversal or attenuation of this catabolic response. Hormonal therapy with insulin and growth hormone has been shown to blunt the catabolic response in certain patients [ 10,111. The ability to attenuate net skeletal muscle proteolysis has also been reported after (Y and /3 adrenergic blockade and after administration of prostaglandin synthesis inhibitors [ 12, 131. Johnson et al. [ 141 have demonstrated that the combination of hypothermia, narcotic anesthesia, and neuromuscular blockade significantly decreases skeletal muscle glutamine and alanine release in patients undergoing open heart surgery. Despite the marked decrease in muscle amino acid release following cardiopulmonary bypass, blood glutamine and alanine levels did not drop as much as one might predict. This observation lead us to hypothesize that the maintenance of blood glutamine and alanine levels in the postoperative cardiac patient might be secondary to an increased production of these amino acids by another organ. Since animal data have demonstrated that the lungs play a key role in maintaining glutamine and alanine homeostasis [X,16], we studied the flux of these two amino acids across the lungs in postoperative cardiac surgical patients at a time when previous studies [14] have shown that skeletal muscle glutamine and alanine release in such patients is significantly attenuated. The data from the present study suggest that the lungs play an important role in amino acid metabolism following cardiopulmonary bypass and hypothermic anesthesia.
Despite the attenuated skeletal muscle proteolysis that occurs following hypothermic anesthesia and open heart surgery, blood amino acid levels are maintained, suggesting enhanced amino acid release by another organ. To investigate the role of the lung in this response, we determined the release of glutamine (Gln) and alanine by the lung, since these two amino acids transport two-thirds of circulating amino acid nitrogen. Three groups of patients were studied: (a) preoperative nonstressed controls; (b) postoperative general surgical patients; and (c) postoperative cardiac surgical patients studied on Postoperative Day 1 following open heart surgery requiring cardiopulmonary bypass and hypothermic anesthesia. In preoperative controls the lung was an organ of glutamine and alanine balance. These exchange rates were unaffected by the stress of an abdominal surgical procedure despite a mild increase in pulmonary blood flow. However, lung Gln release in the cardiac surgical patients was significantly increased (-0.6 + 1.2 pmole/kg/min in controls vs -6.5 * 1.3 pmole/kg/min in postoperative hearts, P < 0.05) and was due exclusively to an increase in the pulmonary artery-systemic arterial concentration difference. Alanine release by the lungs was also increased in the postoperative cardiac surgical patients. The mechanism by which this augmented pulmonary glutamine release occurs following open heart surgery is unclear, but the lungs appear to play a central role in maintaining amino acid homeostasis. This metabolic role of the lungs following hypothermic anesthesia and cardiopulmonary bypass has not been previously described. 0 1991
Academic
Press,
Inc.
INTRODUCTION
Catabolic stresses such as major surgery, trauma, and sepsis are characterized by accelerated skeletal muscle amino acid release [l-6]. Glutamine is the most abun-
MATERIALS Patient
1 Supported by NIH Grants CA 45327 and HL 44986, a Grant from
METHODS
Criteria
Elective surgical patients admitted to the Shands Hospital at the University of Florida or the Gainesville Vet-
the Veterans Administration Merit Review Board, and a Career Development Award from the American Cancer Society. oozz-4804/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Eligibility
AND
82
HERSKOWITZ
ET AL.: LUNG
erans Administration Hospital were studied. The study protocol was approved by Institutional Review Board at the University of Florida and by the Subcommittee for Clinical Investigation at the Veterans Administration Hospital. Three groups of patients were studied. Group 1 consisted of preoperative control patients (n = 14) who required a pulmonary artery catheter (Swan-Ganz 7.5F thermodilution catheter, Model 93-A931H-7.5F, American Edwards) and a radial artery catheter insertedpreoperatively for perioperative monitoring. These catheters were placed the night prior to surgery and the patients were monitored in the surgical intensive care unit (SICU). This group of 14 patients included 5 patients who were to have coronary artery bypass grafting, 3 patients scheduled for carotid artery endarterectomy, 4 patients undergoing abdominal aortic aneurysmectomy, and one patient undergoing aortobifemoral artery bypass, one requiring cholecystectomy, and one needing colectomy. The lung flux data from 6 of these patients was included in a previous control group of patients [ 171. A portable chest roentgenograph was obtained to verify the position of the catheter which had been inserted into the pulmonary artery. Pulmonary artery and arterial blood samples were obtained the morning of surgery following a 12hr fast prior to transporting the patient to the operating room. Eligibility criteria in this group required that these patients have no evidence of pneumonia, cancer, or other acute pathology on the preoperative chest X-ray, as well as no evidence of significant weight loss, insulin-dependent diabetes, impaired renal function, impaired hepatic function, or evidence of metastatic malignant disease. The second group of patients studied were general surgical patients (GS, n = 6) who required pulmonary artery catheter placement intraoperatively for postoperative monitoring. Included in this group were patients undergoing elective abdominal surgical procedures. Patients were excluded from this group if they had metastatic cancer, renal insufficiency, liver disease, or an abnormal postoperative chest roentgenograph. The final group of patients studied were patients undergoing elective coronary bypass grafting (CABG, n = 15). Ten of these patients had pulmonary artery catheters placed immediately following intubation and general anesthesia. Five of the patients had PA catheters inserted preoperatively in the SICU. The anesthesia used during the procedure consisted of fentanyl, oxygen, and a muscle relaxant. The same cardiac anesthesia was used in all the patients undergoing CABG. The patients were then placed on cardiopulmonary bypass and actively cooled to a core temperature of 28°C using a Sarns heater-cooler (Sarns, Inc., Ann Arbor, MI). The coronary bypass procedures were performed under cardioplegic arrest using a standard potassium crystalloid cardioplegic solution. Myocardial temperatures ranged from 10 to 18°C during the procedure. The patients were then actively rewarmed using the Sarns heater-cooler to
GLUTAMINE
FLUX
83
37°C. The mean duration of the pump run was 110 min and the average length of time required to wean off the pump was 10 min. Nonpulsatile flow was used in all patients and was maintained at 2.5 liters/min/m’ throughout the pump run.
Study Procedure Studies were performed between 8:00 and 10~00 AM in the postabsorptive state with 5% dextrose and electrolytes in water administered at a rate of l-2 ml/kg body weight (BW)/hr. Control patients were studied in the SICU on the morning prior to their surgical procedure. The other two groups of patients were studied on Postoperative Day 1 (PODl). All patients were studied in the postabsorptive state. None of the patients had roentgenographic evidence of pneumonia or adult respiratory distress syndrome. Pulmonary artery blood flow (cardiac output) was measured by thermodilution and the average of three measurements was recorded. Immediately prior to determining pulmonary artery blood flow, triplicate pulmonary artery and radial artery blood samples were obtained and analyzed in duplicate for glutamine, alanine, and glutamate.
Sample Collection and Processing The blood samples were immediately placed on ice after collection. Samples were promptly deproteinized with 10% perchloric acid, neutralized to a pH of 6.8 with tripotassium phosphate buffer (0.52 M) and frozen at -20°C for amino acid analysis. Glutamine, glutamate, and alanine concentrations were determined by microfluorometric enzymatic assay adapted from the method described by Bergmeyer [18] using a Turner Model 112 filter flourometer (Sequoia-Turner Corp., Mountain View, CA).
Statistical Analysis Since only five of the cardiac surgical patients had preoperative Swan-Ganz placement (the other patients had PA catheter placement intraoperatively), the number of patients in each group was increased and analysis of variance [19] was used to compare the three independent patient groups. All pulmonary artery-systemic arterial concentration differences were analyzed for statistical difference with zero to determine uptake or release of the amino acid. Substrate exchange (flux) was determined by multiplying pulmonary blood flux by the concentration difference across the lung of the respective amino acids. Flow and flux measurements were divided by the body weight of the patients and expressed per kilogram of body weight. RESULTS
Pulmonary Blood Flow Pulmonary blood flow (Fig. 1) was 61.6 -+ 4.6 ml/kg BW/min in preoperative controls and increased by ap-
84
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
51, NO. 1, JULY
increased by 18% in the (406 + 34 pmole/liter, P concentration was not three groups of patients
(ml/kg BW/min) ‘O0d
1991
postoperative cardiac patients < 0.10). The arterial glutamate significantly different in the studied.
Pulmonary Artery-Systemic Arterial Concentration Differences
Preop Control
POD+OS
Hypothermia
FIG. 1. Pulmonary blood flow (ml/kg BW/min) in preoperative controls, postoperative general surgery patients (PODl-GS), and cardiac surgical patients (hypothermia). *P < 0.05 vs control.
The pulmonary artery-systemic arterial concentration differences (PA-A) for glutamine, glutamate, and alanine were not significantly different than zero in the preoperative controls and the postoperative general surgical patients. In the postoperative cardiac patients the glutamine PA-A concentration difference was -94 +- 19 pmole/liter (P < 0.05 vs control and POD 1 GS patients) while the alanine PA-A concentration difference was -29 f 11 (P < 0.10 vs control).
Lung Amino Acid Flux proximately 20% following a major abdominal operation (77 + 4.4 ml/kg BW/min, P < 0.05). Pulmonary blood flow following cardiopulmonary bypass with hypothermic anesthesia was 70.8 f 3.3 ml/kg BW/min.
Blood Amino Acid Concentrations Whole blood arterial concentrations of amino acids are shown in Table 1. The arterial glutamine concentration in preoperative control patients was 537 + 39 pmole/liter, a value which decreased by nearly 20% in the postoperative general surgical patients (449 _t 23, P < O.lO), but was maintained in the postoperative cardiac patients (508 +- 27 pmole/liter). The arterial alanine concentration in preoperative controls was 344 + 28 pmole/liter. This value was unchanged in the postoperative general surgical patients (358 _+31 pmole/liter), but
TABLE Arterial
Amino acid
Substrate flux across the lungs was significantly altered following cardiopulmonary bypass with hypothermic anesthesia (Table 1). In preoperative controls the lung was an organ of glutamine balance (-0.62 f 1.2 pmole/kg BW/min) and alanine balance (0.83 +- 1.5 pmole/kg B W/min) . These flux rates did not change significantly following a general surgical procedure. In the postoperative cardiac patients following cardiopulmonary bypass with hypothermic anesthesia the lung was an organ of net glutamine release (-6.5 f 1.3 pmole/ kg BW/min, P < 0.05 vs controls and POD1 GS). Those five cardiac surgical patients who had a PA catheter placed preoperatively and therefore served as their own control exhibited a similar increase in lung glutamine release (-0.56 + 0.29 pmole/kg/min vs -6.81 f 1.91, P < 0.01, paired t test). This increased release was due pri-
1
(ART) Concentrations, Pulmonary Artery-Systemic Arterial Concentration Differences, and Lung Flux of Amino Acids Group (4
ART (rmole/liter)
Gln
Pre-op(l4) PODl-GS-(6) PODl-HT(15)
537 f 39 449 k 23 508 + 27
Glu
Pre-op(l4) PODl-GS(6) PODl-HT(15)
50 -c 11 76 + 16 59 + 10
Ala
Pre-op(l4) PODl-GS(6) PODl-HT(15)
344 -t 28 358 -t 31 406 f 34
PA-ART (flmole/liter)
(PA-ART)
Lung flux (pmole/kg BW/min)
-3 + 20 -9-c 7 -94 ? 19*,** o+ -3-c -2*
3 3 3
13 t 20 -6 + 13 -29 5 11*
Note. Data expressed as means * SEM; Flux, net release; Gln, glutamine; Glu, glutamate; studied on PODl; and PODl-HT, cardiac surgical patients studied on PODl. * Significantly different than zero. ** P < 0.05 compared to preop controls and PODl-GS.
Ala, alanine; PODl-GS,
-0.62 + 1.2 -0.72 t 0.6 -6.55 f 1.3** -0.01 f 0.20 -0.25 k 0.24 -0.10 5 0.23 0.83 f 1.5 -0.71 f 1.1 -2.03 r 0.80 general surgical patients
HERSKOWITZ
ET AL.: LUNG GLUTAMINE
marily to an increase in the pulmonary artery-systemic arterial concentration difference. In these same patients, alanine was released by the lungs at a rate of -2.03 -t 0.80 pmole/kg BW/min. Like glutamine, the increased release of alanine by the lungs in postoperative cardiac patients (P < 0.10 vs controls) was due primarily to an increase in the pulmonary artery-systemic arterial concentration difference (Table 1). DISCUSSION The role of the lungs in maintaining glutamine and alanine homeostasis following CABG was studied in order to gain further insight into the alterations in amino acid flux that occur following cardiopulmonary bypass and hypothermic anesthesia. Since these two amino acids collectively transport two-thirds of circulating amino acid nitrogen, they represent the major carriers of nitrogen between tissues. Glutamine is utilized primarily by the gut mucosa and appears to be required for mucosal metabolism, structure, and function [20]. Alanine is consumed primarily by the liver to support gluconeogenesis. Recently, Johnson et al. [14] observed that the expected catabolic response following standard operative stresses did not occur in a group of patients who underwent major cardiac surgical procedures. The intraoperative management of all of these individuals included cardiopulmonary bypass, hypothermia, high-dose narcotic anesthesia, and neuromuscular blockade. To examine this unexpected response, further studies were performed in laboratory dogs in which surgical stress was standardized, allowing comparison of the protein catabolic response following hypothermic anesthesia with that occurring after narcotic or barbiturate administration. In experimental animals, as well as in patients, the combination of hypothermia and anesthesia reliably attenuated the post-traumatic muscle proteolysis that normally develops after major operative procedures. Their flux measurements were made following anesthesia and hypothermia, but at a time when the subjects or experimental animals were euthermic and awake. The persistent decrease in glutamine and alanine e&x suggests that the signal for skeletal muscle proteolysis occurred at the time of the surgical procedure and that this stimulus was prevented or blocked by the anesthetichypothermic technique. Unlike skeletal muscle, which demonstrated an attenuated release of glutamine following hypothermic anesthesia [14], our study showed that net lung glutamine release was significantly augmented. Alanine release by the lungs was also accelerated following hypothermic anesthesia. Although skeletal muscle has traditionally been viewed as the principal organ that releases amino acids, recent evidence suggests that the lungs may play a central role in amino acid exchange and may be a key regulator of glutamine metabolism in normal and cata-
FLUX
85
bolic states [15-171. Welbourne [21] showed that the lung is an organ of net glutamine release and data from our laboratories demonstrate that, in postabsorptive rats, the lung releases significantly more glutamine and alanine than the hindquarter [ 161. The lungs may work together with skeletal muscle to help maintain nitrogen homeostasis by exporting amino acids for other tissues. This is especially true in the septic surgical patient where an accelerated release of glutamine and alanine occurs at a time when skeletal muscle amino acid release is also accelerated [ 171. Thus, the lungs play an active metabolic role in glutamine and alanine metabolism and may be a key regulator in interorgan nitrogen tlux following major injury and infection as well as after open heart surgery. Lung blood flow in this study was measured by the thermodilution principal utilizing an indwelling pulmonary artery catheter. The values obtained in the preoperative control patients, postoperative general surgical patients, and postoperative cardiac patients are similar to those reported by others [22]. Although the lung derives its blood supply from two sources, the contribution of the bronchial circulation total pulmonary blood flow is only about 1% [23] and thus determination of amino acid flux using the substrate concentrations in the pulmonary artery and systemic arterial systems will be accurate. To confirm that radial arterial blood accurately reflects pulmonary venous blood, six postoperative cardiac patients with left atria1 catheters and radial arterial catheters were studied. Blood simultaneously drawn from the left atrium as well as the radial artery showed no difference in amino acid concentrations (data not shown). It is not clear why the lungs should release large amounts of these two amino acids in the immediate postoperative period following open heart surgery. The signals which govern the attenuated skeletal muscle amino acid release are likely to be different than the mediators responsible for the accelerated lung glutamine and alanine release that we observed following open heart surgery. The pulmonary response may not be adaptive at all and may be due in part to an ischemia/reperfusion injury. Regardless of the cause, it is unlikely that this represents accelerated protein catabolism since glutamine comprises only 6-7s of structural proteins [ 241. A more likely explanation is that there is an increase in de rwvo glutamine biosynthesis. Interestingly, the lung contains the prerequisite enzymatic machinery, glutamine synthetase [21], to synthesize glutamine but has received little attention as a potential release site. The accelerated lung glutamine eflux may be designed to supply this key fuel to the intestinal tract. Gut glutamine uptake by the human gastrointestinal tract falls during hypothermia but increases markedly following rewarming [25], suggesting that mucosal glutamine requirements are increased in the postoperative period. The lungs may supply some of this glutamine at a time when muscle
86
JOURNAL
OF SURGICAL
RESEARCH:
glutamine release is diminished. Likewise, the increase in lung alanine release may help supply the liver with its principal gluconeogenic amino acid. The events which mediate these changes in lung glutamine metabolism following open heart surgery may be secondary to ischemia/reperfusion and/or other physiologic and biochemical changes that are mediated by local factors or possibly by the central nervous system. Moreover, the specific cells in the lungs that are responsible for releasing net amounts of glutamine into the blood are unknown. Although the lung is composed of multiple cell populations, the pulmonary endothelial cells (PECs) are by far the most common [26]. Preliminary studies using cultured PECs demonstrate that these cells are avid glutamine consumers [27]. Conceivably the avid glutamine uptake by PECs may be diminished following the hypothermia and/or local hypoxia that is associated with cardiopulmonary bypass, resulting in an increase in lung glutamine release. Further studies will be required to better define the role of the individual cell population in lung glutamine metabolism. The lung which has traditionally been viewed as an organ of gas exchange appears to be an important organ of glutamine metabolism. Additional studies are required to increase our understanding of the mechanisms that regulate lung glutamine metabolism at the whole organ, cellular, and molecular level. REFERENCES 1.
2.
3.
4.
5.
6.
I.
8.
Vinnar, E., Bergstrom, J., and Fur&, P. Influence of the postoperative state on the intracellular free amino acids in human muscle tissue. Ann. Sorg. 182: 665, 1975. Furst, P. Catabolic stress on intracellular amino acid pool. In W. H. Horl and A. Heidland (Eds.), Answers in Experimental Medicine and Biology. New York: Plenum, 1984. Vol. 167, pp. 571-579. Mulbacher, F., Kapadia, C. R., Colpoys, M. F. et al. Effects of glucocorticoids on glutamine metabolism in skeletal muscle. Amer. J. Physiol. 247: E75, 1984. Askanazi, J., Carpentier, Y. A., Michelsen, C. B., et al. Muscle and plasma amino acids following injury: Influence of intercurrent infection. Ann. Surg. 192: 78, 1980. Kapadia, C. R., Muhlbacher, F., Smith, R. J., et al. Alterations in glutamine metabolism in response to operative stress and food deprivation. Surg. Forum 33: 19, 1982. Roth, E., Funovics, J., Muhlbacher, F., et al. Metabolic disorders in severe abdominal sepsis: Glutamine deficiency in skeletal muscle. Clin. Nutr. 1: 25, 1982. Souba, W. W., and Wilmore, D. W. Diet and nutrition in the care of the patient with surgery, trauma and sepsis. In M. Shils and V. Young (Eds.), Modern Nutrition in Health and Disease. Philadelphia: Lea & Febiger, 1988. 7th ed., pp. 1306-1336. Souba, W. W., Wilmore, D. W. Postoperative alteration of arte-
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12.
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riovenous exchange of amino acids across the gastrointestinal tract. Surgery 94: 342, 1983. Souba, W. W. Glutamine Metabolism in Catabolic States: Role of the Intestinal Tract. Thesis, Harvard School of Public Health, Department of Nutritional Biochemistry, June 1984. Wolfson, A. M. J., Healthy, R. V., and Allison, S. P. Insulin to inhibit protein catabolism after injury. N. Engl. J. Med. 300: 14, 1979. Wilmore, D. W., Moylan, J. A., Briston, B. J., et al. Anabolic effects of human growth hormones and high caloric feedings following thermal injury. Surg. Gynecol. Obstet. 138: 875, 1974. Hulton, N. R., Johnson, D. J., and Wilmore, D. W. Hormonal blockage modifies post-trauma protein catabolism. J. Surg. Res.
39: 310,1985. 13.
14.
15.
16.
Rowe, D. J. F., and Gedem, G. Reversal by prostaglandin E2 infusion of the effects of indomethacin on the excretion of nitrogenous compounds in the rat. Brit. J. Enp. Path&. 64: 306, 1983. Johnson, D. J., Brooks, D. C., Pressler, V. M., et al. Hypothermic anesthesia attenuates postoperative proteolysis. Ann. Surg. 204: 419,1986. Souha, W. W., Plumley, D. A., Salloum, R. M., et al. Effects of glucocorticoids on lung glutamine and alanine metabolism. Surgery, 108: 213,199O. Plumley, D. A., Austgen, T. R., Salloum, R. M., et al. The role of the lungs in maintaining amino acid homeostasis. JPEN, 14(6):
569,199O. 17.
Plumley, D. A., Souba, W. W., et al. Accelerated lung amino acid release in hyperdynamic septic surgical patients. Arch. Surg.
18.
Bergmeyer, H. U. (Ed.) Methods of Enzymatic Analysis. New York: Academic Press, 1974. Bhattacharyya, G. K., and Johnson, R. A. Statistical Concepts and Method. New York: Wiley, 1977. Pp. 453-505. Souba, W. W., Klimberg, V. S., Bland, K. I., et al. (Current Research Review) The role of glutamine in maintaining gut structure and function and supporting the metabolic response to injury and infection. J. Surg. Res. 48: 1, 1990. Welbourne, T. C. Role of the lung in glutamine homeostasis. Contr. Nephrol. 63: 178, 1988. Bland, R. D., and Showmaker, W. L. Common physiologic pattern in general surgical patients: Hemodynamic and oxygen transport changes during and after operation in patients with and without associated medical problems. Surg. Clin. North Amer. 66(4): 795, 1985. Shields, T. W. (Ed.) Surgical anatomy of the lungs. In General Thoracic Surgery. Philadelphia: Lea & Fehiger, 1972. Pp. 55-73. Kominz, D. R., Hough, A., Symonds, P., et al. The amino acid composition of actin, myosin, tropomysion, and the meromyosins. Arch. Biochem. Biophys. 60: 148,1954. Herskowitz, K., and Souba, W. W. The effects of hypothermic anesthesia on gut fuel metabolism. Surg. Forum 41: 301,199O. Crapo, J. D., Barry, B. E., Fescue, H. A., et al. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Amer. Rev. Respir. Dis. 122:
126: 57,199o.
19.
20.
21. 22.
23. 24. 25. 26.
123,198O. 27. Herskowitz,
K., Block, E. R., and Souba, W. W. Characterization of L-glutamine transport by cultured pulmonary endothelial cells. Am. J. Physiol. (in press).
OF SURGICAL
RESEARCH
51,82-86
(1991)
Lung Glutamine Flux following Open Heart Surgery KENNETH
HERSKOWITZ, M.D., DONALD A. PLUMLEY, M.D., TOMAS D. MARTIN, M.D., R. DEAN HAUTAMAKI, EDWARD M. COPELAND III, M.D., AND WILEY W. SOUEIA, M.D., Sc.D.l Department
of Surgery, University
of Florida
College of Medicine,
Gainesville,
Florida
M.D.,
32601
Submitted for publication May 11, 1990
dant amino acid in the body and the amino acid most affected by these stress states [2,7,8]. Glutamine depletion in blood and skeletal muscle is commonly observed during critical illness [4,5,6,9]. This catabolic response contributes to a loss of lean body tissue and negative nitrogen balance. A major goal in the nutritional and metabolic care of such patients is the reversal or attenuation of this catabolic response. Hormonal therapy with insulin and growth hormone has been shown to blunt the catabolic response in certain patients [ 10,111. The ability to attenuate net skeletal muscle proteolysis has also been reported after (Y and /3 adrenergic blockade and after administration of prostaglandin synthesis inhibitors [ 12, 131. Johnson et al. [ 141 have demonstrated that the combination of hypothermia, narcotic anesthesia, and neuromuscular blockade significantly decreases skeletal muscle glutamine and alanine release in patients undergoing open heart surgery. Despite the marked decrease in muscle amino acid release following cardiopulmonary bypass, blood glutamine and alanine levels did not drop as much as one might predict. This observation lead us to hypothesize that the maintenance of blood glutamine and alanine levels in the postoperative cardiac patient might be secondary to an increased production of these amino acids by another organ. Since animal data have demonstrated that the lungs play a key role in maintaining glutamine and alanine homeostasis [X,16], we studied the flux of these two amino acids across the lungs in postoperative cardiac surgical patients at a time when previous studies [14] have shown that skeletal muscle glutamine and alanine release in such patients is significantly attenuated. The data from the present study suggest that the lungs play an important role in amino acid metabolism following cardiopulmonary bypass and hypothermic anesthesia.
Despite the attenuated skeletal muscle proteolysis that occurs following hypothermic anesthesia and open heart surgery, blood amino acid levels are maintained, suggesting enhanced amino acid release by another organ. To investigate the role of the lung in this response, we determined the release of glutamine (Gln) and alanine by the lung, since these two amino acids transport two-thirds of circulating amino acid nitrogen. Three groups of patients were studied: (a) preoperative nonstressed controls; (b) postoperative general surgical patients; and (c) postoperative cardiac surgical patients studied on Postoperative Day 1 following open heart surgery requiring cardiopulmonary bypass and hypothermic anesthesia. In preoperative controls the lung was an organ of glutamine and alanine balance. These exchange rates were unaffected by the stress of an abdominal surgical procedure despite a mild increase in pulmonary blood flow. However, lung Gln release in the cardiac surgical patients was significantly increased (-0.6 + 1.2 pmole/kg/min in controls vs -6.5 * 1.3 pmole/kg/min in postoperative hearts, P < 0.05) and was due exclusively to an increase in the pulmonary artery-systemic arterial concentration difference. Alanine release by the lungs was also increased in the postoperative cardiac surgical patients. The mechanism by which this augmented pulmonary glutamine release occurs following open heart surgery is unclear, but the lungs appear to play a central role in maintaining amino acid homeostasis. This metabolic role of the lungs following hypothermic anesthesia and cardiopulmonary bypass has not been previously described. 0 1991
Academic
Press,
Inc.
INTRODUCTION
Catabolic stresses such as major surgery, trauma, and sepsis are characterized by accelerated skeletal muscle amino acid release [l-6]. Glutamine is the most abun-
MATERIALS Patient
1 Supported by NIH Grants CA 45327 and HL 44986, a Grant from
METHODS
Criteria
Elective surgical patients admitted to the Shands Hospital at the University of Florida or the Gainesville Vet-
the Veterans Administration Merit Review Board, and a Career Development Award from the American Cancer Society. oozz-4804/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Eligibility
AND
82
HERSKOWITZ
ET AL.: LUNG
erans Administration Hospital were studied. The study protocol was approved by Institutional Review Board at the University of Florida and by the Subcommittee for Clinical Investigation at the Veterans Administration Hospital. Three groups of patients were studied. Group 1 consisted of preoperative control patients (n = 14) who required a pulmonary artery catheter (Swan-Ganz 7.5F thermodilution catheter, Model 93-A931H-7.5F, American Edwards) and a radial artery catheter insertedpreoperatively for perioperative monitoring. These catheters were placed the night prior to surgery and the patients were monitored in the surgical intensive care unit (SICU). This group of 14 patients included 5 patients who were to have coronary artery bypass grafting, 3 patients scheduled for carotid artery endarterectomy, 4 patients undergoing abdominal aortic aneurysmectomy, and one patient undergoing aortobifemoral artery bypass, one requiring cholecystectomy, and one needing colectomy. The lung flux data from 6 of these patients was included in a previous control group of patients [ 171. A portable chest roentgenograph was obtained to verify the position of the catheter which had been inserted into the pulmonary artery. Pulmonary artery and arterial blood samples were obtained the morning of surgery following a 12hr fast prior to transporting the patient to the operating room. Eligibility criteria in this group required that these patients have no evidence of pneumonia, cancer, or other acute pathology on the preoperative chest X-ray, as well as no evidence of significant weight loss, insulin-dependent diabetes, impaired renal function, impaired hepatic function, or evidence of metastatic malignant disease. The second group of patients studied were general surgical patients (GS, n = 6) who required pulmonary artery catheter placement intraoperatively for postoperative monitoring. Included in this group were patients undergoing elective abdominal surgical procedures. Patients were excluded from this group if they had metastatic cancer, renal insufficiency, liver disease, or an abnormal postoperative chest roentgenograph. The final group of patients studied were patients undergoing elective coronary bypass grafting (CABG, n = 15). Ten of these patients had pulmonary artery catheters placed immediately following intubation and general anesthesia. Five of the patients had PA catheters inserted preoperatively in the SICU. The anesthesia used during the procedure consisted of fentanyl, oxygen, and a muscle relaxant. The same cardiac anesthesia was used in all the patients undergoing CABG. The patients were then placed on cardiopulmonary bypass and actively cooled to a core temperature of 28°C using a Sarns heater-cooler (Sarns, Inc., Ann Arbor, MI). The coronary bypass procedures were performed under cardioplegic arrest using a standard potassium crystalloid cardioplegic solution. Myocardial temperatures ranged from 10 to 18°C during the procedure. The patients were then actively rewarmed using the Sarns heater-cooler to
GLUTAMINE
FLUX
83
37°C. The mean duration of the pump run was 110 min and the average length of time required to wean off the pump was 10 min. Nonpulsatile flow was used in all patients and was maintained at 2.5 liters/min/m’ throughout the pump run.
Study Procedure Studies were performed between 8:00 and 10~00 AM in the postabsorptive state with 5% dextrose and electrolytes in water administered at a rate of l-2 ml/kg body weight (BW)/hr. Control patients were studied in the SICU on the morning prior to their surgical procedure. The other two groups of patients were studied on Postoperative Day 1 (PODl). All patients were studied in the postabsorptive state. None of the patients had roentgenographic evidence of pneumonia or adult respiratory distress syndrome. Pulmonary artery blood flow (cardiac output) was measured by thermodilution and the average of three measurements was recorded. Immediately prior to determining pulmonary artery blood flow, triplicate pulmonary artery and radial artery blood samples were obtained and analyzed in duplicate for glutamine, alanine, and glutamate.
Sample Collection and Processing The blood samples were immediately placed on ice after collection. Samples were promptly deproteinized with 10% perchloric acid, neutralized to a pH of 6.8 with tripotassium phosphate buffer (0.52 M) and frozen at -20°C for amino acid analysis. Glutamine, glutamate, and alanine concentrations were determined by microfluorometric enzymatic assay adapted from the method described by Bergmeyer [18] using a Turner Model 112 filter flourometer (Sequoia-Turner Corp., Mountain View, CA).
Statistical Analysis Since only five of the cardiac surgical patients had preoperative Swan-Ganz placement (the other patients had PA catheter placement intraoperatively), the number of patients in each group was increased and analysis of variance [19] was used to compare the three independent patient groups. All pulmonary artery-systemic arterial concentration differences were analyzed for statistical difference with zero to determine uptake or release of the amino acid. Substrate exchange (flux) was determined by multiplying pulmonary blood flux by the concentration difference across the lung of the respective amino acids. Flow and flux measurements were divided by the body weight of the patients and expressed per kilogram of body weight. RESULTS
Pulmonary Blood Flow Pulmonary blood flow (Fig. 1) was 61.6 -+ 4.6 ml/kg BW/min in preoperative controls and increased by ap-
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increased by 18% in the (406 + 34 pmole/liter, P concentration was not three groups of patients
(ml/kg BW/min) ‘O0d
1991
postoperative cardiac patients < 0.10). The arterial glutamate significantly different in the studied.
Pulmonary Artery-Systemic Arterial Concentration Differences
Preop Control
POD+OS
Hypothermia
FIG. 1. Pulmonary blood flow (ml/kg BW/min) in preoperative controls, postoperative general surgery patients (PODl-GS), and cardiac surgical patients (hypothermia). *P < 0.05 vs control.
The pulmonary artery-systemic arterial concentration differences (PA-A) for glutamine, glutamate, and alanine were not significantly different than zero in the preoperative controls and the postoperative general surgical patients. In the postoperative cardiac patients the glutamine PA-A concentration difference was -94 +- 19 pmole/liter (P < 0.05 vs control and POD 1 GS patients) while the alanine PA-A concentration difference was -29 f 11 (P < 0.10 vs control).
Lung Amino Acid Flux proximately 20% following a major abdominal operation (77 + 4.4 ml/kg BW/min, P < 0.05). Pulmonary blood flow following cardiopulmonary bypass with hypothermic anesthesia was 70.8 f 3.3 ml/kg BW/min.
Blood Amino Acid Concentrations Whole blood arterial concentrations of amino acids are shown in Table 1. The arterial glutamine concentration in preoperative control patients was 537 + 39 pmole/liter, a value which decreased by nearly 20% in the postoperative general surgical patients (449 _t 23, P < O.lO), but was maintained in the postoperative cardiac patients (508 +- 27 pmole/liter). The arterial alanine concentration in preoperative controls was 344 + 28 pmole/liter. This value was unchanged in the postoperative general surgical patients (358 _+31 pmole/liter), but
TABLE Arterial
Amino acid
Substrate flux across the lungs was significantly altered following cardiopulmonary bypass with hypothermic anesthesia (Table 1). In preoperative controls the lung was an organ of glutamine balance (-0.62 f 1.2 pmole/kg BW/min) and alanine balance (0.83 +- 1.5 pmole/kg B W/min) . These flux rates did not change significantly following a general surgical procedure. In the postoperative cardiac patients following cardiopulmonary bypass with hypothermic anesthesia the lung was an organ of net glutamine release (-6.5 f 1.3 pmole/ kg BW/min, P < 0.05 vs controls and POD1 GS). Those five cardiac surgical patients who had a PA catheter placed preoperatively and therefore served as their own control exhibited a similar increase in lung glutamine release (-0.56 + 0.29 pmole/kg/min vs -6.81 f 1.91, P < 0.01, paired t test). This increased release was due pri-
1
(ART) Concentrations, Pulmonary Artery-Systemic Arterial Concentration Differences, and Lung Flux of Amino Acids Group (4
ART (rmole/liter)
Gln
Pre-op(l4) PODl-GS-(6) PODl-HT(15)
537 f 39 449 k 23 508 + 27
Glu
Pre-op(l4) PODl-GS(6) PODl-HT(15)
50 -c 11 76 + 16 59 + 10
Ala
Pre-op(l4) PODl-GS(6) PODl-HT(15)
344 -t 28 358 -t 31 406 f 34
PA-ART (flmole/liter)
(PA-ART)
Lung flux (pmole/kg BW/min)
-3 + 20 -9-c 7 -94 ? 19*,** o+ -3-c -2*
3 3 3
13 t 20 -6 + 13 -29 5 11*
Note. Data expressed as means * SEM; Flux, net release; Gln, glutamine; Glu, glutamate; studied on PODl; and PODl-HT, cardiac surgical patients studied on PODl. * Significantly different than zero. ** P < 0.05 compared to preop controls and PODl-GS.
Ala, alanine; PODl-GS,
-0.62 + 1.2 -0.72 t 0.6 -6.55 f 1.3** -0.01 f 0.20 -0.25 k 0.24 -0.10 5 0.23 0.83 f 1.5 -0.71 f 1.1 -2.03 r 0.80 general surgical patients
HERSKOWITZ
ET AL.: LUNG GLUTAMINE
marily to an increase in the pulmonary artery-systemic arterial concentration difference. In these same patients, alanine was released by the lungs at a rate of -2.03 -t 0.80 pmole/kg BW/min. Like glutamine, the increased release of alanine by the lungs in postoperative cardiac patients (P < 0.10 vs controls) was due primarily to an increase in the pulmonary artery-systemic arterial concentration difference (Table 1). DISCUSSION The role of the lungs in maintaining glutamine and alanine homeostasis following CABG was studied in order to gain further insight into the alterations in amino acid flux that occur following cardiopulmonary bypass and hypothermic anesthesia. Since these two amino acids collectively transport two-thirds of circulating amino acid nitrogen, they represent the major carriers of nitrogen between tissues. Glutamine is utilized primarily by the gut mucosa and appears to be required for mucosal metabolism, structure, and function [20]. Alanine is consumed primarily by the liver to support gluconeogenesis. Recently, Johnson et al. [14] observed that the expected catabolic response following standard operative stresses did not occur in a group of patients who underwent major cardiac surgical procedures. The intraoperative management of all of these individuals included cardiopulmonary bypass, hypothermia, high-dose narcotic anesthesia, and neuromuscular blockade. To examine this unexpected response, further studies were performed in laboratory dogs in which surgical stress was standardized, allowing comparison of the protein catabolic response following hypothermic anesthesia with that occurring after narcotic or barbiturate administration. In experimental animals, as well as in patients, the combination of hypothermia and anesthesia reliably attenuated the post-traumatic muscle proteolysis that normally develops after major operative procedures. Their flux measurements were made following anesthesia and hypothermia, but at a time when the subjects or experimental animals were euthermic and awake. The persistent decrease in glutamine and alanine e&x suggests that the signal for skeletal muscle proteolysis occurred at the time of the surgical procedure and that this stimulus was prevented or blocked by the anesthetichypothermic technique. Unlike skeletal muscle, which demonstrated an attenuated release of glutamine following hypothermic anesthesia [14], our study showed that net lung glutamine release was significantly augmented. Alanine release by the lungs was also accelerated following hypothermic anesthesia. Although skeletal muscle has traditionally been viewed as the principal organ that releases amino acids, recent evidence suggests that the lungs may play a central role in amino acid exchange and may be a key regulator of glutamine metabolism in normal and cata-
FLUX
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bolic states [15-171. Welbourne [21] showed that the lung is an organ of net glutamine release and data from our laboratories demonstrate that, in postabsorptive rats, the lung releases significantly more glutamine and alanine than the hindquarter [ 161. The lungs may work together with skeletal muscle to help maintain nitrogen homeostasis by exporting amino acids for other tissues. This is especially true in the septic surgical patient where an accelerated release of glutamine and alanine occurs at a time when skeletal muscle amino acid release is also accelerated [ 171. Thus, the lungs play an active metabolic role in glutamine and alanine metabolism and may be a key regulator in interorgan nitrogen tlux following major injury and infection as well as after open heart surgery. Lung blood flow in this study was measured by the thermodilution principal utilizing an indwelling pulmonary artery catheter. The values obtained in the preoperative control patients, postoperative general surgical patients, and postoperative cardiac patients are similar to those reported by others [22]. Although the lung derives its blood supply from two sources, the contribution of the bronchial circulation total pulmonary blood flow is only about 1% [23] and thus determination of amino acid flux using the substrate concentrations in the pulmonary artery and systemic arterial systems will be accurate. To confirm that radial arterial blood accurately reflects pulmonary venous blood, six postoperative cardiac patients with left atria1 catheters and radial arterial catheters were studied. Blood simultaneously drawn from the left atrium as well as the radial artery showed no difference in amino acid concentrations (data not shown). It is not clear why the lungs should release large amounts of these two amino acids in the immediate postoperative period following open heart surgery. The signals which govern the attenuated skeletal muscle amino acid release are likely to be different than the mediators responsible for the accelerated lung glutamine and alanine release that we observed following open heart surgery. The pulmonary response may not be adaptive at all and may be due in part to an ischemia/reperfusion injury. Regardless of the cause, it is unlikely that this represents accelerated protein catabolism since glutamine comprises only 6-7s of structural proteins [ 241. A more likely explanation is that there is an increase in de rwvo glutamine biosynthesis. Interestingly, the lung contains the prerequisite enzymatic machinery, glutamine synthetase [21], to synthesize glutamine but has received little attention as a potential release site. The accelerated lung glutamine eflux may be designed to supply this key fuel to the intestinal tract. Gut glutamine uptake by the human gastrointestinal tract falls during hypothermia but increases markedly following rewarming [25], suggesting that mucosal glutamine requirements are increased in the postoperative period. The lungs may supply some of this glutamine at a time when muscle
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glutamine release is diminished. Likewise, the increase in lung alanine release may help supply the liver with its principal gluconeogenic amino acid. The events which mediate these changes in lung glutamine metabolism following open heart surgery may be secondary to ischemia/reperfusion and/or other physiologic and biochemical changes that are mediated by local factors or possibly by the central nervous system. Moreover, the specific cells in the lungs that are responsible for releasing net amounts of glutamine into the blood are unknown. Although the lung is composed of multiple cell populations, the pulmonary endothelial cells (PECs) are by far the most common [26]. Preliminary studies using cultured PECs demonstrate that these cells are avid glutamine consumers [27]. Conceivably the avid glutamine uptake by PECs may be diminished following the hypothermia and/or local hypoxia that is associated with cardiopulmonary bypass, resulting in an increase in lung glutamine release. Further studies will be required to better define the role of the individual cell population in lung glutamine metabolism. The lung which has traditionally been viewed as an organ of gas exchange appears to be an important organ of glutamine metabolism. Additional studies are required to increase our understanding of the mechanisms that regulate lung glutamine metabolism at the whole organ, cellular, and molecular level. REFERENCES 1.
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