Plasma asymmetric dimethylarginine predicts death and major adverse cardiovascular events in individuals referred for coronary angiography

International Journal of Cardiology 153 (2011) 135–140

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International Journal of Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j c a r d

Plasma asymmetric dimethylarginine predicts death and major adverse cardiovascular events in individuals referred for coronary angiography☆ Tse-Min Lu a, b, Ming-Yi Chung c, d, Ming-Wei Lin c, e, Chiao-Po Hsu b, f, Shing-Jong Lin a, b, c,⁎ a

Division of Cardiology, Department of Internal Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, ROC Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, ROC d Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, ROC e Institute of Public Health, National Yang-Ming University, Taipei, Taiwan, ROC f Division of Cardiovascular Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan, ROC b c

a r t i c l e

i n f o

Article history: Received 9 March 2011 Received in revised form 27 May 2011 Accepted 25 June 2011 Available online 26 July 2011 Keywords: Asymmetric dimethylarginine Coronary artery disease Endothelial function Nitric oxide

a b s t r a c t Background: Elevated plasma level of asymmetric dimethylarginine (ADMA) was reported to be associated with endothelial dysfunction and atherosclerotic risk factors. We assessed the prognostic value of plasma ADMA levels in 997 consecutive individuals referred for coronary angiography from July 2006 to June 2009. Methods: ADMA was measured by high performance liquid chromatography. All subjects were followed for a median period of 2.4 years for the occurrence of all-cause mortality, major adverse cardiovascular events (MACE, defined as cardiovascular death, non-fatal myocardial infarction and stroke), and MACE plus clinically-driven target vessel revascularization (TVR). Results: Plasma ADMA levels were significantly higher in patients with significant coronary artery disease (CAD) (≥ 50% stenosis, n = 655) than those with insignificant CAD (20–50% stenosis, n = 272) and normal coronary artery (b 20% stenosis, n = 70) (0.47 ± 0.10 μmol/l vs 0.44 ± 0.10 μmol/l vs 0.42 ± 0.08 μmol/l, p b 0.001). By multivariate analysis, plasma ADMA level was identified as a significant independent risk factor of significant CAD (OR: 1.29, 95% CI: 1.10−1.50; p = 0.002). Moreover, multivariate Cox regression analysis showed that, comparing with the ADMA tertile I, the highest ADMA tertile was a significant independent predictor for all adverse long-term clinical outcomes. Notably, plasma ADMA level remained associated with the long-term outcomes in non-diabetic individuals, but not in those with diabetes (interaction p = 0.04 for MACE plus TVR). Conclusions: Our findings suggest that elevated plasma ADMA level might be a risk factor of significant CAD, and might predict worse long-term clinical outcomes in subjects referred for cardiac catheterization, especially in non-diabetic individuals. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Asymmetric dimethylarginine (ADMA) is characterized as a circulating endogenous inhibitor of nitric oxide (NO) synthase [1,2] by competing with L-arginine as the substrate for NO synthase. It can increase oxidative stress by uncoupling of electron transport between NO synthase and L-arginine, which can lead to decreases in both the production and availability of endothelium-derived NO [3]. Furthermore, elevated ADMA level might inhibit the mobilization, differen☆ Funding: This study was supported by grants from the National Science Council (NSC96-2314-B-075-071-MY3), Taipei, Taiwan, ROC. ⁎ Corresponding author at: Division of Cardiology, Department of Internal Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, ROC. Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan, ROC. Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC. Tel.: + 886 2 28757511; fax: + 886 2 28753580. E-mail address: [email protected] (S.-J. Lin). 0167-5273/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2011.06.120

tiation and function of endothelial progenitor cell [4]. Elevations of plasma ADMA level have been reported to be associated with the presence of endothelial dysfunction [2,5], and have been observed in patients with various risk factors of atherosclerosis, including hypercholesterolemia [5], essential hypertension [6], diabetes and insulin resistance [7,8], hypertriglyceridemia [9] and hyperhomocysteinemia [10]. Taken together, ADMA may be involved in the pathogenesis of endothelial dysfunction and atherosclerosis. Several studies have shown that plasma ADMA level might predict adverse cardiovascular events and mortality in patients with coronary artery disease (CAD) [11,12]. However, these studies enrolled patients with mild, non-obstructive CAD (≥20–30% stenosis in at least 1 major coronary artery) in addition to patients with significantly obstructive CAD (≥50% stenosis). Moreover, although recent studies reported that ADMA might predict cardiovascular mortality in patients with diabetes [13–15], one large community-based study showed recently that ADMA was associated with death only in non-diabetic subjects,

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not in patients with diabetes [16]. Weather type 2 diabetes might modify the prognostic value of ADMA remains unclear. Finally, the end-points of these studies did not always include the long-term revascularization procedure, which may have a potential impact on the long-term prognosis of patients with CAD. Therefore, in this prospective study we aimed to re-assess the prognostic value of plasma ADMA levels, including the end-point of clinically-driven revascularization procedure, in a consecutive cohort referred for coronary angiography. Furthermore, we examined the relation between ADMA and long-term outcomes especially in patients with diabetes. 2. Methods 2.1. Study design and participants From July 2006 to June 2009, we enrolled 997 consecutive individuals who were referred to Taipei-Veterans General Hospital for coronary angiography for chest pain and/or suspected CAD and possible revascularization treatment. Exclusion criteria included patients with liver cirrhosis, end-stage renal disease (estimated glomerular filtration rate b 15 ml/min per 1.73 m2 or under regular hemodialysis), acute or chronic infectious/inflammatory disease, malignancy with expected life span less than 1 year, and unstable hemodynamic status. In contrast, 98 patients presenting as acute coronary syndrome (ACS) and undergoing cardiac catheterization within 7 days after onset of ACS were not excluded if they did not fulfill other exclusion criteria. Thorough medical histories of all patients were recorded. The associated conventional cardiovascular risk factors included age, hypertension, hypercholesterolemia, smoking, and diabetes mellitus. Hypertension was diagnosed according to the Seventh Joint National Committee criteria or if the patients were taking anti-hypertensive drugs. Hypercholesterolemia was defined according to the diagnostic criteria of ATP III-NCEP guidelines. Diabetes mellitus was diagnosed according to the diagnostic criteria defined by the American Diabetes Association or when the patients were taking oral hypoglycemic agents or receiving insulin injection therapy for blood glucose control. All medications, cigarette smoking and beverages containing alcohol or caffeine were withdrawn for at least 12 h. Blood samples were collected before diagnostic coronary angiography, which was then performed by standard procedure. Coronary angiography was independently reviewed by 2 expert angiographers who were unaware of the patient's clinical and analytical data. According to the results of coronary angiography, the study population was divided into patients with obstructive, significant CAD (the presence of ≥50% stenosis in at least one major coronary artery), non-obstructive, insignificant CAD (the presence of b50% but ≥20% stenosis in at least one major coronary artery) and normal coronary artery (b 20% stenosis at all major coronary arteries). Coronary artery bypass surgery or percutaneous coronary intervention (PCI) was performed successfully in all patients with significant CAD. All patients were prospectively followed by office visit monthly or by telephone contact and chart review for the occurrence of first-ever primary endpoints, which included all-cause mortality, cardiovascular death, major adverse cardiovascular events (MACE, defined as cardiovascular death, non-fatal myocardial infarction and stroke), as well as MACE plus clinically-driven target vessel revascularization (TVR), which was defined as repeat clinically driven revascularization of the treated vessels either by PCI or surgery. Follow-up angiography and repeat PCI if needed were performed only by clinical indications. Cardiovascular death was diagnosed as any death with definite cardiovascular cause or any death that was not clearly attributed to a non-cardiovascular cause. Myocardial infarction was defined as the presence of significant new Q waves in at least 2 electrocardiography (ECG) leads or symptoms compatible with myocardial infarction associated with increase in creatine kinase-MB fraction ≥3 times the upper limit of reference range. Stroke with neurologic deficit was diagnosed by a neurologist on the basis of imaging study. The study protocol was approved by the Institutional Review Board at Taipei-Veterans General Hospital and informed written consent was obtained from each participant in accordance with the ethical guidelines of the Declaration of Helsinki. In addition, the authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [17]. 2.2. Laboratory measurements The blood samples were collected using ethylenediaminetetraacetic (EDTA) as an anticoagulant and were centrifuged at 3000 rpm for 10 min at 4 °C immediately after collection. Plasma samples were kept frozen at − 70 °C until analysis. Plasma Larginine and ADMA concentrations were determined by high performance liquid chromatography using precolumn derivatization with o-phthaldialdehyde as described previously [18]. The recovery rate for ADMA was N 90%, and the withinassay and between-assay variation coefficients were not more than 7% and 8%, respectively. Fasting serum creatinine, total and high-density lipoprotein (HDL) — cholesterol, triglycerides, and blood sugar levels were determined by using an autoanalyzer (Model 7600–310, Hitachi, Tokyo). Low-density lipoprotein (LDL) — cholesterol level was calculated according to the Friedewald formula. Estimated glomerular filtration rate (eGFR) was calculated according to the simplified version of the Modification of Diet in Renal Disease Study prediction equation formula,

further modified by Ma et al. for Chinese patients with chronic kidney disease (eGFR = 175 × plasma creatinine −1.234 × age −0.179 × 0.79 [if female]) [19].

2.3. Statistical analysis All continuous data were presented as mean ± standard deviation or with 95% confidence interval (CI). The study population was grouped into tertiles according to the plasma levels of ADMA. The differences of continuous data were compared by twosample t-test/analysis of variance. Post-hoc comparison was performed by Bonferroni test. As the plasma triglyceride levels were not normally-distributed, we logtransformed the plasma triglyceride levels. Categorical data were compared by means of Chi-square test or Fisher's exact test. The significance of trend of categorical data was tested by Chi-square test for trend. Pearson's correlation coefficients were calculated to examine possible correlations between continuous variables. The univariate logistic regression model for significant CAD was performed first for age, gender, hypertension, diabetes, hypercholesterolemia, smoking status, and eGFR, and then those with a p b 0.1 were included into the multivariate logistic regression analysis. The odd ratio (OR) and 95% CI were calculated. Actuarial event-free survival curves were estimated by using the Kaplan–Meier method and compared by log-rank test. Univariate Cox regression model was performed first for age, gender, diabetes, hypertension, hypercholesterolemia, smoking, ADMA, and eGFR as in the logistic regression model and then those with a p b 0.1 were included into the multivariate Cox regression analysis. In addition, we adjusted the Cox model with the variable of clinical presentation as ACS, which may have a potential impact on the long-term outcomes. The plasma ADMA levels were tested by tertiles or as a continuous variable. The hazards ratio (HR) and 95% CI were calculated. A p value of less than 0.05 is considered to be statistically significant. The SPSS 17.0 (SPSS Inc., Chicago, Illinois, US) software package is used for statistical analysis.

3. Results We enrolled 997 consecutive patients (788 male, 79.0%; mean age: 66.9± 12.4 years) from July 2006 to June 2009. For study participants as a whole, plasma ADMA level was significantly positively correlated with age (r = 0.25, p b 0.001) and negatively with eGFR (r = − 0.24, p b 0.001), LDL-cholesterol (r= −0.11, p = 0.001) and total cholesterol (r= −0.11, p = 0.001). Patients with hypertension had higher plasma ADMA level than those without (0.46 ± 0.10 μmol/l versus 0.44 ± 0.10 μmol/l, p = 0.02). In contrast, while there was no significant difference in plasma ADMA level between patients with and without diabetes (0.45 ± 0.11 μmol/l versus 0.46 ± 0.09 μmol/l, p = 0.21), the plasma ADMA level was significant higher in individuals with diabetes under insulin therapy (n= 69) than those using oral hypoglycemic agents only (n= 290) (0.48 ± 0.09 μmol/l versus 0.46 ± 0.09 μmol/l, p = 0.03). We found no statistically significant relationships between ADMA and hypercholesterolemia, body mass index and current smoking status, respectively. According to the results of coronary angiography, there were 655 patients with significant CAD (65.7%), 272 with insignificant CAD (27.3%) and 70 individuals with normal coronary artery (7.0%). The majority of patients with significant CAD were treated with PCI (n= 594, 90.7%), and the remaining patients underwent coronary artery bypass surgery (n = 61, 9.3%). All of the revascularization procedures were performed successfully. Plasma ADMA levels were significantly higher in patients with significant CAD than those with insignificant CAD and normal coronary artery (0.47± 0.10 μmol/l versus 0.44± 0.10 μmol/l versus 0.42 ± 0.08 μmol/l, p b 0.001; post-hoc comparisons: patients with significant CAD versus insignificant CAD and patients with significant CAD versus normal coronary artery: both p b 0.001). In contrast, plasma L-arginine was similar among three groups (p= NS). Notably, the plasma ADMA levels of patients with ACS (n= 98, 15.0%) were significantly higher than those of patients with stable angina (n = 557, 85.0%) (0.49 ± 0.13 μmol/l versus 0.46 ± 0.10 μmol/l, p = 0.036). In the multivariate logistic regression model, plasma ADMA level was identified as a significant independent risk factor of the occurrence of significant CAD (OR/0.1 μmol/l increase of ADMA: 1.29, 95% CI: 1.10–1.50; p = 0.002), in addition to age, gender, diabetes and hypercholesterolemia. In particular, the association of plasma ADMA level and significant CAD remained significant in nondiabetic subgroup (OR/0.1 μmol/l increase of ADMA: 1.29, 95% CI: 1.07–

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1.54; p = 0.006), but this significance disappeared in those with diabetes (p= 0.284). We grouped the whole population into tertiles according to the plasma ADMA levels b0.41 μmol/l, 0.41–0.48 μmol/l and N0.48 μmol/l. The baseline characteristics are summarized in Table 1. The patients in the highest ADMA tertile were older, had lower cholesterol level, worse renal function, and were more likely to have significant CAD, hypertension and insulin-dependent diabetes. 3.1. Plasma ADMA level and long-term clinical events All patients were followed up for a median follow-up period of 2.4 years without any cases loss of follow-up, and during the followup period, there were 64 all-cause mortality (6.4%, including 47 cardiovascular deaths), 81 MACE (8.1%) and 144 MACE plus TVR (14.4%) (Table 2). Patients who suffered from death or adverse cardiovascular events during the follow-up period had significantly higher plasma ADMA levels comparing with those who did not (allcause mortality: 0.52 ± 0.13 μmol/l versus 0.45 ± 0.10 μmol/l; Cardiovascular death: 0.52 ± 0.13 μmol/l versus 0.45 ± 0.10 μmol/l; MACE: 0.52 ± 0.13 μmol/l versus 0.45 ± 0.10 μmol/l; MACE plus TVR: 0.49 ± 0.13 μmol/l versus 0.45 ± 0.10 μmol/l, all p b 0.001 respectively). In addition, Kaplan–Meier analysis showed that the event-free survival from all-cause mortality, cardiovascular death, MACE and MACE plus TVR were significantly associated with ADMA tertiles, with outcomes being the worst in those individuals with highest plasma ADMA tertile, and a marked risk gradient for all-cause mortality and adverse cardiovascular events were noted across the ADMA tertiles (Fig. 1A, B, C, D, Table 2). In multivariate Cox regression analyses the higher plasma ADMA tertile was identified as a significant independent risk

Table 1 Clinical characteristics of study subjects. ADMA tertile I ADMA tertile II ADMA tertile III P (n = 332) (n = 333) (n = 332) value Age (years) Gender (Male/%) BMI (kg/m2) Hypertension (%) Diabetes (%) Insulin-therapy (%) Smoking (%) Hypercholesterolemia (%) Significant CAD ACS (%) Cholesterol (mg/dl) Total HDL-Cholesterol LDL-Cholesterol Log triglyceride (mg/dl) Creatinine (mg/dl) eGFR (ml/min per 1.73 m2) Fasting blood sugar(mg/dl) ADMA (μmol/l) L-arginine (μmol/l) Medication Anti-platelet agents ACE inhibitor/ARB Statins Calcium channel blocker

62.5 ± 12.1a, c 255 (76.6) 25.9 ± 3.6 232 (69.7)c 110 (33.0) 14 (4.2)c 73 (21.9) 137 (41.1) 186 (55.9) 27 (8.1)

a, c

67.2 ± 11.9a, b 260 (78.1) 26.4 ± 3.8 238 (71.5)b 118 (35.4) 20 (6.0)b 60 (18.0) 154 (46.2) 223 (67.0) 33 (9.9)

a

69.5 ± 10.4b, c 273 (82.5) 25.7 ± 3.9 269 (81.3)b, c 131 (39.6) 35 (10.6)b, c 68 (20.5) 141 (42.6) 244 (73.7) 38 (11.5)

c

b0.01 0.15 0.08 b0.01⁎ 0.21 b0.01⁎ 0.44 0.39 b0.01⁎ 0.34

175.1 ± 38.2c 44.2 ± 11.6 110.0 ± 34.6c 2.08 ± 0.24

169.1 ± 34.5 43.1 ± 11.9 103.9 ± 30.2 2.10 ± 0.23

165.5 ± 33.9c 43.4 ± 13.0 100.8 ± 29.5c 2.10 ± 0.23

0.01 0.52 b0.01 0.42

1.1 ± 0.6c 84.7 ± 24.7a, c

1.1 ± 0.4b 75.7 ± 26.6a, b

1.4 ± 1.0b, c 66.5 ± 28.4b, c

b0.01 b0.01

109.5 ± 44.1

107.8 ± 37.7

112.2 ± 44.1

0.36 ± 0.04a, c 91.1 ± 30.2c

0.44 ± 0.02a, b 91.2 ± 31.4b

0.56 ± 0.10b, c 97.1 ± 32.5b, c

267 (80) 73 (22) 131 (39) 80 (24)

247 (74) 68 (21) 140 (42) 91 (27)

255 (77) 85 (26) 150 (45) 93 (28)

0.40 b0.01 0.02 0.16 0.27 0.31 0.48

ACE: angiotensin converting enzyme; ACS: acute coronary syndrome; ARB: angiotensin receptor blocker; BMI: body mass index; CAD: coronary artery disease; and eGFR: estimated glomerular filtration rate. Post-hoc test: a: ADMA tertile I versus ADMA tertile II p b 0.05; b: ADMA tertile II versus ADMA tertile III, p b 0.05; and c: ADMA tertile I versus ADMA tertile III, p b 0.05. ⁎ P value for trend by Chi-square test for trend b0.05.

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Table 2 All-cause mortality and adverse cardiovascular events during follow-up. ADMA tertile I n = 332 All-cause mortality (%) Cardiovascular death (%) MACE (%) MACE + TVR (%)

ADMA tertile II n = 333

ADMA tertile III n = 332

P values*

5(1.5)

19 (5.7)

40 (12.0)

b0.001

3 (0.9)

14 (4.2)

30 (9.0)

b0.001

8 (2.4) 26 (7.8)

25 (7.5) 49 (12.0)

48 (14.5) 69 (20.7)

b0.001 0.004

*By log-rank test. MACE: major adverse cardiovascular events; and TVR: target vessel revascularization.

factor for long-term all-cause mortality, cardiovascular death, MACE and MACE plus TVR (Table 3). Moreover, when the plasma ADMA level was considering as a continuous variable, the plasma ADMA level remained a significant independent predictor for the endpoints of allcause mortality (HR = 1.25, 95% CI = 1.05−1.49, p = 0.01), MACE (HR = 1.23, 95% CI = 1.05−1.44, p = 0.01)and MACE plus TVR (HR = 1.18, 95% CI = 1.03−1.36, p = 0.03), respectively. In the subgroup analysis, plasma ADMA level remained to be a significant predictor for long-term clinical outcome in patients with CAD (significant and insignificant CAD, Table 3). Furthermore, as clinical presentation with ACS was also identified as a strong risk factor for adverse outcomes in our multivariate Cox regression model, we excluded the patients with ACS and found that the Cox regression analyses yielded similar results, with higher ADMA tertile remaining a risk factor for clinical outcomes of non-ACS subgroup (Table 3). Since a recent large-scale study reported that the predictive value of ADMA on long-term mortality was modified by diabetes [16], we did the multivariate Cox regression analyses in diabetic and non-diabetic subgroups respectively and found that higher ADMA tertiles remained to be an independent predictor in the long-term all-cause mortality, cardiovascular death, MACE and MACE plus TVR in non-diabetic individuals (n = 638, Table 3), but not in those with diabetes (n = 359, p = 0.40, 0.70, 0.43, and 0.47 for all-cause mortality, cardiovascular death, MACE and MACE plus TVR respectively; interaction p = 0.04 for MACE plus TVR, interaction p = 0.29, 0.59 and 0.15 for all-cause mortality, cardiovascular death, and MACE). 4. Discussion 4.1. Major findings In this study, our results showed that plasma ADMA level was significantly higher in patients with significant CAD and acute coronary syndrome, and might be a significant independent risk factor of the occurrence of significant CAD in individuals referred for diagnostic coronary angiography, especially in non-diabetic subgroup. In addition, over a follow-up period of 2.4 years, plasma ADMA tertile was identified as an independent predictor of long-term all-cause mortality and cardiovascular events including clinically-driven TVR in the whole population, in those with CAD and those with non-ACS presentation. In contrast, the predictive value of plasma ADMA level for clinical outcomes remained evident in subjects without diabetes, but the predictive value appeared to disappear in those with diabetes. 4.2. ADMA and the risk of CAD Several small studies, including our previous report, have showed that plasma ADMA level might be an independent risk factor of CAD, and might correlate with the severity and extent of CAD [20–22]. In a large population composed of 1011 consecutive subjects undergoing elective diagnostic cardiac catheterization [23], the plasma ADMA levels of significantly obstructive CAD (≥50% stenosis in at least 1 major coronary artery) were significantly higher compared to those

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A. All-cause death

B. Cardiovascular death

C. MACE

D. MACE plus TVR

Fig. 1. Kaplan–Meier survival analyses with all-cause death (A), cardiovascular death (B), major adverse cardiovascular events (MACE) (C) and MACE plus target vessel revascularization (D) during follow-up according to the tertiles of plasma ADMA levels. P values by log-rank test were shown.

Table 3 Cox regression analysis for all-cause death and adverse cardiovascular events. All-cause death HR (95% CI) Age 1.06 (1.03–1.09) ACS 2.69 (1.51–4.80) eGFR 0.89 (0.80–1.00) ADMA tertile II versus I 2.41 (0.97–5.96) III versus I 3.37 (1.40–8.11) CAD subgroup (n = 927) Age 1.06 (1.03–1.09) ACS 2.75 (1.54–4.93) eGFR 0.90 (0.80–1.00) ADMA tertile II versus I 2.30 (0.93–5.71) III versus I 3.01 (1.25–7.25) Non-DM subgroup (n = 638) Age 1.05 (1.01-1.09) ACS 2.93 (1.35-6.35) eGFR 0.90 (0.77–1.04) ADMA tertile II versus I 5.61 (1.28–24.65) III versus I 7.00 (1.62–30.14) Non-ACS subgroup (n = 899) Age 1.06 (1.02–1.09) eGFR 0.91 (0.81–1.03) ADMA tertile II versus I 4.09 (1.20–13.98) III versus I 5.80 (1.74–19.33)

Cardiovascular death

MACE

MACE plus TVR

P

HR (95% CI)

P

HR (95% CI)

P

HR (95% CI)

P

b0.01 b0.01 0.05

1.04 (1.01–1.07) 2.60 (1.34–5.07) 0.83 (0.73–0.95)

0.02 b0.01 b0.01

1.04 (1.01–1.06) 2.84 (1.70–4.75) 0.84 (0.77–0.93)

b 0.01 b 0.01 b 0.01

1.02 (1.00–1.03) 1.95 (1.27–3.00) 0.92 (0.86–0.99)

0.05 b 0.01 0.02

0.06 b0.01

3.07 (1.04–9.10) 3.61 (1.24–10.51)

0.04 0.02

1.96 (0.95–4.03) 2.48 (1.23–5.00)

0.07 0.01

1.38 (0.88–2.15) 1.57 (1.01–2.44)

0.16 0.04

b0.01 b0.01 0.05

1.04 (1.01–1.07) 2.70 (1.38–5.28) 0.83 (0.72–0.94)

0.02 b0.01 b0.01

1.04 (1.01–1.06) 2.87 (1.72–4.79) 0.84 (0.76–0.93)

b 0.01 b 0.01 b 0.01

1.01 (1.00–1.03) 1.88 (1.22–2.90) 0.92 (0.86–0.99)

0.12 b 0.01 0.02

0.07 0.01

2.94 (0.99-8.72) 3.12 (1.07-9.13)

0.05 0.04

1.89 (0.92–3.89) 2.25 (1.11–4.54)

0.09 0.02

1.34 (0.86–2.09) 1.45 (0.93–2.25)

0.20 0.10

0.02 b0.01 0.15

1.02 (0.99-1.06) 3.11 (1.29-7.58) 0.84 (0.70-1.00)

0.29 0.01 0.05

1.04 (1.01–1.08) 4.01 (2.02–7.96) 0.88 (0.76–1.01)

0.02 b 0.01 0.07

1.01 (0.99–1.03) 1.94 (1.03–3.64) 2.27 (1.25–4.12)

0.24 0.04 b 0.01

0.03 b0.01

4.85 (1.08-21.80) 5.09 (1.14-22.76)

0.04 0.03

2.23 (0.81–6.18) 3.36 (1.27–8.90)

0.12 0.02

1.72 (0.92–3.21) 2.26 (1.22–4.17)

0.09 b 0.01

b0.01 0.14

1.03 (0.99–1.06) 0.86 (0.74–0.99)

0.14 0.04

1.03 (1.00–1.06) 0.85 (0.76–0.95)

0.03 b 0.01

1.01 (0.99–1.02) 0.94 (0.87–1.01)

0.35 0.07

0.03 b0.01

5.91 (1.35–25.92) 6.11 (1.40–26.73)

0.02 0.02

2.64 (1.14–6.13) 2.67 (1.16–6.19)

0.02 0.02

1.61 (1.00-2.60) 1.61 (0.99-2.63)

0.05 0.06

HR for age: per increase of 1 year. HR for eGFR: per increase of eGFR of 10 (ml/min per 1.73 m2); ACS: acute coronary syndrome; and eGFR: estimated glomerular filtration rate.

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without significantly obstructive CAD (p = 0.003), which were similar to our present results [21]. In contrast, Meinitzer et al. reported that although the plasma ADMA levels in 2543 patients with CAD (≥20% stenosis in at least 1 of 15 coronary segments) were marginally higher than those of 695 non-CAD individuals (p = 0.022) and the plasma ADMA levels correlated significantly with the severity of CAD, these differences became insignificant on multivariate analysis [12]. Furthermore, Wang et al. measured plasma ADMA levels in 75 patients with triple vessels CAD and 70 patients with normal coronary artery, and found that the reported ADMA elevation in CAD patients may be due to an associated reduction in renal function and to smoking habit [24]. In the present study, we adjusted the eGFR and smoking status together with other conventional risk factors in the multivariate regression model, and though the plasma ADMA were closely correlated inversely with eGFR, plasma ADMA level remained a significant independent risk factor of the risk of CAD. Nevertheless, difference in definition of CAD, exclusion of patients with acute coronary syndrome or not, and ethnic difference may account for the discrepancies of these findings, and the relation between ADMA and CAD deserves further investigations.

Infusion of ADMA elevated systolic blood pressure and caused medial thickening and perivascular fibrosis in mice coronary microvessels, accompanied by angiotensin-converting enzyme protein upregulation and increased cardiac oxidative stress via both endothelial NO synthase-dependent and -independent pathways [35,36]. On the other hand, transgenic mice over-expressing dimethylarginine dimethylaminohydrolase 1(DDAH1, the enzyme responsible for the degradation of ADMA in human) had lower plasma ADMA level [37], and was associated with accelerated endothelial regeneration and reduced myointimal hyperplasia after endothelial denudation and vascular injury [38]. Furthermore, in addition to inhibiting the mobilization, differentiation and function of endothelial progenitor cell, endogenous ADMA was also reported to inhibit angiogenesis and cell motility in endothelial cell from DDAH1 knockout mice [4,39]. These evidences altogether suggested that ADMA might increase vascular superoxide formation, be an endogenous atherogenic molecule, and might be involved in the process of vascular repair and restenosis after PCI.

4.3. ADMA and long-term clinical outcomes

In a large community study enrolling 3320 Framinghan Offspring Study participants, Böger et al. demonstrated that plasma ADMA level was associated with all-cause mortality in a follow-up period of 10.9 years [16]. In particular, it is surprising to find that this association was not evident in subgroup with diabetes, in contrast to the results of several studies which reporting that elevated plasma ADMA levels might be found in the subjects with insulin resistance and diabetes [7,8], and might predict adverse cardiovascular events in patients with type 1 and 2 diabetes [13,14]. Cavusoglu et al. reported recently that, in 170 diabetic men undergoing coronary angiography, elevated baseline levels of ADMA was a strong independent predictor of cardiovascular outcomes at 2 years [15]. In contrast, the results of our study were similar to that of Böger et al. [16], showing similar plasma ADMA levels between diabetic and non-diabetic groups, and that the significant association of ADMA with long-term prognosis disappeared in diabetic subgroup. In another large study by Meinitzer et al., the plasma ADMA level of patients with type 2 diabetes was only marginally higher than those without (0.83 μmol/l versus 0.82 μmol/l, p = 0.032) [12]. The underlying mechanisms of these conflicting results about the association of diabetes and ADMA remained unclear. Diabetes mellitus is associated with increased oxidative stress [40,41], and some studies suggested that increased NO synthase-derived free radical production may be one of the resources of oxidative stress in diabetes [42,43]. Sibal et al. reported recently that the plasma ADMA levels in patients with early type 1 diabetes without macrovascular disease or macroalbuminuria were even significantly lower compared to healthy controls. In addition, the plasma ADMA levels were not associated with impaired flow-mediated dilatation of brachial artery in these early diabetic patients [44]. Therefore, it might be speculated that inhibition of uncoupled endothelial NO synthase by ADMA with resulting paradoxical reduction of oxidative stress might be a possible explanation for the conflicting association of ADMA with cardiovascular events, especially in patients with uncomplicated diabetes [45]. The relation between ADMA and diabetes seems to be more complex and remains to be elucidated.

Several large studies have addressed the prognostic value of plasma ADMA level on long-term clinical outcomes in patients with CAD [11,12]. In a large-scale prospective study involving 1908 patients with CAD (N30% stenosis in at least 1 major coronary artery), Schnabel et al. showed that the baseline concentration of ADMA, along with C-reactive protein and B type natriuretic peptide, predicted independently future risk of cardiovascular death and nonfatal myocardial infarction during a mean follow-up of 2.6 ± 1.2 years [11]. Meinitzer et al. reported that, in individuals scheduled for coronary angiography and followed for 5.45 years, plasma ADMA level independently predicted all cause and cardiovascular mortality in 2543 individuals with CAD [12]. However, both studies provided no data about the treatment of these patients with significant CAD, which might have a potential impact on the long-term prognosis [25,26], especially when considering the end-point of repeat revascularization. Furthermore, when comparing with both large studies, our study subjects were older, with lower BMI, and more likely to have diabetes, suggesting our cohort may be composed of relatively higher risk population. Nevertheless, our results showed that in addition to all-cause mortality and MACE, plasma ADMA remained a significant independent risk factor in the combined end-points of MACE and TVR, which was similar to the results of our previous report and a recent study investigating the role of plasma ADMA level and restenosis after elective coronary angioplasty [18,27]. The relation of ADMA and restenosis after PCI deserves further detailed investigation in larger cohort. 4.4. ADMA predicts long-term outcomes — possible pathophysiologic mechanisms Endothelial dysfunction, characterized by impaired NO bioavailability, is present in the initial stage of atherosclerosis and has been reported to predict acute and long-term cardiovascular events in patients with CAD [28,29]. ADMA was reported to be associated with endothelial dysfunction [2,5,30], probably via its effects on reducing NO bioavailability and/or increasing oxidative stress. Böger et al. reported that ADMA might increase endothelial superoxide radical elaboration and NF-κB activation, resulting in enhanced monocyte chemotactic protein-1 expression and endothelial adhesiveness for monocytes [31]. In addition, ADMA has been reported to accelerate foam cell formation [32], and might induce apoptosis of endothelial cell by increasing intracellular oxidant production [33]. Elevated plasma ADMA level might also induce tissue factor expression in monocytes via NF-κB-dependent pathway, which might contribute to the procoagulant state and lead to adverse clinical events [34].

4.5. ADMA and diabetes

4.6. Limitations There are several limitations in this study. First, our study subjects were enrolled from population referred for coronary angiography in a single center, the sample size was relatively small and the follow-up period was relatively short compared with other large-scale studies. Although the calculated study power would be 0.94 and 0.87 for the endpoints of MACE and MACE plus TVR, respectively (alpha level: 0.05; SD of plasma ADMA level= 0.10 μmol/l. MACE: event rate = 8.1%, log

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HR= 4.0; MACE plus TVR: event rate = 14.4%, log HR= 2.60), confirmation in larger cohort may be mandatory. In addition, the sample size of insignificant CAD group and normal coronary artery group in this study were small compared with patients with CAD. Further studies with larger control group composed of completely normal coronary artery may be warranted. Finally, as our patients received clinicallydriven follow-up coronary angiography which was performed in 276 patients (42.1%), potential bias related to incomplete angiographic follow-up could not be excluded completely. In conclusion, the results of this prospective study suggested that higher plasma ADMA level was not only a significant independent risk factor of the occurrence of significant CAD, but also was associated with long-term all-cause mortality and adverse cardiovascular events. This association was noted in subjects without diabetes, but it seemed to disappear in diabetic subgroup. Measurement of plasma ADMA level might be beneficial in the risk stratification before diagnostic coronary angiography. Acknowledgments The authors are indebted to Ms. Shu-Chuan Lin and Ms. Pei-Chen Chiang for their excellent technical assistance. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [17]. References [1] Kakimoto Y, Akazawa S. Isolation and identification of NG, NG− and NG, NG dimethyl-arginine, Ne-mono-, di- , and trimethyllysine, glucosylgalactosyl- and galactosyl-delta-hydroxylysine from human urine. J Biol Chem 1970;245:5751–8. [2] Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339:572–5. [3] Leiper J, Vallance P. Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 1999;43:542–8. [4] Thum T, Tsikas D, Stein S, et al. Suppression of endothelial progenitor cells in human coronary artery disease by the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine. J Am Coll Cardiol 2005;46:1693–701. [5] Böger RH, Bode-Böger SM, Szuba A, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998;98:1842–7. [6] Perticone F, Sciacqua A, Maio R, et al. Asymmetric dimethylarginine, L-arginine and endothelial dysfunction in essential hypertension. J Am Coll Cardiol 2005;46: 518–23. [7] Abbasi F, Asagmi T, Cooke JP, et al. Plasma concentrations of asymmetric dimethylarginine are increased in patients with type 2 diabetes mellitus. Am J Cardiol 2001;88:1201–3. [8] Stühlinger MC, Abbasi F, Chu JW, et al. Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. JAMA 2002;287:1420–6. [9] Lundman P, Eriksson MJ, Stühlinger M, Cooke JP, Homsten A, Tornvall P. Mild-tomoderate hypertriglyceridemia in young men is associated with endothelial dysfunction and increased plasma concentrations of asymmetric dimethylarginine. J Am Coll Cardiol 2001;38:111–6. [10] Stühlinger MC, Tsao PS, Her JP, Kimoto M, Balint RF, Cooke JP. Homocysteine impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine. Circulation 2001;104:2569–75. [11] Schnabel R, Blankenberg S, Lubos E, et al. Asymmetric dimethylarginine and the risk of cardiovascular events and death in patients with coronary artery disease — results from the AtheroGene study. Circ Res 2005;97:e53–9. [12] Meinitzer A, Seelhorst U, Wellnitz B, et al. Asymmetrical dimethylarginine independently predicts total cardiovascular mortality in individuals with angiographic coronary artery disease (The Ludwigshafen risk and cardiovascular health study). Clin Chem 2007;53:273–83. [13] Lajer M, Teerlink T, Tarnow L, Parving H, Jorsal A, Rossing P. Plasma concentration of asymmetric dimethylarginine (ADMA) predicts cardiovascular morbidity and mortality in type 1 diabetic patients with diabetic nephropathy. Diabetes Care 2008;31:747–52. [14] Krzyzanowska K, Wolzt M, Mittermayer F, Schernthaner G. Asymmetric dimethylarginine predicts cardiovascular events in patients with type 2 diabetes. Diabetes Care 2007;30:1834–9. [15] Cavusoglu E, Ruwende C, Chopra V, et al. Relation of baseline ADMA levels to cardiovascular morbidity and mortality at two years in men with diabetes referred for coronary angiography. Atherosclerosis 2010;210:226–31. [16] Böger RH, Sullivan LM, Schwedhelm E, et al. Plasma asymmetric dimethylarginine and incidence of cardiovascular disease and death in the community. Circulation 2009;119:1592–600. [17] Shewan LG, Coats AJ. Ethics in the authorship and publishing of scientific articles. Int J Cardiol 2010;144:1–2.

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