Relations between plasma asymmetric dimethylarginine (ADMA) and risk factors for coronary disease

Atherosclerosis 184 (2006) 383–388

Relations between plasma asymmetric dimethylarginine (ADMA) and risk factors for coronary disease Jun Wang a , Ah Siew Sim a , Xing Li Wang c , Chris Salonikas b , Daya Naidoo b , David E.L. Wilcken a,∗ a

c

Department of Cardiovascular Medicine, University of New South Wales, Prince of Wales Hospital, Sydney, Australia b Department of Clinical Chemistry, University of New South Wales, Prince of Wales Hospital, Sydney, Australia Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA Received 19 December 2004; received in revised form 27 April 2005; accepted 4 May 2005 Available online 6 June 2005

Abstract Background: Elevated plasma levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide production, are reported to be associated with coronary artery disease (CAD). Methods: We measured plasma levels of ADMA and related compounds, nitrate + nitrite (NOx ), total homocysteine (tHCY) and assessed renal function and lipid profiles in 145 patients—75 with triple vessel coronary disease and 70 with no detectable coronary disease. Results: Levels of ADMA, l-arginine, l-arginine/ADMA and plasma NOx were not different in the two groups but smokers with triple vessel disease had higher ADMA and lower NOx levels than the non-smokers, relationships also present for all smokers and non-smokers in the two groups combined. In all 145 patients ADMA, symmetric dimethylarginine (SDMA) and tHCY levels were significantly higher in patients with glomerular filtration rate (GFR) <81 mL/min/1.73 m2 than in patients with GFR ≥ 81 mL/min/1.73 m2 . There was a modest positive correlation between tHCY and ADMA and both were strongly correlated with SDMA which is excreted by the kidney. ADMA, SDMA and tHCY were negatively correlated with GFR. Conclusions: We suggest that the reported ADMA increases in CAD patients are due to an associated reduction in renal function and to smoking habit. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Asymmetric dimethylarginine; Homocysteine; Coronary artery disease; Smoking; Renal function; Glomerular filtration rate

1. Introduction Asymmetric dimethylarginine (ADMA), the product of asymmetric methylated protein, is an endogenous inhibitor of endothelial nitric oxide synthase (NOS) [1]. Vallance et al. first reported that an elevation of plasma ADMA is associated with increased risk of atherosclerosis in chronic renal failure patients [2]. Chronic renal failure constitutes a major risk for coronary artery disease and mild renal impairment is also as∗ Corresponding author at: The Professorial Suite, Prince of Wales Hospital, Barker St., Randwick, Sydney, NSW 2031, Australia. Tel.: +61 2 93824835; fax: +61 2 93824921. E-mail address: [email protected] (D.E.L. Wilcken).

0021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2005.05.002

sociated with significant increase in coronary artery disease risk [3]. Cooke et al. showed that ADMA decreased NO production by inhibiting NOS in a dose-dependent manner [4]. ADMA also increases superoxide production independently resulting in activation of redox-regulated transcriptional factors such as NF-␬B resulting in concomitant up-regulation of endothelial adhesion molecules [5]. Hyperhomocysteinemia is a putative risk factor for coronary artery disease (CAD) [6]. It is associated with impaired endothelium dependent flow-mediated dilatation of the brachial artery [7] which is mediated largely by endothelium derived nitric oxide (NO) [8]. Total homocysteine has been shown to increase endothelial cell generation of ADMA by inhibiting the activity of the enzyme dimethylarginine

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dimethylamniohydrolase (DDAH) [9] which is responsible for the metabolism of ADMA [4]. It is suggested that elevated levels of ADMA are associated with the severity of CAD [10]. To explore this, in the present study we measured levels of ADMA in patients with severe CAD and in those without CAD documented angiographically. We also explored in these patients possible interactions between ADMA and other risk factors including total homocysteine, the lipid profile, renal function, smoking and gender.

current medications including lipid-lowering drugs. Body mass index (BMI) was measured during admission to the hospital. Patients who had never smoked or had not smoked for at least 5 years were classified as non-smokers, as the risk of a major coronary event reduces to the level of non-smokers at approximately 5 years after stopping smoking [11]. Smokers were defined as those who had regularly smoked at least five cigarettes per day for at least 5 years. Patients with intermediate smoking habits were not included. Ex-smokers were defined as those who had ceased smoking for over 12 months.

2. Materials and methods 2.4. Lipid profile and renal function 2.1. The patient population We studied 145 patients aged 75 years and less, both men and women (mean age 61.9 ± 8.5 years, range 40–74 years) referred to the Eastern Heart Clinic at the Prince of Wales Hospital, Sydney for coronary angiography because of chest pain from January 2001 to June 2002. Patients were included in the present study who had angiographically demonstrated triple vessel disease (see below) or normal coronary arteries. Patients with glomerular filtration rate (GFR) less than 45 mL/min/1.73 m2 were excluded from the study. The study was conducted in accordance with the Australian National Health and Medical Research Council (NH&MRC) Guidelines and was approved by the University of New South Wales Ethics Committee. Written consent was obtained from every patient. 2.2. Blood sample collection Patients were fasted for at least 6 h before venous blood samples were drawn into 10 mL EDTA vacuum tubes prior to the coronary angiography. Blood samples were separated in a refrigerated centrifuge within 15 min of collection. Plasma was divided into small aliquots, snap-frozen in liquid nitrogen, and stored at −70 ◦ C until analysis. 2.3. Documentation of CAD severity and atherosclerosis risk factors The severity of CAD was determined by the number of significantly stenosed coronary arteries (≥50% luminal obstruction). The angiograms were assessed by two cardiologists who were unaware that the patients were to be included in the study. Each angiogram for the patients included was classified as either revealing ≥50% luminal stenoses in three major epicardial coronary arteries, or with no detectable coronary lesions. To enhance the difference between two groups, patients with lesions in only one or two coronary arteries were not included. The medical history of each patient was obtained using a structured questionnaire with standardized choices of answers to be ticked during the interview. We recorded current and past health conditions, risk factors for heart disease and

Total cholesterol (TC), HDL-cholesterol (HDL-C) and triglyceride (TG) levels were measured by the hospital’s Clinical Chemistry Department using standard enzymatic methods. The LDL-cholesterol (LDL-C) levels were calculated using the Friedewald formula. Creatinine levels were measured in non-fasting plasma by the Jaffe method, adapted for autoanalyzers [12] and we also estimated glomerular filtration rate based on the formula [(140 − age) × weight (kg)]/[0.814 × creatinine (␮mol/L)] × 0.85 for women. 2.5. Plasma ADMA assay Plasma levels of ADMA, symmetrical dimethylarginine (SDMA) and arginine were measured by high performance liquid chromatography as described by Teerlink et al. [13]. Briefly, 0.2 mL plasma was mixed with 0.1 mL of a 40 ␮mol/L solution of the internal standard L-NMMA and 0.7 mL phosphate-buffered saline. This mixture was applied to Oasis MCX solid-phase extraction columns (Waters, Sydney, Australia) for extraction of basic amino acids. The columns were consecutively washed with 1.0 mL 100 mM HCl and 1.0 mL methanol. Analytes were eluted with 1.0 mL ammonia/water/methanol (10/40/50). After evaporation of the solvent under nitrogen, the amino acids were derivatized with o-phthaldialdehyde reagent containing 3mercaptopropionic acid. The derivatives were separated by isocratic reversed-phase chromatography on a Waters Symmetry C18 column (3.9 mm × 150 mm; 5 ␮m particle size). Potassium phosphate buffer (50 mM; pH 6.5) containing 8.7% acetonitrile was used as the mobile phase at a flow rate of 1.0 mL/min at room temperature. Fluorescence detection was performed at excitation and emission wavelengths of 340 and 455 nm, respectively. After elution of the last analyte, strongly retained compounds were quickly eluted by a strong solvent flush with 30% acetonitrile. The intra- and inter-assay coefficients of variation for each of these latter measurements were <3.5%. 2.6. Plasma nitrate and nitrite (NOx ) assay Plasma nitrate-plus-nitrite, the metabolic end products of NO, were measured as described by Granger et al. [14]. The

J. Wang et al. / Atherosclerosis 184 (2006) 383–388

intra- and inter-assay coefficients of variation were 1.5 and 2.4%, respectively. 2.7. Statistical analysis In this study, we planned to examine the hypothesis that ADMA levels are higher in patients with triple vessel disease than in those with no significantly diseased vessels. Based on published data, we anticipate that at least a 10% difference in the ADMA levels between age-matched patients with, and without, coronary disease would carry biological significance [10,15]. In our preliminary analyses, we observed that variations or standard deviations in levels were approximately 20% of the mean levels. Taking α = 0.05, we estimated that 70 patients with triple vessel disease and 70 patients with no significantly diseased vessels would be needed to have 80% power to detect significant differences in ADMA levels between these two populations. The Kolmogorov–Smirnov test of normality was used to test the distribution of variables. We have presented normally distributed data as mean ± S.D. and skewed variables as median and interquartile ranges. We used an unpaired student t-test or a Mann–Whitney U-test to compare the difference between two groups, as appropriate. Categorical variables are presented by frequency of counts, and inter-group comparisons analysed by a χ2 analysis. ANOVA was used to compare the difference for more than two groups. When we analyzed the quantitative relationships between ADMA levels and other factors bivariate correlation coefficients were calculated, with Pearson’s for parametric

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and Spearman’s for non-parametric data. We used univariate ANOVA (linear regression analysis) to explore possible effects of smoking and GFR on ADMA levels. Two-tailed Pvalues are reported. Statistical analysis was performed using the statistical package SPSS for Windows, Version 12.1.

3. Results 3.1. Clinical characteristics and coronary risk factors The clinical characteristics of the patients studied are shown in Table 1. The patients with triple vessel disease were slightly older than the patients with no detectable coronary lesions (63 ± 7 years versus 60 ± 10 years); the BMI levels were not different. The patients with triple vessel disease, all of whom were receiving lipid lowering statin therapy, had lower total cholesterol levels (3.8 ± 0.8 mmol/L versus 4.2 ± 0.9 mmol/L, P = 0.006) and LDL levels (2.0 ± 0.8 mmol/L versus 2.4 ± 0.9 mmol/L, P = 0.009); but the triple vessel disease patients also had lower HDL levels (1.0 ± 0.3 mmol/L versus 1.2 ± 0.3 mmol/L, P = 0.002). Thus, there was no difference between patients with triple vessel disease and no significant vessel disease in the post-treatment total cholesterol/HDL ratio (3.9 ± 1.2 mmol/L versus 4.1 ± 2.1 mmol/L, P = 0.62), a more accurate marker for CAD risk [16]. Total homocysteine levels were not different between these two groups [median (interquartile) levels, 8.9 (7.6–11.3) ␮mol/L versus 8.6

Table 1 Clinical and biochemical characteristics in patients with and without angiographically documented coronary artery disease Zero vessel disease (n = 70)

Triple vessel disease (n = 75)

P-values

Age (years) Gender (male/female) BMI (kg/m2 ) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) TC/HDL-C ADMA (␮mol/L) SDMA (␮mol/L) l-Arginine (␮mol/L) l-Arginine/ADMA NOx (␮mol/L) tHCY (␮mol/L) Creatinine (␮mol/L) GFR (mL/min/1.73 m2 )

60 ± 10 42/28 27.4 ± 4.5 4.2 ± 0.9 1.2 (0.8–1.6) 1.2 ± 0.3 2.4 ± 0.9 4.1 ± 2.1 0.45 ± 0.07 0.37 (0.33–0.42) 64.3 ± 12.5 145.2 ± 38.1 37.8 (24.9–47.4) 8.6 (6.9–11.7) 84.5 (69.8–101.2) 102.2 (80.4–123.9)

63 ± 7 46/29 28.3 ± 5.0 3.8 ± 0.8 1.3 (1.0–1.8) 1.0 ± 0.3 2.0 ± 0.8 3.9 ± 1.2 0.44 ± 0.08 0.37 (0.31–0.44) 62.8 ± 17.3 146.2 ± 38.3 36.9 (24.2–51.4) 8.9 (7.6–11.3) 84.0 (65.8–104.0) 83.1 (65.6–99.5)

0.004∗ 0.52** 0.62∗ 0.006∗ 0.09*** 0.002∗ 0.009∗ 0.62∗ 0.72∗ 0.51*** 0.61∗ 0.8∗ 0.97*** 0.54*** 0.74*** <0.001***

Smoking status Never smoked Ex-smoker Current smoker

34 24 12

44 13 18

0.22** 0.02** 0.31**

Data are mean ± S.D., median (interquartile range), or frequency count, as appropriate. BMI, body mass index; TC, total cholesterol; TG, triglyceride; HDL-C, high density lipoprotein; LDL, low density lipoprotein; TC/HDL, total cholesterol/high density lipoprotein ratio; NOx , NO2 + NO3 ; tHCY, total homocysteine; GFR: glomerular filtration rate. * Student’s t-test. ** χ2 -test. *** Mann–Whitney U-test.

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Table 2 The effect of smoking on the plasma levels of ADMA, SDMA, tHcy, arginine, NOx and GFR

Non-smoker Current-smoker Ex-smoker P-value

N

ADMA (␮mol/L)

SDMA (␮mol/L)

Arginine (␮mol/L)

tHCY (␮mol/L)

NOx (␮mol/L)

GFR (mL/min/1.73 m2 )

78 30 37

0.43 ± 0.07∗ 0.47 ± 0.08∗ 0.45 ± 0.08 0.03

0.37 (0.31–0.43)** 0.38 (0.29–0.45)** 0.36 (0.33–0.42) 0.58

62.4 ± 15.1∗ 67.2 ± 17.1∗ 62.9 ± 13.2 0.15

8.4 (7.3–10.8)** 10.4 (7.3–13.1)** 9.6 (7.0–12.2) 0.2

37.0 (26.37–48.15)** 26.1 (16.8–46.14)** 43.5 (28.28–52.2) 0.03

87.5 (71.8–105.2)** 106.0 (74.1–120.9)** 90.4 (73.7–118.7) 0.09

ADMA, SDMA are the asymmetric and symmetric dimethylarginine, respectively; tHCY, total homocysteine; NOx , nitrate plus nitrite; GFR, glomerular filtration rate. * Student’s t-test. ** Mann–Whitney U-test.

(6.9–11.7) ␮mol/L, P = 0.54]. The patients with triple vessel disease had a significantly lower glomerular filtration rate (GFR) than the patients without coronary artery disease [median (interquartile) levels, 83.1 (65.6–99.5) mL/min/1.73 m2 versus 102.2 (80.4–123.9) mL/min/1.73 m2 , P < 0.001]. 3.2. Plasma ADMA and coronary artery disease severity The ADMA level was not different between patients with triple vessel disease and patients without coronary artery disease (0.44 ± 0.08 ␮mol/L versus 0.45 ± 0.07 ␮mol/L, P = 0.72). The SDMA level was also not different between these two groups [median (interquartile) levels, 0.37 (0.31–0.44) versus 0.37 (0.33–0.42), P = 0.51]. The plasma concentration of l-arginine was the same in the two groups (62.8 ± 17.3 ␮mol/L versus 64.3 ± 12.5 ␮mol/L, P = 0.61), as were the plasma levels of nitrate and nitrite [median (interquartile) levels 36.9 (24.2–51.4) ␮mol/L versus 37.8 (24.9–47.4) ␮mol/L, P = 0.97]. Not surprisingly, the l-arginine/ADMA ratio was also not related to the severity of coronary artery disease (146.2 ± 38.3145 versus 145.2 ± 38.1, P = 0.8). In order to control for confounding effects, we also analysed associations between the biochemical findings in the triple and zero vessel disease patients using a univariate ANOVA model in which smoking status, age and gender were entered as fixed factors. There was still no association between ADMA and coronary artery disease in these patients. 3.3. Smoking and plasma ADMA There were 30 current smokers, 37 ex-smokers and 78 non-smokers in the patients studied (Table 2). There was a modest association between smoking and increased ADMA levels (non-smoker versus current smoker, 0.43 ± 0.07 ␮mol/L versus 0.47 ± 0.08 ␮mol/L, P = 0.03) and lower levels of nitrate plus nitrite [non-smoker versus current smoker, median (interquartile) levels, 37.0 (26.3–48.1) versus 26.1 (16.8–46.1), P = 0.03]. The effect of smoking on ADMA was also found when patients with triple vessel disease were considered separately (current smoker versus non-smoker, 0.47 ± 0.08 ␮mol/L versus 0.42 ± 0.07 ␮mol/L, P = 0.03). There were similar trends for higher levels of ADMA and lower levels of nitrate plus nitrite in the

current smokers for both patient groups. With univariate ANOVA analysis in which ADMA or nitrate plus nitrite was a dependent variable, the effect of smoking on ADMA and nitrate plus nitrite levels was independent of age and gender, which were entered as covariates. The elevated ADMA in current smokers was independent of renal impairment. As shown in Table 2, GFR tended to be higher in the current smokers than in the non-smokers although it did not reach statistical significance [median (interquartile) levels, 106.0 (74.1–120.9) mL/min/1.73 m2 versus 87.5 (71.8–105.2) mL/min/1.73 m2 , P = 0.09]. With univariate ANOVA analysis in which log-GFR is the dependent variable and smoking status, age and gender as predictive factors, we found that age is significantly associated with the log-GFR (F = 34.109, P = 0.0001); however, smoking status and gender had no effect on log-GFR. The higher GFR in the current-smokers is related to their younger age (54 ± 12 years versus 64 ± 6 years, P < 0.001). And for all patients age was negatively correlated with GFR (r = −0.56, P < 0.01). 3.4. Renal function and plasma ADMA To explore the relation between plasma ADMA and renal function we assessed this in all the patients as a single group. The patients in the study all had plasma creatinine levels < 140 ␮mol/L and GFR > 45 mL/min/1.73m2 as we had excluded those with marked impairment of renal function. As shown in Table 3, there was a significant relationship between total homocysteine and glomerular filtration rate (r = −0.22, P = 0.005) for all the study subjects. There was a highly significant negative relation between SDMA and glomerular filtration rate (r = −0.45, P < 0.001) which is consistent with the excretion of SDMA by the kidney. Levels of both total homocysteine and SDMA were posiTable 3 The correlation between relevant variables in all the study subjects (n = 145)

Total homocysteine ADMA SDMA Creatinine

r r r r

ADMA

SDMA

GFR

0.25∗

0.48**

– – –

0.50** – 0.06

−0.22** −0.21∗ −0.45** −0.17∗

ADMA, SDMA are the asymmetric and symmetric dimethylarginine. * P < 0.05. ** P < 0.01.

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Table 4 The effect of renal impairment on plasma levels of ADMA, SDMA, tHCY, arginine and NOx

GFR < 81 mL/min/1.73 m2 GFR ≥ 81 mL/min/1.73 m2 P-value

N

ADMA (␮mol/L)

SDMA (␮mol/L)

tHCY (␮mol/L)

Arginine (␮mol/L)

NOx (␮mol/L)**

l-Arginine/ADMA

52 93

0.46 ± 0.07∗ 0.43 ± 0.07∗ 0.01

0.41 (0.37–0.49)** 0.34 (0.31–0.39)** <0.001

9.9 (7.7–13.4)** 8.4 (6.9–10.8)** 0.04

64.1 ± 16.2∗ 63.2 ± 14.6∗ 0.75

37.2 (25.2–52.3)** 37.3 (24.8–47.2)** 0.39

140.1 ± 41.7∗ 147.5 ± 35.1∗ 0.29

ADMA, SDMA are the asymmetric and symmetric dimethylarginine, respectively; tHCY, total homocysteine; NOx , nitrate plus nitrite. * Student’s t-test. ** Mann–Whitney U-test.

tively correlated with the level of ADMA (r = 0.25, P = 0.02 and r = 0. 5, P < 0.001, respectively). GFR was negatively correlated with the ADMA level (r = −0.21, P < 0.05). It has been shown that below 81 mL/min/1.73 m2 , each 10unit reduction in the GFR is associated with a 10% increase in the relative risk of death and nonfatal cardiovascular complications [3]. Among the 145 patients, there were 52 who had GFR < 81 mL/min/1.73 m2 and 93 who had GFR ≥ 81 mL/min/1.73 m2 . As shown in Table 4, patients with GFR < 81 mL/min/1.73 m2 had significantly higher ADMA (0.46 ± 0.07 ␮mol/L versus 0.43 ± 0.07 ␮mol/L, P = 0.01) and higher SDMA [median (interquartile) levels, 0.41 (0.37–0.49) ␮mol/L versus 0.34 (0.31–0.39) ␮mol/L, P < 0.001] and somewhat higher homocysteine [median (interquartile) levels, 9.9 (7.7–13.4) ␮mol/L versus 8.4 (6.9–10.8) ␮mol/L, P = 0.04]. With univariate ANOVA analysis, the effects of GFR on ADMA, SDMA and homocystine were independent of gender, age and smoking status. Those with triple vessel disease were more likely to have mild renal impairment than those with no detectable vessel disease (35/75 versus 17/70, P = 0.005). The levels of total cholesterol, LDL-C, triglyceride and HDL-C were not related to ADMA levels, but the total cholesterol/HDL ratio, a more accurate marker of lipid related coronary risk, was modestly correlated with the level of ADMA (r = 0.22, P < 0.01).

4. Discussion Whilst a number of studies have reported increased plasma ADMA levels in patients with CAD [17], this was not confirmed in a large Scandinavian study by Jonasson et al. [18]. Levels of ADMA were not elevated in their patients with ischaemic heart disease. They suggested that the previously reported increase in ADMA in coronary patients could be due to a concomitant modest impairment of renal function in patients who already have established ischemic heart disease. The present results are consistent with this conclusion, in that the elevations of ADMA were confined to the sub-group of patients with modest impairment of renal function. Our study found no difference overall in plasma ADMA levels between patients with angiographically documented severe triple vessel coronary artery disease and those with

no detectable coronary artery lesions. There was no difference between the two groups in the l-arginine/ADMA ratio which may be a more sensitive measure of NOS inhibition than plasma ADMA alone [19]. In support of these findings the plasma levels of nitrate plus nitrite in the two groups overall were also not different although there were differences between smokers and non-smokers. We concluded from these results that ADMA levels are not elevated in patients with severe coronary disease but preserved renal function. The findings of our study and those of the recent Scandinavian study [18] are inconsistent with results in the studies reviewed recently by Boger [17]. We suggest that undocumented impairment of renal function in some of these studies could be one of the reasons for the inconsistent findings. An interesting finding in our study was the effect of cigarette smoking on levels of ADMA and also on levels of nitrate plus nitrite. Smoking was associated with an increase in ADMA and a decrease in nitrate plus nitrite. Studies have shown that smokers have reduced eNOS expression and hence impaired endothelium-mediated vessel dilatation. However, although molecular mechanisms for this inhibition are not clear, excess production of free radicals generated by the cigarette smoking could be one mediator [20]. Our findings suggest that the inhibitory effect in smokers could be mediated by elevated ADMA levels. How smoking may influence ADMA metabolism requires further investigation. In this patient population, we also found that plasma levels of total homocysteine were not different in the two patient groups. This could be due to the fact that many of these patients were already taking oral vitamins. However, even within the narrow range of the total homocysteine levels, the levels of total homocysteine and ADMA were significantly correlated as was total homocysteine and renal function. Homocysteine is an inhibitor of the enzyme DDAH which is responsible for the metabolism of ADMA and these findings suggest that this may be occurring at only modest plasma levels of total homocysteine [21]. In summary, in the present study of patients with triple vessel coronary disease and normal or modestly impaired renal function we did not find an increase in ADMA or a decrease in the l-arginine/ADMA ratio when we compared the levels with those in patients of similar age who had no detectable coronary disease. However, we did find increased ADMA in smokers and also a positive association between

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