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Ann Thorac Surg 1999;68:169-172
© 1999 The Society of Thoracic Surgeons

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Original Articles

Time course of endothelin-1 and adrenomedullin after the Fontan procedure

^a Department of Pediatric Cardiac Surgery, Tokyo Women’s Medical University, Heart Institute of Japan, Tokyo, Japan
^b Department of Cardiology, Tokyo Women’s Medical University, Heart Institute of Japan, Tokyo, Japan

Address reprint requests to Dr Hiramatsu, Department of Pediatric Cardiac Surgery, Tokyo Women’s Medical University, Heart Institute of Japan, 8-1, Shinjuku-ku, Tokyo, 162-8666 Japan

	Abstract

Top Abstract Introduction Patients and methods Results Comment References

 
Background. The endothelium-derived vasoconstrictor endothelin (ET)-1 might contribute to the physiology of blood flow regulation and play a role in cardiovascular disease. Adrenomedullin (AM) is a potent vasodilator peptide that has major effects on cardiovascular function and has multiple biologic effects involved in cardiovascular homeostasis. Although pulmonary vascular resistance is known to be one of the most important factors to determine the indications for a Fontan procedure, the time course of the plasma cytokine before and after the Fontan procedure is not known.

Methods. Sixteen patients were divided into two groups, 8 patients (1 to 14 years) who had the Fontan procedure (atriopulmonary connection) and 8 age-matched controls who had biventricular repair with normal central venous pressure. Plasma ET-1 and adrenomedullin levels were measured in both groups immediately before cardiopulmonary bypass, immediately after cardiopulmonary bypass, and 6 and 24 hours after cardiopulmonary bypass. A thermodilution catheter was inserted during the operation, and mean pulmonary arterial pressure, pulmonary wedge pressure, and cardiac output were measured, and pulmonary vascular resistance was calculated at the same time points. Correlation between the plasma ET-1 levels and pulmonary vascular resistance data were obtained in the Fontan group.

Results. Plasma ET-1 levels in the Fontan group were elevated after operation and were higher than the control group at 6 and 24 hours after cardiopulmonary bypass. Plasma adrenomedullin in the Fontan group was lower than in the control group at 6 and 24 hours after cardiopulmonary bypass. A significant positive correlation was obtained between the plasma ET-1 and pulmonary vascular resistance data (r = 0.475).

Conclusions. Imbalance between increased ET-1 and relatively decreased adrenomedullin after cardiopulmonary bypass in the Fontan procedure could contribute to dominant effects of ET-1, which might induce vasoconstriction after the Fontan procedure. ET-1 might play an important role in maintaining vasoconstriction after the Fontan procedure.

	Introduction

Top Abstract Introduction Patients and methods Results Comment References

 
Endothelin (ET) is an endothelium-dependent contracting factor, and it has been proposed that ET contributes to the normal physiology of blood flow regulation and has an effect on cardiovascular disease. There are three ET isoforms (ET-1, ET-2, and ET-3), and the ETs are thought to be involved in the pathogenesis of hypertension, heart failure, bronchial asthma, renal failure, and vasospasm [1]. Regarding the pulmonary circulation, prior studies reported an elevated ET-1 level in patients with pulmonary hypertension and suggested that endothelium-derived vasoconstrictor ET-1 might be involved in the pathophysiology of pulmonary hypertension [2].

Conversely, adrenomedullin (AM) is a potent vasodilator peptide that has major effects on cardiovascular function, initially isolated from human pheochromocytoma tissue. Adrenomedullin consists of 52 amino acids with an intramolecular disulfide bond forming a ring structure of six residues, and it shares slight homology with calcitonin gene-related peptide, a potent hypotensive peptide. Like calcitonin gene-related peptide, intravenous injection of AM elicits a strong and long-lasting hypotensive effect [3]. Adrenomedullin is biosynthesized in a wide variety of organs, and cells and vascular endothelial and smooth muscle cells also actively secrete AM. Adrenomedullin has multiple biologic effects involved in cardiovascular homeostasis [4]. Plasma AM concentration is high in patients with cardiopulmonary diseases such as hypertension, congestive heart failure, renal failure, and septic shock. Vascular endothelial cells are known to produce nitric oxide, prostacyclin, and C-type natriuretic peptide, but AM is noted to be a strong and long-lasting vasoactive peptide involved in regulating vascular tone and the control of body fluids [5].

Recently the Fontan procedure was extended to more complex congenital heart defects with poor indication and marginal criteria. Pulmonary factors, especially pulmonary vascular resistance (PVR), are known to be crucial to deciding on the Fontan procedure [6]. Classically, the Fontan procedure was limited to tricuspid atresia with good hemodynamic condition. However, the indication (or criteria) extended to more complex (or challenging) cases recently, such as with higher pulmonary vascular resistance or ATV valve regurgitation, which were thought to be inoperable, previously. Therefore, cytokines that influence pulmonary circulation also seem to influence the hemodynamics after the Fontan procedure. There have been few studies to explore the plasma cytokine levels of ET-1 and AM before and after the Fontan procedure. In the present study we examined the time course of ET-1 and AM and explored the influence of ET-1 and AM on pulmonary vascular tone and the correlation between ET-1 and PVR in congenital heart disease before and after the Fontan procedure.

	Patients and methods

Top Abstract Introduction Patients and methods Results Comment References

 
Eight children who had the Fontan procedure (connection of the right atrial appendage to the pulmonary artery) (tricuspid atresia in 1 patient, double outlet right ventricle in 1, transposition of great arteries with pulmonary stenosis in 1, and single right ventricle in 5) were selected as the Fontan group. Eight other age-matched children who had biventricular repair with normal central venous pressure (CVP) were selected as the control group (secundum type atrial septal defect in 2 patients, ventricular septal defect in 4, aortic regurgitation in 1, and mitral regurgitation in 1). The children were 1 to 14 years old, and there was no significant difference in the mean age between two groups (5.5 ± 3.8 years old in the Fontan group versus 4.7 ± 2.5 years old in the control group). Central venous pressure after operation was significantly higher in the Fontan group compared with the control group (16.9 ± 5.1 cm H₂O in the Fontan group versus 8.0 ± 3.2 cm H₂O in the control group).

Study protocol
Anesthesia was induced by intravenous infusion of diazepam (0.2 mg/kg) with muscle relaxants and morphine used for maintenance. No inhalation agents were used. Immediately before cardiopulmonary bypass (CPB), 5 mL of venous blood was drawn to measure the plasma ET-1 and AM levels preoperatively, and mean pulmonary pressure and left atrial pressure were measured directly. Pulmonary blood flow was measured by magnetic flowmeter and pulmonary vascular resistance (PVR) was calculated. Inspiratory oxygen concentration was kept at 40% during the measurements. The conventional CPB was started with one episode of aortic cross-clamping and multiple-dose cardioplegia (glucose-insulin-potassium solution). Perfusion flow rate was 2.2 to 2.4 L/m² per minute with moderate hypothermia (28 to 30°C rectal temperature). The hematocrit value was kept between 18% and 28% during CPB. The duration of CPB ranged from 30 to 211 minutes (118.7 ± 28.0 minutes in the Fontan group versus 90.4 ± 62.8 minutes in the control group) and aortic cross-clamping time was 0 to 131 minutes (77.2 ± 6.4 minutes in the Fontan group versus 49.7 ± 49.6 minutes in the control group), and there were no statistically significant differences in the CPB time and cross-clamp time between groups. Dopamine at a dose of 4 to 10 µg/kg per minute was continuously infused after CPB.

Blood samples were also taken from the central venous line immediately after cardiopulmonary bypass, and at 6 and 24 hours after cardiopulmonary bypass. A thermodilution catheter was introduced during the operation, and cardiac index, mean pulmonary arterial pressure, and mean pulmonary wedge pressure were measured and then pulmonary vascular resistance (PVR) was calculated at the same time points.

Correlation coefficients between the plasma ET-1 levels and the mPAP data or the PVR data were calculated using a least squares regression equation.

Blood samples were placed in the ice box immediately and collected blood was put into tubes containing 7.5 mM EDTA (ethylenediamenetetraacetic acid) and centrifuged at 2,000 g for 10 minutes at 4°C. The plasma was immediately separated and stored at -70°C to minimize its degradation.

Bioassay of plasma endothelin-1 and adrenomedullin
Plasma ET-1 concentration was measured by commercially available ET-1, 21 specific radioimmunoassay system (SRL, Tokyo, Japan). Briefly, blood collected into tubes containing 7.5 mmol/L EDTA was centrifuged at 2,000 g for 10 minutes at 4°C and the plasma was immediately frozen at -20°C to minimize ET degradation. ET was extracted from 1 or 2 mL of acidified plasma using Amprep 500-mg C2 columns (Amersham, Arlington Heights, IL). After solvent removal by centrifugal evaporation, samples were reconstituted in 250-µL assay buffer (0.02 M sodium borate, pH 7.4). Duplicate 100-µL sample aliquots plus 100-µL aliquots of synthetic ET-1 of known concentration (for standardization) were incubated with rabbit antiendothelin antiserum for 4 hours at 4°C. A radioactive tracer, iodine-125 ET-3, was added and incubated for 16 to 24 hours at 4°C. The antibody-bound ET was separated from free ET using a donkey anti-rabbit antiserum coupled to magnetic beads followed by magnetic separation (Amerlex-M Separator; Amersham). The radioactive counts retained by the magnetic separator were measured in a gamma scintillation counter (Packard Cobra Auto-Gamma, Downer’s Grove, IL). The concentration of ET-1 was determined by plotting the average of duplicate sample counts against the standard curve of known ET-1 concentrations according to the manufacturer’s instructions.

Plasma AM concentration was measured by specific radioimmunoassay after extraction and purification (SRL, Tokyo, Japan). Briefly, 2 mL of Okasna was applied to a conditioned Sep-Pak C18 cartridge (Millipore Corp, Waters Chromatography, Milford, MA), and the column was sequentially washed with 5 mL of isotonic saline, 5 mL 0.1% (vol/vol) trifluoroacetic acid, and 5 mL of 20% (vol/vol) acetonitril in 0.1% trifluoroacetic acid. Then, the absorbed material was eluted with 4 mL of 50% (vol/vol) acetonitril, and the eluate was lyophilized. The residue was dissolved in 0.3 mL of 50 mmol/L phosphate buffer (pH 7.4) and was submitted to radioimmuno assay (RIA) using the radiolabeled AM and antiserum raised against synthetic AM in rabbits.

All values are expressed as mean ± standard deviation and were analyzed using SPSS (SPSS Inc, Chicago, IL). One-way analysis of variance and repeated measures two-way analysis of variance were used to compare the differences in recovery between groups. Data were compared using Student-Newman-Keuls test if the analysis of variance was significant. A p value less than 0.05 was considered to be significant.

	Results

Top Abstract Introduction Patients and methods Results Comment References

 
Time course of the plasma ET-1 levels is shown in Figure 1. The plasma ET-1 levels increased after CPB, peaked at 6 hours after CPB, and decreased 24 hours after CPB in both groups. However, the plasma ET-1 level in the Fontan group showed a significantly higher value compared with the control group at 6 and 24 hours after CPB.

View larger version (18K): [in this window] [in a new window]   Fig 1. Plasma endothelin level before and after operation. *p < 0.05 versus non-Fontan. (non-F = non-Fontan; op. = operation.)

View larger version (19K): [in this window] [in a new window]   Fig 2. Plasma adrenomedullin level before and after operation. *p < 0.05 versus non-Fontan. (non-F = non-Fontan; op. = operation.)

View larger version (11K): [in this window] [in a new window]   Fig 3. Correlation between endothelin-1 (ET-1) and pulmonary vascular resistance (PVR). (Um2 = Wood · unit · m2.)

	Comment

Top Abstract Introduction Patients and methods Results Comment References

 
Unique hemodynamic characteristics after the Fontan procedure differ from those after other types of repair for congenital heart defects in that higher CVP is necessary to maintain cardiac output postoperatively compared with the biventricular repair. High CVP easily causes systemic venous congestion and often leads to water retention, pleural effusion, ascites, and peripheral edema postoperatively. Multiple hemodynamic factors have been examined to decide the proper indications and criteria for doing the Fontan procedure. Pulmonary vascular resistance is known to be one of the most important factors in determining whether to do a Fontan procedure. Elevated PVR has been known to increase the risk of the Fontan procedure, especially when PVR is greater than 2 to 4 units · m² [7].

Although the hemodynamic characteristics after the Fontan procedure were well known, time course of the plasma cytokine levels has not been examined. Recently novel cytokines were discovered and suggested to have an important effect on cardiopulmonary homeostasis. Vascular endothelial cells produce endothelin, nitric oxide, prostacyclin, and C-type natriuretic peptide. Adrenomedullin is produced and secreted by the vascular endothelial cells and vascular smooth muscle cells. These cytokines might participate in the control of circulation as the vascular regulators, and interaction among these cytokines might influence local control of vascular tone. Although hemodynamic status after the Fontan procedure can depend on multiple factors, the plasma cytokines seem to exert an important effect on hemodynamic status after the Fontan procedure. In the present study, the initial increase of plasma ET-1 after CPB in both groups seems to be influenced by CPB, as we have already shown the increased plasma ET-1 levels in congenital heart diseases after CPB [8]. Similarly, the plasma AM levels increased after the operation in both groups, which might be a counterbalance against the initial increase of the plasma ET-1 after CPB. However, the plasma ET-1 levels were significantly elevated and AM levels were significantly lower at 6 and 24 hours after CPB in the Fontan group compared with the control group. Those results suggest that high CVP could cause an imbalance between elevated plasma ET-1 and relatively low AM levels and that this imbalance could contribute to vasoconstriction after the Fontan procedure. Moreover, significant positive correlation was obtained between the plasma ET-1 levels and PVR data in the Fontan group, which suggests that ET-1 might help maintain the vasoconstriction after the Fontan procedure. The homeostatic mechanism might help prevent pulmonary and peripheral fluid retention when CVP is increased purposely.

Another possible function of ET-1 is to compensate for the relatively lower cardiac output after the Fontan procedure. The cardiac output after the Fontan procedure is known to be reduced to about half the preoperative systemic blood flow plus pulmonary blood flow levels. Then high CVP is necessary to maintain cardiac output and could cause the shear stress on endothelium of the cardiovascular system and increased plasma ET-1. The pulmonary and peripheral vascular system might be vasoconstrictive so as to respond to relatively lower cardiac output after the Fontan procedure.

Regarding the vasoconstrictive status after the Fontan procedure, Kelly and colleagues [9] found that resting plasma norepinephrine level was elevated in Fontan subjects, which indicates diminished venous vascular capacitance or increased venous tone in patients with univentricular hearts who had the Fontan procedure. We also measured the plasma norepinephrine level in several patients after the Fontan procedure, and it similarly was elevated postoperatively (data not shown). Therefore, the hemodynamic status after the Fontan procedure also seemed to be vasoconstrictive from the view point of plasma norepinephrine levels.

Nishikimi and colleagues [10] demonstrated a significant and proportionate increase in plasma levels of AM in patients with moderate to severe heart failure and significant correlation between plasma levels of AM, norepinephrine, and atrial natriuretic peptide. They concluded that the increased plasma volume and an activated sympathetic nervous system might be involved in the increased levels of circulating AM and that increased AM in heart failure might function as a defense mechanism against further elevation in peripheral vascular resistance [10]. However, the plasma level of AM was not increased after the Fontan procedure in the present study. We have already shown significant elevation of the plasma atrial natriuretic peptide level after the Fontan procedure and its significant correlation with CVP [11]. From these results, both atrial natriuretic peptide and AM might be involved in the following protective mechanism after the Fontan procedure: elevated atrial natriuretic peptide might control body fluid levels to avoid massive ascites and pleural effusion under high CVP circumstance, and relatively suppressed AM might regulate the vascular tone to increase the pulmonary and peripheral vascular resistance when cardiac output is relatively low.

The limitation of this study is that all plasma cytokine data were obtained in a Fontan circulation established by an atriopulmonary connection and not by a total cavopulmonary connection. The plasma atrial natriuretic peptide levels have the direct effects on atrial wall stretch in an atriopulmonary connection. However, regarding the plasma ET-1 and AM levels, there could be little difference between the two procedures because both cytokines are secreted mainly by vascular endothelial cells or smooth muscle cells, not by the atrial wall.

	References

Top Abstract Introduction Patients and methods Results Comment References

 

1. Nayler W.G. The endothelins. Berlin, Heidelberg: Springer-Verlag, 1990:1-188.
2. Yoshibayashi M., Nishioka K., Nakao K., et al. Plasma endothelin concentrations in patients with pulmonary hypertension associated with congenital heart defects. Circulation 1991;84:2280-2285.[Abstract/Free Full Text]
3. Kitamura K., Kangawa K., Kawamoto M., et al. Adrenomedullin. Biochem Biophys Res Commun 1993;192:553-560.[Medline]
4. Sugo S., Minamino N., Kangawa K., et al. Endothelial cells actively synthesize and secrete adrenomedullin. Biochem Biophys Res Commun 1994;201:1160-1166.[Medline]
5. Ishimitsu T., Nishikimi T., Saito Y., et al. Plasma levels of adrenomedullin, a hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest 1994;94:2158-2161.
6. Mayer J.E., Jr, Helgason H., Jonas R.A., et al. Extending the limits for modified Fontan procedures. J Thorac Cardiovasc Surg 1986;92:1021-1028.[Abstract]
7. Mayer J.E., Jr, Bridges N.D., Lock J.E., et al. Factors associated with marked reduction in mortality for Fontan operation in patients with single ventricle. J Thorac Cardiovasc Surg 1992;103:444-452.[Abstract]
8. Hiramatsu T., Imai Y., Takanashi Y., et al. Time course of endothelin-1 and nitrate anion after cardiopulmonary bypass in congenital heart defects. Ann Thorac Surg 1997;63:648-652.[Abstract/Free Full Text]
9. Kelly J.R., Mack G.W., Fahey J.T. Diminished venous vascular capacitance in patients with univentricular hearts after the Fontan operation. Am J Cardiol 1995;76:158-163.[Medline]
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