An early and critical event in the pathogenesis of cardiovascular disease (CVD) is endothelial (vasodilator) dysfunction. Normal endothelial function depends on adequate levels of nitric oxide (NO), which acts as a vasodilator, inhibits the excessive proliferation of vascular smooth muscle cells [
NO is synthesized from the amino acid L-arginine by a family of NO synthase enzymes (NOS). Asymmetric dimethylarginine (ADMA) acts as an inhibitor of NOS and thus decreases the synthesis and availability of NO. A high plasma level of ADMA is regarded as an independent predictor of CVD and is also associated with end stage renal disease [
Altered activity of the ADMA metabolizing enzymes, dimethylarginine dimethylaminohydrolase I and II (DDAH-I and DDAH-II), has been suggested as a possible cause for plasma ADMA accumulation. DDAH activity is directly downregulated by reactive oxygen species (ROS) generated by high glucose levels [
Studies have revealed altered DDAH activity through activation of peroxisome proliferator-activated receptor
n-3 PUFAs include the plant-derived
Studies investigating the association between n-3 PUFA and ADMA are scant and inconsistent. A randomized intervention trial, among men with long-standing hyperlipidemia, revealed no differences in ADMA levels after n-3 PUFA supplementation [
The aim of the present study was to investigate if n-3 PUFA influences the association between ADMA levels and risk of AMI in patients with coronary heart disease, hypothesizing that the relationship would be the strongest in patients with impaired n-3 PUFA status.
The Bergen coronary angiography cohort (BECAC) includes 3718 patients who underwent coronary angiography for suspected CAD during 2000–2004. The majority (92%) had stable angina. The present study included 1364 initial patients recruited to BECAC during 2000-2001. More than half of these patients (
Information about medical history, risk factors and medications were provided through a self-administered questionnaire completed by each patient as previously reported [
The participants were followed from angiography in 2000 or 2001 and until they experienced an acute AMI or throughout December 31, 2006.
Information on clinical events was collected from hospitals and from the Norwegian Cause of Death Registry. AMI definition, published in 2000 [
Serum samples were collected before angiography and stored at −80°C until analysis. Serum apolipoprotein A-I, apolipoprotein B, and lipoprotein (a) were measured on the Hitachi 917 system (Roche Diagnostics, GmbH, Mannheim, Germany). C-reactive protein (CRP) was determined using a latex, high sensitive assay (Behring Diagnostics, Marburg, Germany). Serum fatty acid methyl esters were extracted by treatment of serum with 2% (v/v) of sulfuric acid in methanol [
Continuous variables are presented as means (±SD) and categorical variables as counts (percentage). Mean trends over plasma ADMA quartiles were estimated using linear regression for continuous variables and logistic regression for binary variables.
Hazard ratios of AMI events over quartiles of plasma ADMA and for ADMA as a dichotomous variable (cutoff at 90th percentile) were estimated with Cox proportional hazard models. Nonlinear effects were additionally investigated with GAM plots using penalized smoothing splines for the functional form of the covariate [
All probability values are 2-tailed and were considered significant when <0.05. Statistical analyses were performed with SPSS 18 (SPSS Inc., Chicago, IL, USA) and R 2.14.2 (the R Foundation for Statistical Computing, Vienna, Austria).
Baseline characteristics of the 1364 participants, according to quartiles of plasma ADMA concentrations, are presented in Table
Baseline characteristics of participants by quartiles and in the upper decile of plasma ADMA concentration1.
Quartiles | Upper decile | |||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 | |||
0.46 (0.10, 0.50) | 0.54 (0.50, 0.59) | 0.63 (0.59, 0.70) | 0.80 (0.70, 1.71) |
|
0.89 (0.82, 1.71) | |
|
|
|
|
|
||
Male sex, |
287 (83.9) | 261 (77.0) | 249 (72.8) | 222 (65.1) | <0.001 | 90 (66.2) |
Age (years), mean (±SD) | 58 (±9.7) | 61 (±9.7) | 62 (±10.7) | 64 (±11.0) | <0.001 | 65.5 (±11.2) |
BMI (kg/m2), mean (±SD) | 27.1 (±3.55) | 26.6 (±3.51) | 26.4 (±4.15) | 26.5 (±3.84) | 0.02 | 26.3 (±4.08) |
Fasting, |
44 (14.2) | 58 (18.5) | 53 (16.0) | 32 (9.5) | 0.05 | 10 (7.4) |
| ||||||
Cardiovascular history, |
||||||
| ||||||
Previous AMI | 130 (38.0) | 143 (42.2) | 130 (38.0) | 150 (44.0) | 0.20 | 62 (45.6) |
Previous CBV | 12 (3.5) | 18 (5.3) | 28 (8.2) | 28 (8.2) | 0.11 | 15 (11.0) |
Previous PVD | 30 (8.8) | 25 (7.4) | 34 (9.9) | 47 (13.8) | 0.09 | 23 (16.9) |
Previous PCI | 80 (23.4) | 63 (18.6) | 43 (12.6) | 53 (15.5) | 0.01 | 22 (16.2) |
Previous CABG | 32 (9.4) | 41 (12.1) | 36 (10.5) | 25 (7.3) | 0.05 | 14 (10.3) |
| ||||||
Cardiovascular risk factors, |
||||||
| ||||||
Hypercholesterolemia4 | 212 (65.2) | 199 (61.8) | 189 (58.2) | 155 (49.8) | <0.001 | 48 (40.0) |
Hypertension | 154 (45.0) | 159 (46.9) | 163 (47.7) | 174 (51.0) | 0.87 | 71 (52.2) |
Impaired LVEF5 | 40 (11.7) | 31 (9.1) | 35 (10.2) | 45 (13.2) | 0.45 | 18 (13.2) |
Diabetes6 | 41 (12.0) | 38 (11.2) | 29 (8.5) | 32 (9.4) | 0.04 | 12 (8.8) |
Current smoker | 118 (34.5) | 113 (33.3) | 123 (36.0) | 103 (30.2) | 0.22 | 41 (30.1) |
Ex-smoker | 249 (72.8) | 254 (74.9) | 251 (73.6) | 267 (78.3) | 0.86 | 58 (42.6) |
Never smoked | 74 (22.5) | 94 (27.7) | 78 (22.9) | 101 (29.6) | 0.31 | 37 (27.2) |
Family history of CAD7 | 123 (36.4) | 112 (33.3) | 114 (34.0) | 86 (25.8) | 0.02 | 34 (25.4) |
| ||||||
Clinical diagnosis before BCA, | ||||||
| ||||||
Stable angina pectoris | 288 (84.2) | 318 (93.8) | 330 (96.5) | 337 (98.8) | <0.001 | 135 (99.3) |
Acute coronary syndrome | 54 (15.8) | 21 (6.2) | 12 (3.5) | 4 (1.2) | <0.001 | 1 (0.7) |
| ||||||
Extent of CAD at BCA, | ||||||
| ||||||
No significant CAD | 15 (4.4) | 8 (2.4) | 55 (16.1) | 84 (24.6) | <0.001 | 33 (24.3) |
1 vessel disease | 112 (32.7) | 118 (34.8) | 83 (24.3) | 55 (16.1) | <0.001 | 16 (11.8) |
2 vessel disease | 107 (31.3) | 99 (29.2) | 88 (25.7) | 65 (19.1) | <0.001 | 25 (18.4) |
3 vessel disease | 98 (28.7) | 102 (30.1) | 97 (28.4) | 106 (31.1) | 0.29 | 51 (37.5) |
| ||||||
Medication following BCA, | ||||||
| ||||||
Acetylsalicylic acid | 318 (93.0) | 314 (92.6) | 277 (81.0) | 266 (78.0) | <0.001 | 106 (77.9) |
Statins | 306 (89.5) | 299 (88.2) | 264 (77.2) | 238 (69.8) | <0.001 | 83 (61.0) |
|
270 (79.2) | 271 (79.9) | 241 (70.5) | 240 (70.6) | 0.001 | 89 (65.4) |
ADP receptor blocker | 132 (38.6) | 97 (28.6) | 56 (16.4) | 37 (10.9) | <0.001 | 15 (11.0) |
Anticoagulants (warfarin) | 4 (1.2) | 8 (2.4) | 21 (6.1) | 23 (6.7) | <0.001 | 5 (3.7) |
ACE inhibitors | 61 (17.8) | 64 (18.9) | 62 (18.1) | 85 (24.9) | 0.08 | 43 (31.6) |
Angiotensin II receptor antagonist | 41 (12.0) | 37 (10.9) | 22 (6.4) | 34 (10.0) | 0.06 | 13 (9.6) |
Loop diuretics | 22 (6.4) | 29 (8.6) | 34 (9.9) | 60 (17.6) | 0.001 | 28 (20.6) |
| ||||||
CR following BCA, | ||||||
| ||||||
PCI | 214 (62.6) | 193 (56.9) | 140 (40.9) | 91 (26.7) | <0.001 | 37 (27.2) |
CABG | 51 (14.9) | 53 (15.6) | 61 (17.8) | 64 (18.8) | 0.34 | 29 (21.3) |
ACE: angiotensin converting enzyme; ADP: adenosine diphosphate; BCA: baseline coronary angiography; BMI: body mass index; CABG: coronary artery bypass graft surgery; CAD: coronary artery disease; CBV: cerebrovascular disease; CR: coronary revascularization; LVEF: left ventricular ejection fraction; PCI: percutaneous coronary intervention; PVD: peripheral vascular disease.
1Median (range) plasma ADMA concentrations (
2
3Not having ingested any food 6 hours prior to blood samples were collected.
4≥6.5 mmol/L.
5<50%.
6Includes diabetes type 1 and 2.
7Includes those reporting to have at least one 1st degree relative suffering from CAD before the age of 55 for men and 65 for women.
Patients with high ADMA levels were less likely to have been treated with PCI, having hypercholesterolemia, DM, or family history of CAD. Patients with low ADMA were more often diagnosed with ACS and significant CAD at angiography. However, the prevalence of 3-vessel disease did not differ across ADMA quartiles.
Because the majority was diagnosed with significant CAD, most patients were discharged with various medications. Antiplatelet therapy (acetylsalicylic acid and ADP receptor blockers), statins, and
FA and biochemical markers, relevant for CAD, by quartiles of ADMA are presented in Table
Serum fatty acids and biochemical markers by ADMA quartiles and in the upper decile of plasma ADMA concentration1.
Quartiles | Upper decile | |||||
---|---|---|---|---|---|---|
1 | 2 | 3 | 4 |
|
||
0.46 (0.10, 0.50) |
0.54 (0.50, 0.59) |
0.63 (0.59, 0.70) |
0.80 (0.70, 1.71) |
0.89 (0.82, 1.71) |
||
Fatty acids | ||||||
| ||||||
TFAs (mg/L) | 4277 (4087, 4467) | 4024 (3839, 4209) | 4115 (3932, 4297) | 4373 (4192, 4554) | 0.73 | 4265 (3980, 4550) |
Total n-3 PUFA (wt%)3 | 7.57 (7.23, 7.91) | 7.29 (7.00, 7.59) | 7.70 (7.38, 8.01) | 7.82 (7.51, 8.13) | 0.55 | 7.23 (6.74, 7.73) |
ALA (wt%) | 0.73 (0.71, 0.76) | 0.72 (0.70, 0.74) | 0.74 (0.72, 0.76) | 0.75 (0.73, 0.78) | 0.74 | 0.77 (0.73, 0.80) |
n-3 LCPUFA (wt%)4 | 6.90 (6.57, 7.23) | 6.52 (6.19, 6.84) | 6.86 (6.54, 7.18) | 6.84 (6.52, 7.15) | 0.54 | 6.47 (5.97, 6.97) |
| ||||||
Lipid related parameters | ||||||
| ||||||
ApoA1 (g/L) | 1.36 (1.33, 1.39) | 1.35 (1.32, 1.38) | 1.36 (1.33, 1.39) | 1.36 (1.34, 1.39) | 0.51 | 1.33 (1.29, 1.37) |
ApoB (g/L) | 0.94 (0.91, 0.97) | 0.91 (0.88, 0.94) | 0.95 (0.92, 0.97) | 0.95 (0.92, 0.98) | 0.59 | 0.93 (0.89, 0.97) |
Total Ch. (mmol/L) | 5.26 (5.13, 5.40) | 5.10 (4.96, 5.23) | 5.29 (5.16, 5.42) | 5.31 (5.17, 5.44) | 0.58 | 5.17 (4.96, 5.38) |
LDL Ch. (mmol/L) | 3.19 (3.07, 3.30) | 3.14 (3.02, 3.25) | 3.32 (3.20, 3.43) | 3.30 (3.19, 3.41) | 0.71 | 3.23 (3.05, 3.40) |
HDL Ch. (mmol/L) | 1.30 (1.26, 1.34) | 1.28 (1.24, 1.32) | 1.32 (1.28, 1.36) | 1.33 (1.29, 1.37) | 0.41 | 1.28 (1.21, 1.34) |
Non HDL (mmol/L) | 1.30 (1.26, 1.34) | 1.28 (1.24, 1.32) | 1.32 (1.28, 1.36) | 1.33 (1.29, 1.37) | 0.42 | 3.89 (3.68, 4.10) |
TG (mmol/L) | 1.96 (1.80, 2.12) | 1.73 (1.58, 1.89) | 1.66 (1.51, 1.81) | 1.77 (1.62, 1.92) | 0.06 | 1.72 (1.49, 1.96) |
Lp(a) (mmol/L) | 0.37 (0.33, 0.41) | 0.37 (0.33, 0.41) | 0.37 (0.33, 0.41) | 0.39 (0.35, 0.43) | 0.23 | 0.40 (0.39, 0.47) |
| ||||||
Other parameters | ||||||
| ||||||
Glucose (mmol/L) | 6.46 (6.18, 6.74) | 6.27 (6.00, 6.55) | 6.23 (5.96, 6.49) | 6.22 (5.95, 6.48) | 0.15 | 6.28 (5.87, 6.70) |
HbA1c (mmol/L) | 5.97 (5.82, 6.12) | 5.79 (5.64, 5.93) | 5.91 (5.77, 6.05) | 6.40 (6.25, 6.54) | <0.001 | 6.56 (6.33, 6.78) |
Arginine ( |
76.5 (73.8, 79.2) | 80.9 (78.3, 83.5) | 70.6 (68.0, 73.1) | 53.6 (51.1, 56.2) | <0.001 | 49.9 (45.7, 54.0) |
Creatinine ( |
85.4 (83.8, 89.4) | 86.6 (83.8, 89.4) | 88.3 (85.6, 91.0) | 95.2 (92.5, 97.5) | <0.001 | 104.0 (99.8, 108.2) |
GFR (mL/min) | 90.3 (88.8, 91.7) | 88.7 (87.2, 90.1) | 87.1 (85.6, 88.5) | 82.6 (81.2, 84.0) | <0.001 | 78.0 (75.8, 80.3) |
CRP (mg/L) | 6.33 (5.19, 7.48) | 4.10 (2.98, 5.22) | 3.75 (2.65, 4.85) | 4.09 (3.00, 5.19) | 0.99 | 3.72 (2.00, 5.44) |
ALA:
1Median (range) plasma ADMA concentrations (
2
3Combination of ALA, EPA, DPA, and DHA.
4Combination of EPA, DPA, and DHA.
During the follow-up period (mean 63 (SD 20) months), a total of 129 patients experienced an AMI, of which 44 were fatal. The relationship between ADMA levels and subsequent risk of AMI after angiography was evaluated across ADMA quartiles using lower quartile 1 as reference, and for the upper decile compared to ADMA below the upper decile.
ACS and extent of CAD were strongly associated with ADMA and were included in a multivariate adjusted survival model together with other important risk factors for AMI. Hazard ratios (HR (95% CI)) for AMI according to ADMA levels are presented in Table
Risk of acute myocardial infarction across quartiles and upper decile of ADMA.
Model | Quartiles | Upper decile | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
2 | 3 | 4 |
|
|||||||
HR | 95% CI | HR | 95% CI | HR | 95% CI | HR | 95% CI |
|
||
Univariate | 1.26 | (0.75, 2.12) | 1.23 | (0.73, 2.07) | 1.47 | (0.89, 2.42) | 0.16 | 2.24 | (1.45, 3.47) | <0.001 |
Sex, age adjusted | 1.22 | (0.72, 2.07) | 1.16 | (0.69, 1.96) | 1.35 | (0.80, 2.25) | 0.32 | 2.06 | (1.33, 3.21) | 0.001 |
Multivariate adjusted1 | 1.22 | (0.72, 2.07) | 1.27 | (0.74, 2.18) | 1.44 | (0.84, 2.47) | 0.20 | 2.11 | (1.34, 3.32) | 0.001 |
HR: hazard ratio; CI: confidence interval.
Hazard ratios for the quartile groups are compared to first quartile; hazard ratio for plasma ADMA levels > 90th percentile is compared to plasma ADMA levels < 90th percentile.
1The model includes age (continuous), sex, acute coronary syndrome (yes/no), diabetes mellitus (yes/no), hypertension (yes/no), current smoking (yes/no), extend of coronary artery disease (0–3), and left ventricular ejection fraction (continuous).
Possible effect modifications of n-3 PUFA on the relationship between ADMA and risk of AMI were evaluated by repeating the survival analyses after stratifying the study population according to median levels of TFA concentration or wt% of ALA, n-3 LCPUFA (Table
Risk of acute myocardial infarction for the upper decile of ADMA in strata of TFAs and n-3 PUFA.
Fatty acids | Below median | Above median |
|
---|---|---|---|
HR (95% CI) | HR (95% CI) | ||
TFAs (mg/L) | |||
Model 12 | 2.60 (1.41, 4.80) | 1.67 (0.83, 3.36) | 0.29 |
Model 23 | 2.57 (1.25, 5.29) | 1.49 (0.56, 3.93) | 0.35 |
Total n-3 PUFA (wt%)4 | |||
Model 1 | 1.89 (0.98, 3.63) | 2.25 (1.17, 4.34) | 0.72 |
Model 2 | 2.36 (1.05, 5.33) | 1.97 (0.91, 4.30) | 0.99 |
ALA (wt%) | |||
Model 1 | 3.12 (1.64, 5.93) | 1.49 (0.77, 2.88) | 0.07 |
Model 2 | 2.42 (1.13, 5.16) | 1.57 (0.69, 3.55) | 0.11 |
n-3 LCPUFA (wt%)5 | |||
Model 1 | 2.05 (1.08, 3.89) | 2.11 (1.08, 4.15) | 0.96 |
Model 2 | 2.81 (1.28, 6.16) | 1.74 (0.78, 3.90) | 0.78 |
ALA:
1
2Model 1: hazard ratios of acute myocardial infarction for plasma ADMA > 90th percentile with plasma ADMA levels < 90th percentile as reference. The model included age (continuous), sex, acute coronary syndrome (yes/no), diabetes mellitus (yes/no), hypertension (yes/no), current smoking (yes/no), extend of coronary artery disease (0–3), left ventricular ejection fraction (continuous).
3Model 2: hazard ratios of acute myocardial infarction for plasma ADMA levels > 90th percentile with plasma ADMA levels < 90th percentile as reference. The model included age (continuous), sex, acute coronary syndrome (yes/no), diabetes mellitus (yes/no), hypertension (yes/no), current smoking (yes/no), extend of coronary artery disease (0–3), left ventricular ejection fraction (continuous), hypercholesterolemia (yes/no), HbA1c (continuous), and glomerular filtration rate (continuous).
4Combination of ALA, EPA, DPA, and DHA.
5Combination of EPA, DPA, and DHA.
Association between plasma ADMA levels (
In this prospective cohort study, we identified plasma ADMA levels in the upper decile to be moderately associated with risk of AMI. No serum n-3 PUFA was related to plasma ADMA concentration. However, the risk of AMI associated with elevated ADMA was particularly strong among patients with ALA concentration below median, whereas similar effect modification for n-3 LCPUFA was only observed after additional adjustment.
Plasma ADMA levels in healthy individuals appears to lie in the range of 0.4–0.6
An experimental animal study demonstrated that plasma ADMA levels were reduced by EPA and DHA supplementation [
Previous studies have revealed a positive relation between serum glucose levels and ADMA [
A high level of total cholesterol is associated with increased production of oxLDL which has the potential to inhibit ADMA degradation [
Reduced renal function is associated with elevated plasma ADMA levels. Adding eGFR to our multivariate survival model strengthened the association between ADMA and AMI in patients with below median levels of n-3 LCPUFA.
FAs of the n-3 PUFA family have anti-inflammatory properties [
This study is based on a large, well-characterized population with complete followup with respect to clinical endpoints. However, despite the clear differences in risk associations observed, even this cohort was too small to demonstrate significant effect modification. Limitations also include the single baseline measurement of FAs and biomarkers, which may have introduced underestimated associations (regression dilution bias) [
The association between plasma ADMA and risk of AMI was influenced by serum n-3 PUFA and primarily ALA. Additional research is needed to further elucidate the clinical implications of these findings and whether the relationship between ADMA and AMI is modified by other FAs.
The authors thank all WENBIT coworkers at Haukeland and Stavanger University Hospitals. The authors also thank the staff at the Department of Nutrition, University of Oslo, for help with extracting the dietary data. They are grateful to Liv Kristine Øysæd, Kari Helland Mortensen, Randi Sandvik, and Marte Aanestad for excellent technical assistance during FA composition analyses, and Gry Kvalheim and her staff at BEVITAL AS for the analyses of ADMA and HbA1C.