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: inga@mini.li.ttu.ee

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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References

1. Ursell PC, Mayes M. The majority of nitric oxide synthase in

pig heart is vascular and not neural. Cardiovasc Res. 1993;/27:/

1920�4.

2. Balligand JL, Kobzik L, Han X, Kaye DM, Belhassen L,

O’Hara DS, et al. Nitric oxide-dependent parasympathetic

signaling is due to activation of constitutive endothelial (type

III) nitric oxide synthase in cardiac myocytes. J Biol Chem.

1995;/270:/14582�6.

3. Leiper J, Vallance P. Biological significance of endogenous

methylarginines that inhibit nitric oxide synthase. Cardiovasc

Res. 1999;/43:/542�8.

4. Boger RH, Bode-Boger SM, Szuba A, Tsao PS, Chan JR,

Tangphao O, et al. Asymmetric dimethylarginine (ADMA): A

novel risk factor for endothelial dysfunction: Its role in

hypercholesterolemia. Circulation. 1998;/98:/1842�7.

5. Tahepold P, Valen G, Starkopf J, Kairane C, Zilmer M, Vaage

J. Pretreating rats with hyperoxia attenuates ischemia- reper-

fusion injury in the heart. Life Sci. 2001;/68:/1629�40.

6. Tahepold P, Elfstrom P, Eha I, Kals J, Taal G, Talonpoika A,

et al. Exposure rats to hyperoxia enhances relaxation of

isolated aortic rings and reduces infarct size of isolated hearts.

Acta Physiol Scand. 2002;/175:/271�7.

7. Felaco M, Grilli A, Gorbunov N, Di Napoli P, De Lutiis MA,

Di Giulio C, et al. Endothelial NOS expression and ischemia-

reperfusion in isolated working rat heart from hypoxic

and hyperoxic conditions. Biochim Biophys Acta. 2000;/

1524:/203�11.

8. Sydow K, Munzel T. ADMA and oxidative stress. Atheroscler

Suppl. 2003;/4:/41�51.

9. Ogawa T, Kimoto M, Sasaoka K. Occurrence of new enzyme

catalyzing the direct conversion of NG,NG-dimethyl-L-argi-

nine to L-citrulline in rats. Biochem Biophys Res Comm.

1987;/148:/671�7.

10. Amrani M, Chester AH, Jayakumar J, Schyns CJ, Yacoub

MH. L-Arginine reverses low coronary reflow and enchances

postischemic recovery of cardiac mechanical function. Car-

diovasc Res. 1995;/30:/200�4.

11. Jones SP, Girod WG, Palazzo AJ, Granger DN, Grisham MB,

Jourd’Heuil D, et al. Myocardial ischemia-reperfusion injury

is exacerbated in absence of endothelial cell nitric oxide

synthase. Am J Phys. 1999;/276(5 Pt2):/H1567�73.

12. Bae SW, Stuhlinger MC, Yoo HS, Yu KH, Park HK, Choi

BY, et al. Plasma asymmetric dimethylarginine concentrations

in newly diagnosed patients with acute myocardial infarction

or unstable angina pectoris during two weeks of medical

treatment. Am J Card. 2005;/95:/729�33.

13. Schulze F, Maas R, Freese R, Schwedhelm E, Silberhorn E,

Boger RH. Determination of a reference value for NG, NG-

dimethyl-L-arginine in 500 subjects. Eur J Clin Invest. 2005;/

35:/622�6.

14. Leiper J, Murray-Rust J, McDonald N, Vallance P. S-

Nitrosylation of dimethylarginine dimethylaminohydrolase

regulates enzyme activity: Further interactions between nitric

oxide synthase and dimethylarginine dimethylaminohydro-

lase. Proc Nat Acad Sci USA. 2002;/99:/13527�32.

ADMA levels during coronary surgery 367

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use

onl

y.