Download Increased Plasma Adrenomedullin

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.
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Received 3 October 1997/28 January 1998: accepted 10 February 1998
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