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Clinical Science (1998) 94, 585-590 (Printed in Great Britain) 585 Increased plasma adrenomedullin concentrations during cardiac surgery Toshio NISHIKIMI*, Yukio HAYASHlt, Gentaro IRIBUt, Shuichi TAKISHITA*, Yoshio KOSAKAIS, Naoto MINAMINOS, Atsuro MIYATAS, Hisayuki MATSUOS, Masakazu KUROt and Kenji KANGAWAS *Division of Hypertension, National Cardiovascular Center, 5-7- I , Fujishirodai, Suita, Osaka 565, japan, tDivision of Anesthesiology. National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565, japan. *Division of Cardiovascular Surgery, National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565, Japan, and §Research Institute, National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565. japan 1. Adrenomedullin (AM), a potent hypotensive peptide, was originally isolated from human phaeochromocytoma. Plasma AM concentrations are elevated in hypertension, heart failure and renal failure in proportion to the severity of the disease. This study was performed to investigate the pathophysiological significance of AM during cardiac surgery. 2. Serial blood samples were obtained from patients undergoing cardiac surgery and plasma AM concentrations were determined by specific radioimmunoassay. 3. Plasma AM concentrations did not increase with anaesthesia or surgery (n = 9). Plasma AM concentrations gradually increased during cardiopulmonary bypass and after pulmonary reperfusion. After pulmonary reperfusion, plasma AM concentrations increased further. In addition, we measured plasma AM concentrations in the pulmonary vein (n = 8) and coronary sinus (n = 8) to examine the contribution of the lungs and heart to the increase in circulating AM concentrations after cardiopulmonarybypass. However, no significant differences were seen in plasma AM Concentrations of the pulmonary vein or the coronary sinus and the aorta. Peak AM concentrations during cardiac surgery correlated with duration of surgery. Elevated plasma AM levels during and after surgery began to decline next day after surgery and returned to normal levels 7 days after surgery. 4. These results demonstrate that plasma AM concentrations increase during cardiac surgery and that the duration of surgery may be related to the changes in AM concentrations. Taken together with recent findings that vascular endothelial cells and vascular smooth muscle cells actively produce AM, these results suggest that plasma AM during cardiac surgery may act as a vasodilatory hormone. INTRODUCTION Adrenomedullin (AM), which was originally isolated from human phaeochromocytoma, is a novel vasodilatory peptide consisting of 52 amino acid residues [l]. AM, which has slight similarity to calcitonin gene-related peptide, causes a potent and sustained hypotensive effects in rats [l]. Previous studies have demonstrated that vascular smooth muscle cells contain specific AM receptors that are functionally coupled to adenylate cyclase [2,3]. Furthermore, Sugo et al. [4] have demonstrated that cultured vascular endothelial cells and vascular smooth muscle cells from various species express abundant amounts of AM mRNA and release AM into media. AM release is augmented by tumour necrosis factor-a and interleukin-6, suggesting that vessel wall is a primary source of circulating AM [5]. ImmunoreactiveAM has been detected in human plasma by specific radioimmunoassay [11, and plasma AM concentrations increase in patients with essential hypertension, chronic renal failure and heart failure in proportion to clinical severity [6-81. In addition, Lainchbury et al. [9] recently demonstrated that AM infusion significantly decreased mean arterial pressure with minor elevation of plasma AM levels in healthy human subjects. These results suggest that AM may be involved in the regulation of the cardiovascular system. Several reports have shown that plasma endothelin, a potent vasoconstrictor peptide produced by vascular endothelium, is affected by cardiac surgery and anaesthesia [lo-121. The gene expression and release of AM in cultured vascular endothelial cells is similar to endothelin [4]. These observations led us to speculate that synthesis and release of AM may also be affected by anaesthesia and cardiac surgery. Indeed, Nagata et al. [13] recently reported that plasma AM levels increased in a small number of patients who underwent cardiac surgery. However, it is not fully understood how the plasma AM levels change over time in patients who undergo cardiac surgery, or whether the levels are altered in relation to clinical parameters. The present study was performed to investigate whether plasma AM changes during anaesthesia or surgery. Furthermore, we studied whether the heart and lung relate to the increase in circulating AM levels after cardiopulmonary bypass (CPB). Key words: adrenomedullin, anaesthesia, cardiac surgery, cardiopulmonary bypass. Abbreviations: AM. adrenomedullin; ANOVA. analysis of variance; CPB, cardiopulmonary bypass; TFA. trifluoroacetic acid. Correspondence: Dr T. Nishikimi. 586 T. Nishikimi and others during CPB, immediately after pulmonary reperfusion, and 4 h after surgery. Plasma AM concentrations were measured in all samples. METH0DS Informed consent was obtained from each patient and the protocol was approved by the ethics committee of our institute. Anaesthesia was induced and maintained with fentanyl, diazepam, midazolam and isoflurane. CPB was introduced with gradually increasing pump flow, and 100 % flow was established within the first minute. During CPB, a non-pulsatile pump flow of 2-3 litres min-' rn-, was maintained using a membrane oxygenator and an arterial filter. Moderate hypothermia (28-32 "C) was induced and the PaCO, was not corrected for temperature. Study 2: pulmonary reperfusion To study the effect of pulmonary reperfusion, plasma AM concentrations were measured in eight patients undergoing cardiac surgery at the following time points : the day before surgery, after induction of anaesthesia, 30 min after the start of surgery, 30 min before pulmonary reperfusion, immediately after pulmonary reperfusion, and 0.5, l, 2 and 4 h after pulmonary reperfusion. In addition, to assess the contribution of AM produced by the lungs to the increase in circulating AM after pulmonary reperfusion, simultaneous blood samples were obtained from the pulmonary vein by direct puncture using a 22 G needle and from the sampling device in the pump circuit for measurement of plasma AM. PROTOCOL Study I :surgical time course To investigate the time course for changes in the plasma concentrations of AM during induction of anaesthesia, cardiac surgery and CPB, serial blood samples were withdrawn in nine patients undergoing cardiac surgery. Blood was obtained on the day before surgery, before induction of anaesthesia, 15 min after the start of anaesthesia, 30min after the start of surgery, immediately before institution of CPB, immediately after the initial drop in blood pressure during CPB, after stabilization of blood pressure Table I Study 3: time course during CPB Blood samples were obtained in eight patients before CPB (n = 8), and 1 (n = 8), 2 (n = 8), 3 ( n = 6), 4 (n = Clinical characteristics of the patients in studies I t o 5 Values are meansfS.E.M. BSA. body surface area; CPB, cardiopulmonary bypass; CABG. coronary-aorto bypass graft; ASD, atrial septal defect patch closure; MVR. mitral valve replacement; MVP, mitral valve plasty; AVR, aortic valve replacement; TAP, tricuspid annulus plasty; VSD. ventricular septal defect patch closure. Operation (n) Gender (M/F) Age (years) BSA (m') Duration of surgery (min) Duration of CPB (min) CABG (5) ASD (2) MVR (I) MVP (I) 712 56f4 1.61f O . 0 6 350f44 162f29 AVR+MVR+maze (3) AVR (2) MVR ( I ) AVR+MVR ( I ) CABG ( I ) 414 61 +4 I .54f 0.05 393f 49 190f43 AVR+MVR+maze (3) CABG (2) MVR maze (2) Bentall (I) 3/5 58f4 1.52f0.06 367L-60 213+36 MVR+maze (3) MVP+ maze (2) AVR MVR +maze (2) AVR MVR +TAP maze ( I ) 513 64*3 1.55fO.06 432k55 273 f 38 MVR (2) MVR maze (2) MVR+TAP+maze (2) MVP+TAP+maze (2) VR+AVR+maze (I) VSD+maze (I) 3P 54f3 1.54fO.06 435f30 173f9 Surgical time course (n = 9) Pulmonary reperfusion (n = 8) Time course during CPB (n = 8) + Production of heart during CPB (n = 8) + + + Time course before and after surgery (n = 10) + Adrenomedullin in cardiac surgery 587 5), 5 (n = 2) and 6 h (n = 1) after CPB for the measurement of plasma AM concentrations. immunoassay for AM has been described previously ~41. Study 4: production of AM in the heart during CPB Statistical analysis To investigate the contribution of AM produced by the heart to circulating AM after CPB, blood samples were obtained simultaneously from the aorta and the coronary sinus in eight patients immediately after, and 0.5 and 2 h after pulmonary reperfusion for determination of plasma AM concentrations. All values are expressed as meansfS.E.M. To analyse the effect of time course, we used one-way repeated measures of analysis of variance (ANOVA). Post-hoc test was performed using Dunnett's method. To analyse the results of study 4, two-way repeated measures of ANOVA were used. Post-hoc test was performed using the Newman-Keuls method. Correlation coefficients were calculated by linear regression analysis. P < 0.05 was considered statistically significant. Study 5: time course before and after surgery To investigate the time course of plasma AM concentrations after cardiac surgery, blood samples were obtained in 10 patients before cardiac surgery, and 1, 3, 7 and 21 days after surgery for the measurement of AM. The clinical characteristics of the patients and the surgical procedures performed in the studies described above are presented in Table 1. Patients in this study received infusions of dopamine (86 YO), prostaglandin El (70 %), nitroglycerine (56 %) and noradrenaline (49 %) during cardiac surgery. Blood sampling A radial artery catheter was placed percutaneously to allow continuous monitoring of the systemicarterial pressure and frequent sampling of arterial blood ( 5 ml). Blood samples were obtained through the antecubital vein the day before surgery in studies 1 to 3, and 1, 3, 7 and 21 days after surgery in study 5 . Blood was immediately transferred into chilled glass tubes containing disodium-EDTA (1 mg/ml) and aprotinin (500 units/ml). Blood was immediately centrifuged at 4°C and the plasma was frozen and stored at - 80 "C until assayed. RESULTS Study I :surgical time course The plasma AM concentration the day before surgery was 6.4 0.9 fmol/ml. The plasma concentration of AM in age-matched normal volunteers (n = 18) was 5.0k0.2 fmol/ml. The plasma AM concentrations did not change before or after the induction of anaesthesia (Figure 1). The plasma AM concentrations also did not change 30 min after the start of surgery. Thereafter, the concentrations of AM tended to increase during CPB. After pulmonary reperfusion, plasma AM increased about 8-fold. The plasma AM concentrations increased further 4 h after surgery. Study 2: pulmonary reperfusion The plasma concentrations of AM did not change after the induction of anaesthesia or during surgery compared with the day before surgery, consistent with Adrenomedullin assay Stored plasma samples were extracted before radioimmunoassay. Briefly, Sep-Pak C18 cartridges (Millipore-Waters, Milford, MA, U.S.A.) were preconditioned with 5 ml each of chloroform, methanol, 50 % acetonitrile containing 0.1 % trifluoroacetic acid (TFA), 0.1 YOTFA and saline. Plasma (2 ml) was acidified with 24 p1 of 1 M HCl, diluted with 2 ml of saline, and then loaded on to a Sep-Pak C18 cartridge. The column was then washed with 5 ml each of saline, 0.1 % TFA and 20% acetonitrile containing 0.1 % TFA, and the absorbed materials were eluted with 4 ml of 50 % acetonitrile containing 0.1 % TFA. The eluate was then lyophilized. The lyophilized material was dissolved in radioimmunoassay buffer and the clear solution was radioimmunoassayed. The radio- Figure I Changes in mean plasma levels of AM during time course of cardiac operation All values are expressed as meansfS.E.M. #P < 0.05 compared with the day before operation. The bars for S.E.M. are not shown for smaller sizes than symbols at points before CPB. T. Nishikimi and others 588 Figure 2 Changes in mean plasma levels of A M before and after pulmonary reperfusion All values are expressed as meansfS.E.M. #P < 0.05 compared with the day before operation. The bars for S.E.M. are not shown for smaller sizes than symbols at points before CPB. Figure 5 Time course of changes in plasma levels of AM before and after cardiac surgery Values are expressed as meansf5E.M. #P < 0.05 compared with before surgery. the results of study 1 (Figure 2). However, the plasma AM concentrations increased significantly 30 min before the pulmonary reperfusion. The concentrations of AM increased further immediately after the pulmonary reperfusion. Plasma AM concentrations continued to increase after pulmonary reperfusion. No significant differences were noted between the AM concentrations immediately after reperfusion in the pulmonary vein plasma and plasma from the pump circuit (38 & 9 versus 45 f 10 fmol/ml). pre 1 2 3 J 5 6 E R Duration of CPB (h) Figure 3 Time course of changes in plasma levels of A M before and after induction of CPB in each patient Values are expressed as meansfS.E.M. #P < 0.05 compared with pre-CPB. E, end of CPB; R. reperfusion. Study 3: time course during CPB The mean plasma AM concentration before CPB was 9.4f 4.7 fmol/ml. The concentrations of AM increased by about 4-fold by the end of CPB (Figure 3). After pulmonary reperfusion, the plasma AM concentration increased further. Study 4: production of AM in the heart during CPB Plasma AM concentrations in both the aorta and coronary sinus were elevated immediately after pulmonary reperfusion and increased for 2 h after pulmonary reperfusion (Figure 4). However, there were no differences between the plasma AM concentrations in the aorta and the coronary sinus. Study 5: time course before and after surgery Figure 4 Changes in mean plasma levels of A M from The plasma concentrations of AM peaked at 6 h after surgery and then gradually decreased (Figure 5). Plasma AM levels normalized 7 days after cardiac surgery. The peak plasma AM concentrations in patients in studies 1 and 2 correlated significantly with the duration of surgery (r = 0.62, P < 0.01). aorta ( 0 )and coronary sinus ( 0 )after pulmonary reper- fusion DISCUSSION All values are expressed as meansfS.E.M. #P < 0.05 compared with immediately after pulmonary reperfusion. The present studies demonstrate that plasma AM concentrations do not change during the induction of Adrenomedullin in cardiac surgery anaesthesia before surgery but do increase gradually during and after CPB, and return to normal levels 1 week after surgery. The present studies also show that the heart and lung do not significantly contribute to the increase in circulating AM during cardiac surgery. In the present study, marked increases in the plasma AM occurred in patients undergoing cardiac surgery. This finding is consistent with the recent report by Nagata et al. [13]. They reported that plasma AM levels increased during CPB and decreased after weaning of CPB, and suggested that pulmonary vasculature is a possible source of increased circulating AM after reperfusion of the lungs. However, the exact source of the increased plasma AM during cardiac surgery remains unknown. A previous study reported that AM mRNA is highly expressed in the adrenal gland, heart, kidney and lung [ 11, suggesting that these organs may secrete AM into the bloodstream. In the present study we measured plasma AM concentrations both in the pulmonary vein and pump circuit immediately after pulmonary reperfusion to see if AM is produced in the lungs. However, there was no significant difference between plasma AM concentrations in the pulmonary vein and pump circuit. Furthermore, we also measured plasma AM concentrations in the aorta and the coronary sinus after pulmonary reperfusion to investigate the contribution of AM produced by the heart to circulating AM. Once again, there was no difference between the plasma concentrations of AM from the aorta and the coronary sinus. These results support the previous findings suggesting that the heart and lungs are not the main source of plasma AM in patients with ischaemic heart disease and normal cardiac function [15]. Sugo et al. [4,5] have reported that cultured vascular endothelial cells and vascular smooth muscle cells actively synthesize and secrete AM, suggesting that the vessel wall is a potential site for AM production. They also reported that several cytokines markedly increase the expression of AM mRNA in cultured vascular smooth muscle cells [5]. It has been reported that the levels of several cytokines, including tumour necrosis factor-a and interleukin-lp, increase during surgery [16-1 81. Taken together with the present findings, it seems that the increased AM during cardiac surgery may be produced by the vascular wall which is stimulated by activated monocytes, lymphocytes and leucocytes through cytokines. The physiological role of AM during cardiac operation remains unknown. Previous studies have demonstrated that vascular smooth muscle cells possess specific AM receptors that are functionally coupled to adenylate cyclase [2,3]. Nakamura et al. [191have demonstrated that the vasodilator potency of AM is approximately 10-200-fold greater than sodium nitroprusside and acetylcholine when infused into the brachial artery in humans. These results suggest that increased AM concentrations during cardiac surgery may play a role in regulating peripheral vascular resistance. Moreover, Lainchbury et al. [9] recently demonstrated that a low-dose infusion of AM 589 (8 ng amin-' * kg-') significantlydecreased blood pressure with only about a 1.5-fold increase of plasma AM levels, suggesting that the biological threshold of AM for decreasing blood pressure may be lower than the plasma levels seen in cardiovascular disease. The plasma concentrations of AM during cardiac surgery were approximately 10-fold higher than in normal healthy volunteers. In fact, the concentrations were much higher than those found in patients with severe heart failure (NYHA functional class I11 to IV) or severe chronic renal failure (creatinine > 5 mg/dl) [6-81. In addition, previous studies have demonstrated that intra-arterial infusion of AM into the renal artery at a dose of 0.8 ng.min-'*kg-' body weight has a vasodilatory effect in the dog [20]. This dose translates to a renal artery plasma concentration of 13 fmol/ml, assuming a renal plasma flow of 100 ml/min. Taken together with the present findings, these results suggest that increased AM during cardiac surgery may act as a vasodilatory or a diuretic and natriuretic hormone to counter perioperative increases in systemic vascular resistance or oliguria. Plasma AM concentrations did not increase during anaesthesia or during the initial 2 h of surgery. In contrast, significant increases in the plasma concentrations of AM were observed after the start of CPB. These results indicate that the increase in plasma AM levels occurs after several hours. The slow increase in plasma AM may be related to its mechanism of secretion. Many classic hormones are stored in large amounts in secretory granules and are secreted via regulated pathways. However, previous reports showed that AM production in the vessel wall is regulated at the level of gene expression. Furthermore, it has been demonstrated that AM is secreted via constitutive pathways without intermediate storage in secretory granules [4]. It is therefore likely that there is a lag between the onset of intracellular production of AM after stimulation and the increase in systemic plasma AM concentrations. In addition, significant correlations were seen between peak plasma AM concentration and the duration of surgery. These results suggest that a relatively long-term stimulus is required to induce AM gene expression. In the present study plasma AM levels further increased after CPB; however, Nagata's report showed that plasma AM levels decreased after weaning from CPB, suggesting that CPB plays an important role in increase of AM during cardiac surgery. We have seen that plasma AM levels increased even during and after general surgery, indicating that increased plasma AM is not specificfor CPB (T. Nikishima and K. Kangawa, unpublished work). Because secretion of AM is regulated by gene expression, increased levels of AM gene expression may continue for a few hours after weaning of CPB. Discrepancy between our findings and those of Nagata et al. [13] may be due, in part, to different patients, protocol and operation. In conclusion, we demonstrated that concentrations of plasma AM increase during cardiac surgery and that the duration of surgery and CPB may be im- T. Nishikimi and others 590 portant factors in its production. Increased AM after surgery may act as a defence mechanism for increased systemic vascular resistance during cardiac surgery. Further studies are necessary to determine the exact role of increased plasma AM concentrations during cardiac surgery. ACKNOWLEDGMENT We authors thank Nobuo Shirahashi for helpful advice on the statistical analysis. We also wish to thank Ms Yoko Saito for her technical assistance.This work was supported in part by Special Coordination Funds for Promoting Science and Technology (Encouragement System of COE) from the Science and Technology Agency of Japan, grants from the Ministry of Health and Welfare, and the Human Science Foundation of Japan, and Scientific Research Grantsin-Aid 09670776 from the Ministry of Education, Science and Culture of Japan. REFERENCES I. Kitamura K. Kangawa K. Kawamoto M. et al. (1993) 2. 3. 4. 5. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192: 553-60. Eguchi S. Hirata Y, Kano H. et al. (1994) Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells. FEBS Lett 3 4 : 226-30. lshizaka Y, lshizaka Y. Tanaka M. et al. (1994) Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells. Biochem Biophys Res Commun 200: 642-6. Sugo S, Minamino N. Kangawa K, et al. 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