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Changes of plasma asymmetric dimethylarginine levels after coronaryartery bypass grafting
INGA KARU1,2,3, KERSTI ZILMER1, JOEL STARKOPF2 & MIHKEL ZILMER1
1Insitute of Biochemistry, University of Tartu, Estonia, 2Clinic of Anaesthesiology and Intensive Care, University of Tartu,
Estonia, and 3Clinic of Anaesthesiology, North Estonia Regional Hospital, Estonia
AbstractObjectives . We investigated whether coronary artery bypass grafting affects plasma asymmetric dimethylarginine (ADMA)concentrations and whether precardioplegic hyperoxia influences ADMA release from the heart. Design . Twenty twopatients were randomized into control (n�/11) and hyperoxia (n�/11, ventilated with �/96% oxygen before cardiopul-monary bypass) groups. Arterial and coronary sinus blood was sampled before cardioplegia and during early reperfusion.Arterial samples were drawn 60 min after declamping of the aorta, and on the first postoperative day. Results . Baselinearterial values of ADMA were not different between groups (0.599/0.18 mmol/l control, 0.639/0.13 mmol/l hyperoxiagroup). Negligible release of ADMA into coronary sinus was detected 20 min after cardioplegia. A significant decrease ofarterial ADMA was observed by the first postoperative morning (0.429/0.16 mmol/l in control, and 0.389/0.07 in hyperoxiagroup, pB/0.01 compared to baseline). Conclusions . CABG with cardioplegia is associated with decrease of ADMA by thefirst postoperative morning. Reperfusion of cardioplegic heart did not result in significant release of ADMA. Pretreatmentwith hyperoxia had no influence on myocardial release and arterial levels of ADMA.
Key words: Asymmetric dimethylarginine, ADMA, hyperoxia, preconditioning, coronary artery bypass
Endothelium plays a crucial role in maintenance of
vascular tone and structure. One of the major
vasoactive mediators, derived from endothelium is
nitric oxide (NO), formed in the endothelial cells
by isoforms of nitric oxide synthase (NOS). In the
heart, most of NO is produced by endothelial
NOS, present in the endothelium of coronary
vessels and myocardium (1,2). Asymmetric di-
methylarginine (ADMA) is an endogenous compe-
titive inhibitor of NOS and thereby modulates NO
production (3). Elevated level of circulating
ADMA has been suggested as a marker of
endothelial dysfunction (4), leading to increased
resting vascular tone and decreased myocardial
vasodilatory capacity.
Cardiac surgery with cardiopulmonary bypass
(CPB) is associated with myocardial ischemia during
cardioplegia, followed by reperfusion upon declamp-
ing of the aorta. As a result, ischemia-reperfusion
(IR) injury, including damage of both myocardial
and endothelial cells, develops. Whether increased
ADMA levels interfere with postischemic endothelial
dysfunction is not clear. Furthermore, to the best of
our knowledge, it is not known whether coronary
artery bypass grafting (CABG) with CPB has an
influence upon myocardial release or circulating
levels of ADMA.
In our previous experimental studies we have
shown that preischemic exposure of experimental
animals to low-grade oxidative stress, induced by
hyperoxia, can reduce both myocardial and endothe-
lial injury (5,6). In the model of IR injury a decrease
in endothelial NOS expression following chronic
hyperoxia has been reported (7). Whether the
exposure to hyperoxia influences levels of circulating
ADMA is not known.
In present preliminary report we describe the
changes in arterial and coronary sinus concentrations
of ADMA during CABG procedure. Additionally we
investigated the effect of hyperoxic pretreatment on
plasma levels of ADMA.
Material and methods
Patients
The study design was approved by the Ethics Review
Committee on Human Research of the University of
Tartu and written informed consent was obtained
Correspondence: Inga Karu, North Estonian Regional Hospital, Clinic of Anaesthesiology 19, Sutiste Str., 13419 Tallinn, Estonia. Tel: �/372 697 1196. Fax:
�/372 697 1150. E-mail: [email protected]
Scandinavian Cardiovascular Journal. 2006; 40: 363�367
(Received 20 June 2006; accepted 24 August 2006)
ISSN 1401-7431 print/ISSN 1651-2006 online # 2006 Taylor & Francis
DOI: 10.1080/14017430600983648
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from all patients. Twenty two adult patients sched-
uled for isolated primary elective aorto-coronary
bypass grafting with at least three distal anastomoses
were recruited and randomized into the control
(n�/11) and hyperoxia (n�/11) groups. The exclu-
sion criteria included preoperative ejection fraction
of the heart below 40%; unstable angina or elevated
markers for myocardial necrosis; treated diabetes
mellitus; hepatic, renal or pulmonary disease. All
medications, except salicylates were allowed up to
the day before surgery.
Study protocol
After induction of anesthesia and intubation of the
trachea the patients were ventilated until the begin-
ning of CPB with either 40% or �/96% oxygen.
After CPB mixture of oxygen and air was adjusted to
obtain normal paO2 levels. Arterial blood gases were
analyzed (Radiometer ABL 700 series, Ratiometer
Medical A/S, Denmark) 15 min after randomization
and verified again before CPB.
Anaesthesia and operative procedure
Intravenous anaesthesia with midazolam, fentanyl
and pancuronium was used in all cases, no volatile
anesthetics were added. CPB was performed with
roller pump (Stockert Instrumente GmbH, Munich,
Germany) and membrane oxygenator (Dideco,
Mirandola, Italy) under mild hypothermia (naso-
pharyngeal temperature 33�358C). Manually
inflatable 15 Fr coronary sinus cannula (Medtronic
Inc., Minneapolis, USA) was inserted transatrially.
For achieving cardioplegia cold crystalloid solution
(St.Thomas’ II) was infused antegradely into the
aortic root and retrogradely into the coronary sinus.
Infusion technique of cardioplegia was standardized
in all cases. Coronary sinus pressure was carefully
monitored and kept between 20 and 40 mmHg not
to cause coronary venous injury. Infusion was
repeated after completion of each anastomosis or at
least every 20 min. Both distal and proximal anasto-
moses were performed under a single cross-clamping
period. All operations were performed by the same
surgical team.
ADMA measurement
Blood samples were collected from coronary sinus
and radial artery cannulae before CPB and at 5 and
20 min after declamping. The balloon of the cor-
onary sinus cannula was manually inflated at these
time points to get blood exclusively from
the coronary sinus. Additional arterial samples
were drawn 60 min after declamping of the aorta,
and on the morning of the first postoperative day.
Blood was centrifuged immediately after collecting
and serum stored at �/808C until analyses.
ADMA was determined using the competitive
ADMA-ELISA (DLD Gesellschaft fur Diagnostika
und Medizinische Gerate mbH; Hamburg, Ger-
many). The kit uses the microtiter plate format,
where ADMA is bound to the solid phase of the
plate. ADMA in the samples is acylated and
competes with the solid phase bound ADMA for a
fixed number of rabbit anti-ADMA anti-serum
binding sites. After the system has reached equili-
brium, free antigen and free antigen-antiserum
complexes are removed by washing. The antibody
bound to the solid phase is then detected by
anti-rabbit-IgG-peroxidase. The substrate tetra-
methylbenzidine-peroxidase reaction is monitored
at 450 nm.
Myocardial release was calculated as the difference
between arterial and coronary sinus concentrations,
thus negative values indicate a release from the heart
during reperfusion.
Statistical analysis
Student?s t-test was used to distinguish differences in
demographic data between groups. Friedman?s AN-
OVA followed by Dunnet?s test was applied to locate
significant differences over time. Differences be-
tween groups were assessed by the analysis of
variance for repeated measures. Correlation is ex-
pressed as Pearson correlation coefficient. Data are
represented as mean9/SD, with a value of pB/0.05
considered as significant.
Results
Demographic data
Patients’ demographic data are presented in Table I.
There were no inter-group differences regarding age
of the patients, and ischemic time, but there were
more male patients in the hyperoxia group. Three to
five coronary arteries were bypassed in both groups.
No inotropic support was needed during the perio-
perative period.
Arterial levels of ADMA
The baseline arterial levels of ADMA, measured
before CPB, were similar in both groups (Figure 1).
The minimum and maximum values were 0.34 and
0.80 mmol/l among controls, and 0.36 and
0.76 mmol/l in hyperoxia pretreated patients. During
the first hour of reperfusion no alterations in arterial
concentrations were seen, but a significant decrease
(pB/0.01) was observed by the first postoperative
morning in both groups (mean 0.429/0.16, mini-
364 I. Karu et al.
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mum 0.23, maximum 0.78 mmol/l in the control,
and mean 0.389/0.07, minimum and maximum
values 0.27 and 0.48 in the hyperoxia pretreated
patients; Figure 1).
Myocardial release of ADMA
In the control group, reperfusion of cardioplegic
heart did not result in washout of ADMA (Figure 2).
Some extent of ADMA was released from the
myocardium of the hyperoxia pretreated patients
during 20 min of reperfusion. The values, however,
did not reach statistical difference (Figure 2). The
coronary sinus level of ADMA did not differ from
the baseline values during 20 min of reperfusion. In
the hyperoxia pretreated, but not in the control
patients, the concentration in the coronary sinus
correlated with duration of the cardioplegic arrest
(r�/0.65, p�/0.04, Figure 3).
The duration of preischemic hyperoxic exposure,
arterial paO2 values before CPB and preoperative
cardiac function (EF) had no influence on coronary
sinus levels of ADMA.
Discussion
This study provides the first evidence that CABG
surgery is associated with a significant decrease
of arterial ADMA concentrations by the first
postoperative morning. We demonstrated that a
cardioplegic arrest did not cause a release of
ADMA from the human heart, and ventilation with
hyperoxic gas mixture had no influence on either
arterial or coronary sinus levels of ADMA.
Free ADMA � an endogenous competitive
inhibitor of NOS � is released into the plasma after
proteolytic breakdown, and is eliminated via renal
excretion (8) and hydrolytic degradation by
dimethylarginine dimethylaminohydrolase (DDAH)
(9).
Whether ADMA is involved in reducing NO
levels and causing endothelial dysfunction of
reperfused heart is not fully clear. NO, although
decreased during reperfusion (10), has been shown
to afford cardioprotection, as described by reduced
infarct size and neutrophil accumulation in the
reperfused heart (11). In the present study, no
significant changes in arterial and coronary sinus
Table I. Patient characteristics
Variable Controls (n�/11)
Hyperoxia-pretreated
(n�/11) p-value
Age (years) 63.89/9.6 59.79/5.5 0.23
Gender (male/female) 6/5 9/2 0.17
Preoperative ejection fraction (%) 589/11 539/7 0.23
paO2 15 min after intubation (mmHg) 132.89/39.0 356.59/65.3 B/0.00001
paO2 before cardiopulmonary bypass (mmHg) 124.19/35.9 300.89/118.8 0.0001
Exposure time to oxygen (40% vs �/96%, min) 1359/20 1489/24 0.18
Aortic cross-clamping time (min) 859/18 899/13 0.56
Vessels bypassed 3.79/0.8 4.19/0.7 0.26
Figure 1. ADMA concentrations in arterial plasma of control
(40%) and hyperoxia (�/96%) pretreated patients before cardio-
plegia, during reperfusion and on the first postoperative morning
(1 POP). Data are given as mean9/SD. *pB/0.01 compared to
baseline values.
Figure 2. Myocardial release (arterio-coronary sinus difference)
of ADMA in control (40%) and hyperoxia (�/96%) pretreated
patients before cardioplegia and during immediate reperfusion.
Data are given as mean9/SD.
ADMA levels during coronary surgery 365
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ADMA were detected during the immediate, 20 min
reperfusion of cardioplegic heart. This allows us to
speculate, that other mechanisms rather than altered
metabolism of ADMA are responsible for endothe-
lial dysfunction after cardioplegia. In hyperoxia
pretreated patients we observed the correlation
between coronary sinus ADMA and aortic cross-
clamping time, which could be interpreted as a
relation between the severity of endothelial reperfu-
sion injury and duration of ischemic period. Why
such correlation occurred only in the hyperoxia
pretreated but not in control patients, remains
unclear. It cannot be excluded that additional
oxidative stress induced by hyperoxia aggravated
endothelial dysfunction. Also, a small number of
patients as important limiting factor of the study
should be accounted while interpreting the results.
Despite of careful selection of patients, possible
influence of variations in retrograde infusion of
cardioplegic solution and degree of coronary athero-
sclerosis cannot be ultimately excluded.
Two weeks of medical therapy after acute coronary
syndrome has been previously shown to reduce
ADMA levels (12). We observed attenuation of the
plasma ADMA already to the first post-operative
morning after CABG procedure. The measured
levels were on the very low end and in some cases
even lower than recently proposed reference limits
for ADMA in healthy population (13). Decrease of
ADMA concentration may reflect adaptive changes,
aimed to enhance NO release through better func-
tioning of endothelial NOS. Whether these are only
temporary alterations of immediate postoperative
period, or reflect true improvement of endothelial
functioning after surgery remains to be elucidated.
Number of different powerful stimuli interferes in
the early postoperative period, and therefore, the
changes during this short period of time are difficult
to interpret. Obviously, only persisting changes in
ADMA could reflect the modification of cardiovas-
cular risk in particular patients.
Ischaemic heart disease is characterized by a
mismatch of myocardial oxygen demand and supply.
All our patients presented with diffuse coronary
artery disease as they had 3�5 vessels to bypass
and had concurrent stenoses in smaller arteries.
By ventilation with hyperoxic gas mixture oxygen
supply to the myocardium can be improved, but also
formation of reactive oxygen intermediates may
occur. Oxidative stress may decrease the activity of
DDAH (14) with concomitant increase in ADMA
levels. In our study, exposure to 96% of oxygen
in average for 135 min before bypass did not
cause increased production of ADMA in the heart
(Figure 2). During reperfusion period, there was a
tendency, albeit non-significant, for ADMA release
form hyperoxia pretreated but not from control
hearts (Figure 2).
Conclusion
We have provided the first preliminary evidence
regarding arterial and coronary sinus ADMA con-
centrations after cardioplegia and coronary artery
bypass grafting operations.
CABG surgery with cardioplegia resulted in a
significant decrease in arterial ADMA concentrations
by the first postoperative morning. Pretreatment with
hyperoxia had no influence on either myocardial
release or arterial levels of ADMA.
Acknowledgements
Authors would like to thank Ragnar Loit, MD and
Ants Paapstel, MD for help in collecting blood
samples. This study was supported by grants
No.5327 of Estonian Science Foundation and No.
TARBK 0411 of Estonian Ministry of Education
and Research.
Figure 3. Relation of coronary sinus ADMA level to aortic cross-clamping time in hyperoxia pretreated patients; 5th (panel A) and 20th
minute (panel B) of reperfusion.
366 I. Karu et al.
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ADMA levels during coronary surgery 367
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