By Popov, A F Hinz, J; Liakopoulos, O J; Schmitto, J D; Seipelt, R; Quintel, M; Schoendube, F A
Aim. The angiotensin I-converting enzyme insertion/deletion polymorphism (ACE-I/D), including three genotypes (II, ID, DD), with a known impact on midterm mortality and morbidity in patiente after coronary artery bypass graft surgery (CABG), was studied. Since this polymorphism has been linked with increased vascular response to phenylephrine during cardiopulmonary bypass (CFB), we investigated its possible effect on perioperative hemodynamlcs in patiente undergoing CABG. Methods. Genotyping for the ACE-I/D was performed by polymerase chain reaction (PRC) amplification in 110 patiente who underwent elective CABG with CPB. Patiente were assigned to two groups according to their genotype (group II [II genotype] and group ID/DD [ID and DD genotypes]). Systemic hemodynamics were measured directly before and at 4 h, 9 h, and 19 h after CPB.
Results. Genotype distribution of ACE-I/D was 18%, 57%, and 25% in genotypes II, ID, and DD, respectively. The two groups were similar in age (group II: 66+-6 years, group ID/DD: 66+-8 years), body-mass-index (BMI) (group II: 28+-2, group ID/DD: 29+-5 kg/m^sup 2^), male: female ratio (group II: 16: 4, group ID/DD: 63: 27) and Euroscore (group II: 3.1+-1.9, group ID/DD: 3.5+-2.1). There were no differences in mortality rate or perioperative systemic hemodynamics. The pulmonary vascular resistance before cardiopulmonary bypass was higher in the ID/DD genotypes than in the II genotypes (227+-121 vs 297+-169 dyn-s-1-m2-cm-5). Four hours after CPB no difference remained; at 9 h after cardiopulmonary bypass there was a slight difference in pulmonary vascular resistance between the two groups (247+-134 vs 290+-117 dyn*s^sup – 1^*m^sup 2^*cm^sup -5^) and a significant difference in pulmonary arterial pressure (19+-6 vs 23+-8); at 19 h after CPB the differences were no longer detectable.
Conclusion. ACE-I/D had no influence on perioperative systemic hemodynamlcs. However, transitory differences in pulmonary hemodynamic were observed after CPB. These differences may have been due to changes in serum ACE activity during CPB.
KEY WORDS: Cardiopulmonary bypass – Polymorphism, genetic – Hemodynamics.
Angiotensin I-converting enzyme (ACE), an important enzyme in the renin-angiotensin system, helps to regulate blood pressure, cellular growth and cardiovascular remodeling. Insertion (I) or deletion (D) polymorphism in the ACE gene is due to the presence or absence, respectively, of a 287 Alu repeat sequence in intron 16 of the ACE gene and results in three genotypes (II, ID, DD) of this sequence – 1 Increased ACE levels in plasma and in cardiac tissue have been reported in individuals with the ID and the DD genotype.2 Numerous association studies have investigated possible links of this polymorphism with several cardiovascular disorders.3-7 It is associated with hypertension and cardiovascular diseases (CVD) such as left ventricular hyperthrophy, cardiomyopathy and myocardial infarction.8, 9
Patients with the DD genotype are at an increased risk of midterm mortality and cardiac morbidity after coronary artery bypass graft (CABG) surgery.10 Our study investigated only midterm results during a 2-year period, without taking into account the early influence of this polymorphism on perioperative mortality and morbidity in CABG surgery patients. Patients with the DD genotype are also noted to have an increased vascular response to phenylephrine during cardiopulmonary bypass.11 This polymorphism is associated with impaired endothelial-dependent vasodilation in vitro in human internal mammary arteries.12 Also, in vivo studies have shown that the endothelium-dependent dilation in response to pharmacologie stimulation is impaired in D-allele carriers.13, 14 Lasocki et al. confirmed the impact of this polymorphism on vascular reactivity in humans and found that among patients with cardiopulmonary bypass Dallele carriers have a minor decrease in vascular resistance.15 Against this pathophysiological background we hypothesized that the presence of the D allele may be associated with changes in perioperative hemodynamics during CABG surgery and investigated the influence of this polymorphism on perioperative hemodynamics and mortality in these patients.
Materials and methods
Study population
The study protocol was approved by the ethics committee of the University of Gottingen, and all patients gave written informed consent. A total of 110 patients who underwent elective CABG were included into this prospective study. Patients were excluded who had a previous or concomitant cardiac surgical procedure, age >80 years, acute myocardial infarction (2.0mg/dL) or known neoplasms.
Operative technique
Anesthesia was induced and maintained using sufentanil, midazolam and pancuronium. Coronary artery bypass graft surgery was performed via median sternotomy and moderate hypothermie (32 [degrees]C) non- pulsatile cardiopulmonary bypass was established via cannulation of the ascending aorta and right atrium. Cardioplegic cardiac arrest was achieved by intermittent antegrade/retrograde infusion of cold blood cardioplegia. After rewarming and weaning from cardiopulmonary bypass, systemic anticoagulation was reversed 1: 1 by protamine sulfate administration. Initiation of treatment with inotropic drugs or nitroglycerine was at the discretion of the attending surgeon and anesthesiologist and based on hemodynamics at cardiopulmonary bypass and in the intensive care unit (ICU).
Hemodynamic measurements
After induction of anesthesia, a pulmonary catheter was inserted for hemodynamic measurement by means of the thermodilution technique. Hernodyhamic parameters were heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), mean pulmonary artery pressure (PAP), pulmonary capillary wedge pressure (PCWP). Cardiac index (CI), systemic (SVRI) and pulmonary vascular resistance (PVRI) indices were calculated from standard formulas. Hemodynamics were measured during steady-state conditions at least 20 min before cardiopulmonary bypass and at 4 h, 9 h, and 19 h after cardiopulmonary bypass in the ICU. Postoperative data recording included mean inotropic support, nitroglycerine therapy, markers of myocardial damage (troponin T, creatinine phosphokinaseAMB fraction), creatinine concentration, urine output and drainage blood loss 19 h after cardiopulmonary bypass. Ventilation time, length of ICU stay, length of stay in hospital and in-hospital mortality were also recorded.
Genotyping
DNA was isolated from leucocytes collected from whole blood by standard method.11 Insertion/deletion polymorphism in the ACE gene was identified by the polymerase chain reaction (PCR) according to Rigat et al.1 The following primers were used: forward primer 5′- CTG GAG ACC ACT CCC ATC CTT TCT-3′ (Biomers.net GmbH, Ulm, Germany) and the reverse primer ‘5- GAT GTG GCC ATC ACA TTC GTC AGA T 3′ (Biomers.net GmbH, Ulm, Germany). Genotypes were interpreted according to the length of the PCR products: 190 and 490 base pairs for the deletion and insertion alleles, respectively. In DD homozygotes we confirmed the accuracy of the genotyping results by using an insertion-specific primer pair (5′ TGG GAC CAC AGC GCC CGC CAC TAC 3′; 5′ TCG CCA GCC CTC CCA TGC CCA TAA 3’) (Biomers.net GmbH, Ulm, Germany) (Figure 1).
Figure 1.-Gel electrophoresis of the angiotensin I!-converting enzyme insertion/deletion polymorphism from three different patients. Lane 1: DNA ladder with base pair (bp) marker. Lane 2-3: patient homozygous for insertion polymorphism (II genotype). Lane 4- 5: patient homozygous for deletion polymorphism (DD genotype). Lane 6-7: patients heterozygous for insertion/deletion polymorphism (ID genotype).
Patients were assigned to two groups depending on their genotype: (group II [II genotype], group ID/DD [ID and DD genotype]). Acquisition of hemodynamics and perioperative data was performed in a blinded manner.
Statistical analysis
Statistical analysis was computed using commercial available software (SPSS for Windows, SPSS Inc. Chicago, USA). Pre- and postoperative data were compared using Student’s t-test and the chi^sup 2^ test. The hemodynamic data were compared using ANOVA for repeated measurements followed by post hoc tests. Allele frequencies in the study population were counted and compared with an expected distribution in a normal population by Hardy-Weinberg equilibrium and checked with the chi^sup 2^ test. Only 27 patients were homozygous for the D allele. Genotypes were defined as carriers (ID/ DD) and noncarriers (II) of the D allele. Statistical difference was set at P>0.05.
Results
Out of a total of 110 patients, 20 were homozygous for the insertion in the ACE gene, 63 were heterozygous for the insertion/ deletion, and 27 were homozygous for the deletion. Accordingly, the allele frequency of the insertion allele was 46.8% and 53.2% for the deletion. Genotype distribution of the ACE-I/D polymorphism was confirmed to be in prediction with Hardy-Weinberg equilibrium. The genotype and allelic frequencies of the polymorphism in this study population closely matched those published elsewhere (Table I).3, 8, 10
TABLE I.-Distribution of angiotensin I-converting enzyme insertion/deletion polymorphism genotypes and allele frequencies. TABLE II.-Pre- and intraoperative characteristics of patients (N=110) with angiotensin I-convening enzyme insertion/deletion polymorphism. Patients were grouped according to genotype (II: II genotype, ID/DD: ID and DD genotype).
There were no differences in the preoperative characteristics of the two groups with respect to age, gender, BMI, use of ACE inhibitors, creatinine level, left ventricular ejection fraction, and additive Euroscore. No differences were noted in aortic clamp time, cardiopulmonary bypass time, reperfusion time or cardiopulmonary bypass temperature during cardiopulmonary bypass (Table II).
Hemodynamic measurements and postoperative characteristics
No differences between the groups were noted in perioperative systemic hemodynamics with respect to heart rate, mean arterial pressure, central venous pressure, pulmonary capillary wedge pressure, cardiac index and systemic vascular resistance index.
TABLE III.-Perioperative hemodynamics of patients (N=110) with angiotensin I converting enzyme insertion/deletion polymorphism. Hemodynamic data of patients grouped by genotype (II: II genotype, ID/DD: ID and DD genotype).
TABLE IV.-Postoperative characteristics of patients N=110) with angiotensin I converting enzyme insertion/deletion polymorphism and grouped by genotype (II: II genotype, ID/DD: ID and DD genotype).
Differences in pulmonary hemodynamics were observed, however. The pulmonary vascular resistance index was higher before cardiopulmonary bypass in individuals carrying the D allele (227+- 27 vs 296+18 dyn*s^sup -1^*m^sup 2^*cm^sup -5^; P=0.05). Directly after cardiopulmonary bypass and 4 h later these differences were no longer detectable. At 9 h after cardiopulmonary bypass the Dallele carriers were noted to have a higher pulmonary vascular resistance index (247+-30 vs 290+-12 dyn*s ^sup -1^*m^sup 2^*cm^sup -5^; P=0.19). These differences disappeared at 19 h after cardiopulmonary bypass. The mean pulmonary pressure was comparable during the study period, except at 9 h after cardiopulmonary bypass in the patients carrying the D allele (19+-1 vs 23+-1; P
The postoperative characteristics of the two groups are illustrated in Table IV. No D-allele dependent differences were observed in markers for myocardial damage, renal function, ventilation time, length of ICU stay, length of hospital stay or in- hospital mortality.
Discussion
We investigated the influence of ACE-I/D polymorphism on perioperative hemodynamics and perioperative mortality in patients undergoing CABG surgery. In our study sample, the distribution of genotypes and alleles was comparable to that of a metaanalysis that investigated the ACE-I/D polymorphism in Caucasian populations.8 We observed no perioperative differences in systemic hemodynamics between the two groups.
This result disagrees with previous findings concerning vascular response in patients undergoing cardiopulmonary bypass. Lasocki et al. demonstrated in patients with the DD genotype a modified vascular response and suggested that this reactivity could be a result of increased vascular smooth tone or endothelial dysfunction.15
In our patients, however, those carrying the D allele were noted to have significant differences in pulmonary hemodynamics before and at 9 h after cardiopulmonary bypass. Analysis of the perioperative data revealed higher PVRI and PAP in patients with the D allele. These differences had no early perioperative clinical impact, however, as indicated by the comparable cardiac index and consumption of cardiac inotropia in both groups.
Carriers of the DD genotype are at increased risk of midterm mortality and cardiac morbidity after CABG.10 Plasma and tissue levels of ACE are higher in these patients than in those with the II genotype, while patients with the ID genotype have an intermediate ACE level.1,2 Given the influence of the ACE-I/D polymorphism on plasma and tissue levels of ACE and the important role ACE plays in cardiovascular disease, the DD genotype has been implicated as a genetic marker that predisposes patients to a variety of cardiovascular diseases.10, 16-8
Although our study did not identify any underlying mechanisms, it is known that the expression of ACE increases remodelling19 and atherosclerosis20 and that blood pressure is regulated by the renin- angiotensinaldosterone system. Increasingly, studies are uncovering the genetic influences on the components of this system and their impact on the development of coronary artery disease (CAD). The ACE- I/D polymorphism has engendered numerous reports of possible allelic influences on intermediate and complex phenotypes, such as serum levels of ACE,1, 18, 21 conversion of Angiotensin I to Angiotensin II, and blood pressure,22 on risk factors such as hypertension,6, 23 left ventricular function, 24 and on outcome such as myocardial infarction, 17, 25 ischemic stroke 26 and hypertrophie cardiomyopathy.27
In-hospital mortality was similar in both groups and comparable to 30-day mortality after cardiac surgery.28 Although our patients were distributed according to Hardy-Weinberg equilibrium, our study is limited by the small number of patients in group II, which may have affected hospital mortality rates. Because the study population was selected from patients undergoing elective isolated CABG surgery, the sample size was smaller than in gene polymorphism studies in patients with cardiovascular diseases.1
Another limitation to the study was that only some patients received preoperative ACE inhibitors. However, there were no differences in this distribution across the studied genotypes. Patients treated with ACE inhibitors received various other drugs as well. Licker et al. demonstrated in a cardiopulmonary bypass model that the chronic use of ACE inhibitors attenuates vascular response to norepinephrine.29 But drug-specific or class-specific interactions with the D allele have not been investigated so far, and our analysis showed no association between the use of ACE inhibitors and genotype. In brief, it cannot be ruled out that this may have influenced our study results.
Conclusions
The ACE-I/D polymorphism does not influence perioperative systemic hemodynamics or mortality in patients undergoing elective CABG surgery. Pulmonary hemodynamics was affected by this polymorphism, but this may not have a clinical impact in the perioperative phase. Further study is needed to verify these findings.
References
1. Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I- converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990;86:1343-6.
2. Danser AH, Schalekamp MA, Bax WA, van den Brink AM, Saxena PR, Riegger GA elal. Angiotensin-converting enzyme in the human heart: effect of the deletion/insertion polymorphism. Circulation 1995;92:1387-8.
3. Cambien F, Poirier O, Lecerf L, Evans A, Cambou JP, Arveiler D et al. Deletion polymorphism in the gene for angiotensin-converting enzyme is a potent risk factor for myocardial infarction. Nature 1992;359:641-4.
4. Keavney B, McKenzie C, Parish S, Palmer A, Clark S, Youngman L et al. large-scale test of hypothesized associations between the angiotensin-converting-enzyme insertion/deletion polymorphism and myocardial infarction in about 5 000 cases and 6 000 controls. International Studies of Infarct Survival (ISIS) Collaborators. Lancet 2000;355:434-42.
5. Lindpaintner K, Pfeffer MA, Kreutz R, Stampfer MJ, Grodstein F, LaMotte F et al. A prospective evaluation of an angiotensin- converting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J Med 1995;332:706-11.
6. O’Donnell CJ, Lindpaintner K, Larson MG, Rao VS, Ordovas JM, Schaefer FJ et al. Evidence for association and genetic linkage of the angiotensin-converting enzyme locus with hypertension and blood pressure in men but not women in the Framingham Heart Study. Circulation 1998;97:1773-9.
7. Pinto YM, van Gilst WH, Kingma JH, Schunkert H. Deletion-type allele of the angiotensin-converting enzyme gene is associated with progressive ventricular dilation after anterior myocardial infarction. Captopril and Thrombolysis Study Investigators. J Am Coll Cardiol 1995;25:1622-6.
8. Agerholm-Larsen B, Nordestgaard BG, Tybjaerg-Hansen A. ACE gene polymorphism in cardiovascular disease: meta-analyses of small and large studies in whites. Arterioscler Thromb Vasc Biol 2000;20:484-92.
9. Arnett DK, Borecki IB, Ludwig EH, Pankow JS, Myers R, Evans G et al. Angiotensinogen and angiotensin converting enzyme genotypes and carotid atherosclerosis: the atherosclerosis risk in communities and the NHLBI family heart studies. Atherosclerosis 1998;138:111-6.
10. Volzke H, Engel J, Kleine V, Schwahn C, Dahm JB, Eckel L et al. Angiotensin I-converting enzyme insertion/deletion polymorphism and cardiac mortality and morbidity after coronary artery bypass graft surgery. Chest 2002;122:31-6.
11. Henrion D, Benessiano J, Philip I, Vuillaumier-Barrot S, Iglarz M, Plantefeve G et al. The deletion genotype of the angiotensin I-converting enzyme is associated with an increased vascular reactivity in vivo and in vitro. J Am Coll Cardiol 1999;34:830-6.
12. Buikema H, Pinto YM, Rooks G, Grandjean JG, Schunkert H, van Gilst WH. The deletion polymorphism of the angiotensin-converting enzyme gene is related to phenotypic differences in human arteries. Fur Heart J 1996; 17:787-94.
13 Perticone F, Ceravolo R, Maio R, Ventura G, Zingone A, Perrotti N et al. Angiotensin-converting enzyme gene polymorphism is associated with endothelium-dependent vasodilation in never treated hypertensive patients. Hypertension 1998;31:900-5.
14. Butler R, Morris AD, Burchell B, Struthers AD. DD angiotensin- converting enzyme gene polymorphism is associated with endotheHal dysfunction in normal humans. Hypertension 1999;33:1164-8. 15. Lasocki S, Iglarz M, Seince PF, Vuillaumier-Barrot S, Vicaut E, Henrion D et al. Involvement of renin-angiotensin system in pressure- flow relationship. Anaestesiology 2002; 96:271-5.
16. Lahiri DK, Nurnberger Jr JI. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res 1991;19:5444.
17. Beohar N, Damaraju S, Prather A, Yu QT, Raizner A, Kleiman NS et al. Angiotensin converting enzyme genotype DD is a risk factor for coronary artery disease. J Invest Med 1995;43:275-80.
18. Marian AJ, Yu QT, Workman R, Greve G, Roberts R. Angiotensinconverting enzyme polymorphism in hypertrophie cardiomyopathy and sudden cardiac death. Lancet 1993;342:1085-6.
19. Morishita R, Gibbons GH, Ellison KE, Lee W, Zhang L, Yu H etal. Evidence for direct local angiotensin in vascular hypertrophy: in vivo gene transfer of Angiotensin converting enzyme. J Clin Invest 1994;94:978-84.
20. Pitt B. Angiotensin-converting enzyme inhibitors in patients with coronary atherosclerosis. Am Heart J 1994;128:1328-32.
21. Tiret L, Rigat B, Visvikis S, Breda C, Corvol P, Cambien F et al. Evidence from combined segregation and linkage analysis, that a variant of the angiotensin !-converting enzyme (ACE) gene controls plasma ACE levels. AmJ Hum Genet 1992;51:197-205.
22. Lachurie ML, Azizi M, Guyene TT, Alhenc-Gelas F, Menard J. Angiotensin converting enzyme gene polymorphism has no influence on the circulating rerun angiotensin-aldosterone system or blood pressure in normotensive subjects. Circulation 1995;91:2933-^i2.
23. Harrap SB, Davidson HR, Connor JM, Soubrier F, Corvol P, Fraser R et al. The angiotensin !-converting enzyme gene and predisposition to high blood pressure. Hypertension 1993;21:455-60.
24. Clarkson PB, Prasad N, MacLeod C, Burchell B, MacDonald TM. Influence of the angiotensin converting enzyme I/D gene polymorphisms on left ventricular diastolic filling in patients with essential hypertension. J Hypertens 1997;15:995-1000.
25. Samani NJ, Thompson JR, O’Toole L, Channer K, Woods KL. A meta-analysis of the association of the deletion allele of the angiotensin converting enzyme gene with myocardial infarction. Circulation 1996;94:708-12.
26. Margaglione M, Celentano E, Grandone E, Vecchione G, Cappucci G, Giuliani N etal. Deletion polymorphism of the angiotensin- converting enzyme gene in patients with a history of ischaemic stroke. Arterioscler Thromb Vase Biol 1996; 16:304-9.
27. Lechin M, Quinones MA, Omran A, Hill R, Yu QT, Rakowski H et al. Angiotensin I-converting enzyme genotypes and left ventricular hypertrophy in patients with hypertrophie cardiomyopathy. Circulation 1995;92:1808-12.
28. Osswald BR, Blackstone EH, Tochtermann U, Thomas G, Vahl CF, Hagl S. The meaning of early mortality after CABG. Eur J Cardiothorac Surg 1999;15:401-7.
29. Licker M, Neidhart P, Lustenberger S, Valloton MB, Kalonji T, Fathi M et al. Long-term angiotensin-converting enzyme inhibitor treatment attenuates adrenergic responsiveness without altering hemodynamic control in patients undergoing cardiac surgery. Anaestesiology 1996; 84:789-800.
A. F. POPOV1, J. HINZ2, O. J. LIAKOPOULOS1, J. D. SCHMITTO1, R. SEIPELT1, M. QUINTEL2, R A. SCHOENDUBE1
1 Department of Thoracic Cardiovascular Surgery
University of Gottingen, Gottingen, Germany
2 Department of Anesthesiology
Emergency and Intensive Care Medicine
University of Gottingen, Gottingen, Germany
Acknowledgements.-The project was supported by in part by grants from the Department of Thoracic Cardiovascular Surgery and the Department of Anaesthesiology, Emergency and Intensive Care Medicine, University of Gottingen, Germany. We thank PhD Hilgers, (Department of Medical Statistics, Georg-August University Gottingen, Germany) for his help with the statistical analysis.
Received on February 12, 2008.
Accepted for publication on March 19, 2008.
Address reprint requests to: A. F. Popov, Department of Thoracic and Cardiovascular Surgery, University of Gottingen, Robert-Koch- Strasse 40, 37099 Gottingen, Germany. E-mail: [email protected] goettingen.de
Copyright Edizioni Minerva Medica Apr 2008
(c) 2008 Journal of Cardiovascular Surgery. Provided by ProQuest Information and Learning. All rights Reserved.
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