Heart Vessels (2009) 24:79–83 DOI 10.1007/s00380-008-1086-1

© Springer 2009

ORIGINAL ARTICLE San Bao Chai · Yong Ming Hui · Xue Min Li · Yang Xiao Chao Shu Tang

Plasma levels of copeptin in patients with coronary heart disease

Received: January 23, 2008 / Accepted: July 3, 2008

Abstract The present study was undertaken to investigate alteration in plasma levels of copeptin, a stable fragment derived from provasopressin, in patients with coronary heart disease. We measured plasma level of copeptin in 21 patients with coronary heart disease (CHD) and 12 agematched healthy subjects by radioimmunoassay (RIA). Chi-square test, Student’s t-test and one-way analysis of variance were used for statistical analyses. Correlations between variables were tested by simple linear regression analysis. The plasma level of copeptin was significantly increased in patients (43.07 ± 17.08 vs 11.13 ± 5.73 pmol/l in controls, P < 0.01) and was further increased, by 60%, to 68.71 ± 16.81 pmol/l on day 1 after therapy with percutaneous transluminal coronary angioplasty (PTCA) and stent (P < 0.05). On days 3 and 7 after therapy, the levels were greatly decreased, to 38.82 ± 19.00 and 32.10 ± 14.00 pmol/l, respectively, from that before therapy (all P < 0.05) but were higher, by 249% and 188%, respectively, than that of controls (all P < 0.01). The results suggest that the vasopressin system is activated in patients with CHD as indicated by changes in copeptin level, especially after PTCA and stent therapy. As a potential risk factor for CHD, plasma copeptin activation might have important clinical significance in terms of early intervention in patients with CHD. Key words Coronary heart disease · Arginine vasopressin · Copeptin · Percutaneous transluminal coronary angioplasty

S.B. Chai (*) · Y.M. Hui Department of Cardiovascular Disease, Beijing Fengtai Hospital, Fengtai District Xi An Street 1#, Beijing 100071, PR China Tel. +86-10-6381 1115/5101; Fax +86-10-8280-5222 e-mail: [email protected] X.M. Li Department of Ophthalmology, Eye Center of Beijing University, Beijing, PR China Y. Xiao · C.S. Tang Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, PR China

Introduction Arginine vasopressin (AVP) is released from neurohypophysis to promote renal water conservation, which affects osmoregulation and cardiovascular homeostasis.1,2 Plasma AVP is increased during congestive heart failure and acute myocardial infarction, which suggests that excessive AVP affects the pathogenesis of cardiovascular disease.3,4 Measurement of AVP level has limitations because of a short half-life and cumbersome detection methods.5 Recently, copeptin, a stable 39-amino-acid glycopeptide of 5 kDa derived from the C-terminal of provasopressin,6–8 was found to be stable in level after blood withdrawal, so it can be measured quickly and easily, being secreted in amounts equimolar to vasopressin.9 Copeptin assay may be a useful alternative to direct measurement of AVP concentration. Some authors reported copeptin concentrations elevated in hemorrhagic and septic shock; copeptin level was higher on admission in nonsurvivors than in survivors, which suggests copeptin as a prognostic marker in sepsis.10,11 Khan et al.12 reported that plasma copeptin was highest on admission in patients with myocardial infarction and reached a plateau on days 3–5. Logistic regression analysis revealed copeptin as a significant independent predictor of death or heart failure at 60 days. In addition, copeptin levels are increased in patients with chronic obstructive pulmonary disease13 and heart failure,14,15 copeptin level being closely related to prognosis of patients. Numerous studies have demonstrated that AVP plays an important role in stress reaction and thromboembolism, which underlies the pathophysiology of acute coronary syndrome.16,17 However, the role of AVP in the pathogenesis of coronary heart disease (CHD) remains unclear. To investigate this, we compared the plasma levels of copeptin in 21 patients with CHD who underwent therapy with percutaneous transluminal coronary angioplasty (PTCA) and stent and 12 healthy subjects.

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Materials and methods Subjects The study was reviewed and approved by the Ethics Committee of Feng Tai Hospital, Beijing, PR China, and informed consent was obtained from each subject before the initiation of the study. Subjects were consecutively recruited from among patients with CHD fulfilling the World Health Organization diagnostic criteria. They were admitted to our hospital from November 2006 through May 2007 and were diagnosed as unstable angina pectoris. In addition, all patients underwent PTCA and stent therapy on the basis of results of coronary angiography. Meantime, they were diagnosed as one-, two- and three-vessel disease according to coronary angiography. Patients with ketoacidosis, acute infection, severe liver or renal disease, congestive heart failure, malignant tumor, and disorder of sodium were excluded. Our sample included 21 patients (13 men; mean age 61 years, range 43–80 years). Twelve healthy subjects with no clinical problems (8 men; mean age 62 years, range 40–68 years), who underwent a health checkup in our hospital, served as controls.

Study design Prior to and on days 1, 3, and 7 after PTCA and stent therapy, blood samples were collected from the antecubital vein of subjects after overnight fasting for measuring copeptin and other biochemical factors. All subjects completed a self-administered questionnaire to gather data on smoking, family history of CHD (i.e., a first-degree relative 60 years old or less with clinical evidence of coronary artery disease), use of medications, and medical history (such as evidence of essential hypertension, coronary artery disease). All subjects underwent physical examination, including measurement of height, weight, body mass index (BMI = kg/m2), and blood pressure (repeated three times, and systolic and diastolic pressure recorded). Meanwhile, all patients received routine therapy with, for example, aspirin, clopidogrel sulfate, and simvastatin. When diagnosed with hypertension, subjects were given antihypertensive drugs.

were assayed by use of an RIA kit (Phoenix Pharmaceuticals, Belmont, CA, USA). The IC50 was 13.43 pg/tube and reactivity with human copeptin 100%. No cross-reactivity was found with human angiotensin II, urotensin II, endothelin, adrenomedullin, or neuropeptide Y. The intra- and interassay coefficients of variation for blood samples were <10%. Reverse-phase high-performance liquid chromatography revealed the major peak of immunoreactive copeptin in the plasma detected by RIA identical to that of synthetic human copeptin. Measurement of other parameters The levels of fasting blood glucose (FBG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TGs), total cholesterol (TC), and C-reactive protein (CRP) were assessed by routine biochemistry analysis. Left ventricular ejection fraction (LVEF) of patients was measured by echocardiography on the next day of hospitalization. Each of the intra- and interassay coefficients of variation was <5%. Statistical analysis Results are expressed as mean ± SD. Chi-square test, Student’s t-test, and one-way analysis of variance were used for statistical analyses. Correlations between variables were tested by simple linear regression analysis (Spearman correlation). A value of P < 0.05 was considered significant.

Results Subjects’ profiles The characteristics of the study subjects are shown in Table 1. Patients with CHD and healthy controls did not differ in age, gender, smoking, BMI, FBG level, plasma sodium, diastolic pressure, and LDL level but did differ in having higher plasma levels of systolic blood pressure, TC, TGs, and CRP, and a lower plasma HDL level (all P < 0.05). Data for LVEF, culprit artery, and stenting are also given in Table 1.

Sample collection Alteration in plasma level of copeptin Blood was drawn into tubes containing disodium ethylenediamine tetraacetic acid (1 mg/ml) and aprotinin (500 U/ml; Sigma, St. Louis, MO, USA), then centrifuged immediately at 3500 × g for 10 min at 4°C; plasma was stored at −80°C for assay. Radioimmunoassay for copeptin Copeptin level was measured by radioimmunoassay (RIA). In brief, samples extracted through a Sep-Pak C18 cartridge

The plasma copeptin level was markedly higher, by 287%, in patients with CHD than in controls (43.07 ± 17.08 vs 11.13 ± 5.73 pmol/l, P < 0.01; Fig. 1). Plasma copeptin level was further increased to 68.71 ± 16.81 pmol/l on day 1 after PTCA and stent therapy (P < 0.05 compared with that before therapy). On days 3 and 7 after therapy, the levels were greatly decreased, to 38.82 ± 19.00 and 32.10 ± 14.00 pmol/l, respectively, from that before therapy (all P < 0.05) but were still higher, by 249% and 188%, respectively, than that of controls (all P < 0.01).

81 Table 1. Subjects’ characteristics Variable

Control subjects n = 12

Patients with coronary heart disease n = 21

Male/female Age (years) Height (m) Weight (kg) BMI (kg/m2) Smoker (%) SBP (mmHg) DBP (mmHg) FBG (mmol/l) Total cholesterol (mmol/l) Triglycerides (mmol/l) HDL (mmol/l) LDL (mmol/l) CRP (mg/l) [Na+] (mmol/l) Troponin (ng/ml) LVEF (%) Culprit artery LAD (%) LCx (%) RCA (%) LAD + RCA (%) LAD + LCx (%) RCA + LCx (%) LAD + LM+RCA + LCx (%) DES (%) Bare stent (%)

8/4 62.42 ± 9.27 1.66 ± 0.07 64.58 ± 8.82 23.23 ± 1.41 5 (42) 117.5 ± 8.12 69.17 ± 7.93 4.90 ± 0.52 4.46 ± 0.23 1.55 ± 0.05 1.54 ± 0.05 2.72 ± 0.07 4.28 ± 0.50 138.23 ± 3.10

13/8 61.00 ± 11.00 1.66 ± 0.08 65.62 ± 10.53 24.20 ± 2.89 16 (76) 129.76 ± 21.42* 76.90 ± 12.70 5.53 ± 1.04 5.17 ± 0.73* 2.14 ± 0.94* 1.33 ± 0.30* 3.00 ± 0.89 9.32 ± 3.07* 139.52 ± 2.32 0.02 ± 0.01 58.95 ± 3.19 7 (33) 2 (10) 4 (19) 3 (14) 2 (10) 2 (10) 1 (5) 8 (31) 18 (69)

Data are mean ± SD, unless indicated BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; LDL, low-density lipoprotein; HDL, high density lipoprotein; CRP, C-reactive protein; LVEF, left ventricular ejection fraction; LAD, left anterior descending artery; LCx, left circumflex artery; RCA, right coronary artery; LM, left main; DES, drug-eluting stent * P < 0.05 compared to controls 120

100

*# 100

*

*#

60

*#

40 20

copeptin (pmol/L)

copeptin (pmol/L)

80

80

60

40

0 control

CHD

1st day

3rd day

7th day

Fig. 1. Comparison of plasma copeptin levels in controls and patients with coronary heart disease (CHD). *P < 0.01 vs control, #P < 0.05 vs CHD

20

0 50

60

70

80

90

100

110

120

DBP (mmHg)

Relation of plasma copeptin level with other parameters Plasma copeptin level was positively correlated with diastolic blood pressure (r = 0.53, P = 0.01; Fig. 2) but not systolic blood pressure, FBG, cholesterol contents (TC, TGs, HDL, LDL), LVEF, plasma sodium, and CRP level (Table 2).

Fig. 2. Correlation between plasma copeptin and diastolic blood pressure (DBP) in patients with coronary heart disease. r = 0.53, P = 0.01

Discussion Arginine vasopressin (AVP), also termed antidiuretic hormone, is a nonapeptide produced in the hypothalamus

82 Table 2. Spearman correlation coefficients for copeptin level and other risk factors in patients with coronary heart disease Copeptin

LVEF

SBP

DBP

FBG

TC

TG

HDL

LDL

CRP

[Na+]

Troponin

R P

−0.33 0.15

0.27 0.24

0.38 0.03*

−0.09 0.71

−0.17 0.47

0.52 0.12

−0.17 0.46

−0.08 0.72

−0.08 0.72

0.01 0.97

0.48 0.08

LVEF, left ventricular ejection fraction; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; TC, total cholesterol; TG, triglycerides; HDL, high-density lipoprotein; LDL, low-density lipoprotein; CRP, C-reactive protein * P < 0.05

and contributes to osmoregulation and cardiovascular homeostasis. Watanabe et al.,18 in 93 patients who underwent coronary angiography and left ventriculography, found that left ventricular end-diastolic volume index and left ventricular stroke volume index increased with increased plasma AVP level, which suggests that AVP is involved in the volume load at regional sites in the heart. Moreover, plasma AVP levels were significantly increased with severity of New York Heart Association classification and negatively correlated with cardiac index in congestive heart failure. So plasma AVP level can indicate the presence and severity of congestive heart failure.19 In addition, AVP has a close relationship with the content of intracellular calcium.20 Since AVP is unstable and rapidly cleared in blood and complex preanalytical requirements and lack of readily available, fast AVP assays have limited the clinical use of AVP measurement, assay of copeptin, the stable C-terminal glycopeptide of the AVP prohormone and stoichiometrically secreted from the posterior pituitary, might be useful in direct measurement of AVP concentration.21 In the present study, we observed a 2-fold increase in plasma copeptin level in patients with coronary heart disease as compared with controls (P < 0.01). As well, on days 1, 3, and 7 after PTCA and stent therapy, plasma levels of copeptin were higher, by 5-, 2.5-, and 2-fold, respectively, than that of controls (all P < 0.01). The level was increased by 60% on day 1 after therapy but was lower on days 3 and 7 than that before therapy (all P < 0.05). Such marked changes in copeptin level, especially after PTCA and stent therapy, suggest copeptin level as a potential risk factor for CHD. Plasma copeptin levels were positively correlated with diastolic blood pressure (r = 0.53, P = 0.01) but not systolic blood pressure, FBG, cholesterol contents (TC, TGs, HDL, LDL), LVEF, CRP level, and plasma sodium. Schäfer et al.4 documented a substantial increase in AVP during early reperfusion in acute myocardial infarction, and found positive correlations between increased AVP and mean arterial and diastolic pulmonary blood pressure. Recently, observation of AVP gene expression in the myocardium and release of this peptide in the coronary effluent in isolated rat hearts exposed to acute wall stress suggested a cardiac source of AVP.22 The production and synthesis of AVP may be enhanced at regional sites of the heart. In addition, AVP has a close relation with the regulation of water and sodium balance. Nakamura et al.19 demonstrated a marked increase in plasma AVP level with congestive heart failure. Plasma

AVP level was negatively correlated with the cardiac index and positively correlated with norepinephrine level. A persistent increase in vasoconstrictor tone, which reduces cardiac function and impairs myocardial blood flow in the infarct-related and noninfarct-related artery, has recently been reported during primary percutaneous coronary intervention in acute myocardial infarction.23 Myocardial ischemia is known to cause an increased activity of sympathetic afferents and pain fibers, which consequently enhances central sympathetic nerve activity and, finally, amplifies the peripheral sympathetic outflow.24 In addition, CHD has a close relationship with inflammation and multiple predisposing genes.25,26 Our study showed that copeptin plasma concentration in patients with CHD was much higher than that of controls. Notably, the highest level of plasma copeptin in patients with CHD was on day 1 after PTCA and stent therapy. On day 3 after therapy, plasma copeptin level decreased significantly from that before therapy. The removal of the total occlusion obtained either by thrombolysis or by primary angioplasty is followed by the ischemia–reperfusion sequelae. The presence of a reperfusion-induced intrinsic activated cardiocardiac reflex might explain this increase in vasoconstrictor tone. Meanwhile, acute left ventricular wall stress during ischemia– reperfusion is suggested to be the stimulus for an instantaneous AVP release from the heart. Thus early elevation of plasma copeptin level after PTCA and stent therapy could be associated with increased vascular tone and ischemia–reperfusion damage. With improved vascular tone and corrected ischemia–reperfusion damage, the plasma level of copeptin recovered gradually. Meanwhile, we failed to find a relation of plasma copeptin levels with LVEF in patients with CHD but without clinical symptoms of heart failure. In addition, patients with PTCA and stent therapy routinely received drug therapy such as aspirin, clopidogrel sulfate, and simvastatin, but we failed to search related reports about the effect of these drugs on the expression of copeptin. In conclusion, our study shows increased plasma copeptin level in patients with CHD, and therapy with PTCA and stent affected this level, which suggests that early intervention in plasma copeptin activation might have an important clinical significance for patients with CHD. Acknowledgments This work was supported by the National Natural Science Foundation of the People’s Republic China (No. 30670847) and the Major State Basic Research Development Program of the People’s Republic of China (No. 2006CB503807).

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