Targeted Metabolomic Evaluation of Arginine Methylation and Cardiovascular Risks
Potential Mechanisms Beyond Nitric Oxide Synthase Inhibition
Background— We examine the relationship of related posttranslational modification products of arginine methylation and coronary artery disease (CAD) phenotypes.
Methods and Results— Plasma was isolated from 1011 consecutive consenting subjects undergoing elective diagnostic cardiac catheterization, and future major adverse cardiac events (MACE, including myocardial infarction, stroke, and death) at 3 years were investigated. Plasma levels of asymmetrical dimethylarginine (ADMA, endogenous nitric oxide synthase [NOS] inhibitor), symmetrical dimethylarginine (SDMA, which lacks NOS inhibitory activity), N-mono-methylarginine (MMA, a potent NOS inhibitor), methyl-lysine (Methyl-Lys, an unrelated methylated amino acid), arginine, and its major catabolites (citrulline and ornithine) were quantified simultaneously by stable isotope dilution HPLC with online electrospray ionization tandem mass spectrometry and adjusted for traditional risk factors, C-reactive protein, and estimated creatinine clearance. High SDMA levels (adjusted odds ratio [OR] 1.6, 95%CI, 1.1 to 2.6, P<0.001), low MMA (adjusted OR 0.5, 95%CI 0.4 to 0.8, P=0.007), but not ADMA (adjusted OR 1.3, 95%CI 0.88 to 2.0, P=0.177) were associated with increased prevalence of significantly obstructive CAD. Elevated levels of SDMA (adjusted Hazard Ratio [HR] 2.4, 95%CI 1.2 to 4.6, P=0.009), ADMA (adjusted HR 2.2, 95%CI 1.2 to 4.0, P=0.015), as well as an integrated index of arginine methylation [ArgMI=(ADMA+SDMA)/MMA] (adjusted HR 2.4, 95%CI 1.3 to 4.5, P=0.006) were significant independent predictors of incident MACE. ArgMI was predictive of incident MACE even following adjustments for global arginine bioavailability, particularly within secondary prevention patients.
Conclusions— ADMA, SDMA, and the integrated quantification of arginine methylation (in the form of a methylation index) provided independent risk prediction for both significantly obstructive CAD and incident MACE in stable patients undergoing cardiac evaluation. These results suggest that factors beyond direct NOS inhibition contribute to the clinical associations between methylarginines and CAD outcomes.
Nitric oxide (NO), produced by the oxidation of arginine by NO synthases of endothelial cells, plays an important antiatherogenic role in the development of cardiovascular disease. NO promotes many beneficial effects in the vasculature, including vasodilation, enhanced fibrinolysis, and inhibition of multiple atherothrombotic biological processes including platelet aggregation, leukocyte adhesion, endothelin generation, and smooth muscle cell proliferation.1 Asymmetrical dimethylarginine (ADMA) has been demonstrated in a variety of clinical settings to serve as an independent risk factor for long-term adverse cardiovascular events.2–7 The mechanistic explanation has been attributed to its role as an endogenous inhibitor of nitric oxide synthase (NOS), reducing the production of NO thereby diminishing vascular reactivity and leading to endothelial dysfunction and vasculopathy.8
Recent identification of a family of alternative methyl-amino acid derivatives generated by related methylation pathways have provided insight into the overall protein methylation pathways that generate ADMA and other derivatives that may possess similar biological activities.9 For example, N-mono-methylarginine (MMA), the immediate precursor to ADMA, is a more potent albeit less abundant endogenous NOS inhibitor. Symmetrical dimethylarginine (SDMA), an alternative methylation product of MMA and a stereoisomer of ADMA, lacks NOS inhibitory activity, but like ADMA, is a weak inhibitor of arginine transporters.10 These derivatives are released from the methylated protein arginines by proteolysis (Figure 1). In contrast, methylation of other amino acids such as the production of methyl-lysine occurs via alternative methyltransferases and has no known relationship with arginine-NO metabolic pathways. The methyltransferases responsible for arginine methylation, the proteases involved in liberation of the free methylargnine derivatives, and the catabolic dimethylarginine dimethylaminohydrolases involved in metabolism of ADMA are all apparently influenced by conditions associated with inflammation and oxidative stress,11 raising the possibility that some of the observed associations between ADMA levels and cardiovascular disease may occur in part via mechanisms independent of endogenous NOS inhibition. Apart from ADMA, the relationship between other methylated arginine metabolites and prevalence of significantly obstructive coronary artery disease (CAD) and incident adverse cardiac events such as myocardial infarction (MI), stroke, and death, as well as their interactions with measures of global arginine bioavailability (previously identified as predictive of adverse long-term outcomes in other cohorts9,12,13) have not yet been explored. Herein, by simultaneous measurements of multiple amino acid methylation derivatives, we sought to examine the prognostic value of ADMA relative to SDMA, MMA, as well as the unrelated methylated amino acid methyl-lysine (Methyl-Lys) in patients undergoing evaluation for coronary artery disease.
All plasma specimens were obtained from subjects who were prospectively enrolled in GeneBank, a large (n=10 000) and well-characterized clinical repository with clinical and longitudinal data comprised from consenting subjects undergoing elective diagnostic cardiac catheterization at the Cleveland Clinic. All GeneBank participants gave written informed consent, and the Institutional Review Board of the Cleveland Clinic approved the study protocol. For our study, we evaluated 1011 consecutive consented patients, with clinical and demographic information collected at the time of catheterization and for whom long-term (3-year) incident outcome data were available. The Framingham Risk Score was calculated for each subject. An estimate of creatinine clearance (CrCl) was calculated using the Cockcroft–Gault equation.14 High sensitivity C-reactive protein (hsCRP), creatinine, and fasting blood glucose and lipid profiles were measured on the Abbott ARCHITECT platform (Abbott Diagnostics). Adjudicated outcomes were ascertained over the ensuing 3 years for all subjects after enrollment.
End Point Definitions
Significantly obstructive CAD was defined as any clinical history of myocardial infarction (MI), percutaneous coronary intervention, coronary artery bypass surgery, or angiographic evidence of CAD (≥50% stenosis) in 1 or more major coronary arteries. Major adverse cardiovascular event (MACE) was defined as death, nonfatal myocardial infarction, or nonfatal cerebrovascular accident.
Measurements of Amino Acids
Plasma analyzed was isolated from fasting whole blood collected in EDTA tubes that had been maintained at 4°C immediately after phlebotomy, processed typically within 2 hours (4 hours max) of blood draw, and stored at −80°C until use. Arginine and the arginine metabolites ornithine, citrulline, MMA, ADMA, and SDMA were quantified in plasma by stable-isotope-dilution HPLC with online tandem mass spectrometry. Briefly, [13C6] arginine (10 μmol/L final) was initially added to plasma as internal standard and proteins then precipitated by addition of 4 volumes of methanol. Supernatant (20 μL) was injected onto a Phenyl column (4.6×250 mm, 5 μm Rexchrom Phenyl; Regis) at a flow rate of 0.8 mL/min. Separation was performed using a gradient starting from 10 mmol/L ammonium formate aqueous solution over 0.5 minutes, then linearly to 25% methanol containing 0.1% formic acid and 5 mmol/L ammonium formate over 3 minutes, followed by isocratic run with the same solvent composition for 15 minutes. The HPLC column effluent was introduced into an API 365 triple quadrupole mass spectrometer with Ionics EP 10+ upgrade (Concord) interfaced to a Cohesive Technologies Aria LX Series HPLC multiplexing system. Analyses were performed using electrospray ionization in positive-ion mode with multiple reactions monitoring of parent and characteristic daughter ions specific for components monitored. The transitions monitored were mass-to-charge ratio (m/z): m/z 133.2→70.2 for ornithine; m/z 175.1→70.0 for arginine; m/z 176.1→70.1 for citrulline; m/z 189.3→70.1 for MMA; m/z 203.2 →70.3 for SDMA and ADMA; and m/z 181→74 for [13C6]Arg. The calibration curves for quantification of ornithine, arginine, citrulline, MMA, ADMA, and SDMA were prepared by spiking different concentrations of each individual analyte to control plasma. All analytes were baseline resolved and showed unique retention times. A S/N of 3 was used as minimal for limit of detection. For ornithine, arginine, citrulline, MMA, SDMA, and ADMA, the interassay CVs (%) were 12.1, 4.4, 6.3, 6.7, 6.5, 5.5 and the intraassay CVs (%) were 4.8, 3.3, 8.6, 8.3, 5.5, 6.7, respectively. Assay performance characteristics included average spike and recovery of 94% (range from 84% to 110%) for all analytes monitored in plasma matrix, and assay precision of <10% across all concentration ranges monitored for all analytes under the assay conditions used.
Separate analyses examined the methylation index for arginine (ArgMI), an integrated quantification of products generated from the arginine methylation pathways, and is estimated by the ratio of the known dimethylated arginine posttranslational modifications (ADMA+SDMA) to the immediate mono-methylated precursor, MMA [ie, ArgMI=(ADMA+SDMA)/MMA]. Global arginine bioavailability ratio (GABR) has previously been defined as the ratio of substrates (arginine) and catabolic products (ornithine and citrulline) of arginine metabolism.9,12,13
Summary data were presented in mean±SD for parametric variables or median (interquartile ranges) for nonparametric variables. The Student t test or Wilcoxon Rank sum test for continuous variables and χ2 test for categorical variables were used to examine the difference between the groups. Levels of MMA, SDMA, ADMA, methyl-Lys, and ArgMI were divided into quartiles for analyses. Unadjusted trends for all-cause mortality rates as well as nonfatal MI/stroke rates with increasing quartiles of methylated derivatives were evaluated with the Cochran-Armitage trend test. Logistic regression models were developed to calculate odds ratios (ORs) and 95% confidence intervals (95%CI) of the prevalence of coronary artery disease for the second, third, and the highest quartiles of analytes (or ArgMI) compared with the lowest quartile. Adjustments were made for individual traditional cardiac risk factor or Framingham Risk Score, log-transformed hsCRP, and CrCl to predict incident 3-year MACE risks. Kaplan–Meier analysis with Cox proportional hazards regression was used for time-to-event analysis to determine Hazard ratio (HR) and 95% confidence intervals (95%CI) for MACE. Levels of analytes were then adjusted for traditional CAD risk factors in a multivariable model including Framingham Risk Score, CrCl, and log-transformed hsCRP, as well as incorporating GABR into the model.
To determine whether ArgMI has an additional predictive value compared to a model including Framingham Risk Score, we calculated the concordance indexes (C statistic) with and without ArgMI in separate multivariable models. The improvement in predictability was assessed by calculating the difference in the C statistics. Bootstrapping was used to generate 95% CI. A 1-sample t test was performed to determine whether the difference was equal to zero. We defined primary and secondary prevention cohorts as those with and without the following characteristics, respectively: history of significantly obstructive CAD by coronary angiography, history of coronary revascularization (surgical or percutaneous), or history of MI or stroke.
All analyses were performed using SAS version 8.2. Probability values <0.05 were considered statistically significant.
Table 1 illustrates the baseline characteristics of our study population, which included 608 subjects with significantly obstructive CAD classified according to our end point criteria at time of enrollment. As expected, subjects with significantly obstructive CAD were older, more likely to have diabetes and hypertension, and have lower CrCl, though well within the normal range. Patients with significantly obstructive CAD had higher levels of hsCRP, triglyceride, and lower levels of (HDL) cholesterol compared to those without significantly obstructive CAD (Table 1).
Methylation Derivatives of Amino Acids and Prevalence of Coronary Artery Disease
Patients with significantly obstructive CAD had significantly higher median plasma levels of ADMA (1.09 μmol/L versus 0.97 μmol/L, P=0.003), SDMA (0.71 μmol/L versus 0.59 μmol/L, P<0.001), and lower levels of MMA (0.06 μmol/L versus 0.07 μmol/L, P=0.002) compared to those without significantly obstructive CAD (Table 1). In contrast, there was no difference between the 2 groups in the level of methyl-lysine (2.21 μmol/L versus 2.17 μmol/L, P=0.45).
For further analysis, ADMA, SDMA, MMA, and methyl-lysine were divided into quartiles. Unadjusted odds ratio for increasing ADMA and SDMA quartiles were associated with significantly obstructive CAD (Table 2). In contrast, unadjusted odds ratios for increasing MMA quartiles were paradoxically associated with lower prevalence of CAD compared to the lowest quartile (Table 2). In view of the opposite trends of association among products (positive for ADMA, SDMA) and precursors (negative for MMA) of arginine methylation with prevalence of significantly obstructive CAD, we further examined the relationship between increasing quartiles of the Arginine Methylation Index (ArgMI, defined as [ADMA+SDMA]/MMA) and significantly obstructive CAD in the cohort. Interestingly, among the methylated arginine parameters examined (ie, ADMA, SDMA, MMA, and ArgMI), the integrated methylation index, ArgMI, remained the most robust independent predictor of significantly obstructive CAD status after adjustments for traditional risk factors, renal function, and hsCRP, demonstrating odds ratios of (adjusted OR [95% CI]: 1.63 [1.09 to 2.46], P=0.019) for the third quartile and (adjusted OR [95% CI]: 2.64 [1.71 to 4.08], P<0.001) for fourth quartile (Table 2). Interestingly, although SDMA remained a significant predictor of significantly obstructive CAD status after adjustments (adjusted OR[95%CI]: 1.64 [1.06 to 2.56]), ADMA no longer remained significantly associated with CAD status (adjusted OR[95%CI]: 1.33 [0.88 to 2.03]; Table 2).
Methylation Derivatives of Amino Acids and Incident Cardiovascular Risks
Among the 1011 subjects, there were 56 patients lost to follow up. Of the remaining 955 patients, 824 patients had no MACE (nonfatal MI, stroke or death) over the 3 years after GeneBank enrollment, whereas 131 patients experienced at least 1 MACE. Patients who experienced MACE were older and more likely to have a history of diabetes and hypertension. Patients with risk for MACE also had increased CRP level, higher triglyceride, lower HDL, lower CrCl, and higher Framingham Risk Score. Plasma levels of ADMA (1.27 [0.95, 1.69] versus 0.97 [0.76, 1.40] μmol/L, P<0.001), SDMA (0.89 [0.60, 1.35] versus 0.63 [0.46, 0.93] μmol/L, P<0.001), and ArgMI (33.3 [25.3, 53.9] versus 24.4 [19.0, 40.0], P<0.001) were significantly higher in patients who experienced a MACE over the ensuing 3 years after enrollment compared with those who did not experience a MACE. In contrast, the alternative methylated amino acid methyl-lysine (2.11 [1.40, 4.76] versus 2.18 [1.36, 5.56], P=0.37) and MMA (0.07 [0.05, 0.08] versus 0.06 [0.05, 0.08], P=0.95) levels were not significantly different between the 2 groups.
The analytes were divided into quartiles for further analyses of risk prediction, and the rates of nonfatal MI or stroke, all-cause mortality, as well as the composite MACE, are presented in Figure 2. Interestingly, although a trend between increasing quartiles of ADMA levels and incident rates of MI or stroke failed to reach significance, systemic levels of SDMA were significantly associated with incident MI or stroke frequency (Figure 2). In particular, there was statistically significant increased unadjusted risk for all-cause mortality at 3 years across increasing quartiles of SDMA (HR [95% CI]: 5.3 [2.5 to 11.3]) and ADMA (HR [95% CI]: 5.3 [2.3 to 11.8]). In contrast, there was no association between quartiles with all-cause mortality or MACE for either MMA (P trend=0.88) or methyl-lysine (P trend=0.75). Also with each increasing quartile of SDMA (HR [95% CI]: 1.63 [1.4 to 1.9]) and ADMA (HR [95% CI]: 1.46 [1.2 to 1.7]), there was statistically significant increased unadjusted risk for future MACE.
Kaplan–Meier survival curves for each analyte and prospective MACE are shown in Figures 3 and ••. Dose-dependent increases in systemic levels of both ADMA and SDMA within subjects were strongly associated with increased risk for experiencing a MACE over the 3-year monitoring period after enrollment (Table 2). After adjusting for Framingham Risk Score, hsCRP, and renal function (CrCl), subjects with higher levels of ArgMI (third quartile HR [95% CI]: 2.5[1.3 to 4.7], P=0.005; fourth quartile HR [95% CI]: 2.4[1.3 to 4.5], P=0.006; all compared to the first quartile) showed the greatest risk for experiencing an incident MACE over the ensuing 3 year period. Moreover, C-statistic analyses demonstrated significant incremental prognostic value in the highest ArgMIquartile plus risk factors for prediction of incident 3-year MACE risk compared to that of risk factors alone (AUC 0.78 versus 0.55, P<0.001).
Several recent studies have reported that the Global Arginine Bioavailability ratio (GABR, defined as [Ornithine+Citrulline]/Arginine) shows prognostic value for prediction of incident risk for major adverse cardiac events.9,12,13 To gain a better appreciation of the relationship between the targeted arginine metabolome and cardiovascular disease within the same cohort, we examined the prognostic value of various arginine methylation products as comprehensively monitored in the ArgMI and the GABR. ArgMI and GABR each retained independent prognostic value in multilogistic regression models simultaneously incorporating ArgMI, GABR, and other traditional cardiovascular risk factors (Table 3). Remarkably, when further stratified according to primary versus secondary prevention cohorts, ArgMI was found to be highly predictive of incident 3-year MACE risk particularly in the secondary prevention cohort, with a trend toward predictive of incident 3-year MACE risk in the primary prevention cohort (Table 3). In contrast, GABR showed a stronger predictive value among primary prevention subjects compared with secondary prevention subjects (Table 3).
There are several key findings regarding the role of methylated derivatives of amino acids in this large well-characterized patient population. First, we confirmed the potential prognostic utility of ADMA in significantly obstructive CAD severity and related events in a large cohort of patients undergoing elective diagnostic cardiac catheterization. More importantly, we identified for the first time that in addition to ADMA, plasma levels of other products of arginine methylation such as SDMA and MMA are also strongly associated (inversely in the case of MMA; Table 2) with the presence of significantly obstructive CAD and incident risks for MACE over the ensuing 3-year period. It is also important to recognize that these associations remained robust even after adjustments for renal function as well as both traditional risk factors and hsCRP. Known as a “uremic toxin,” a potential confounding factor on plasma levels of ADMA and other methylarginine is the presence of renal insufficiency and underlying inflammation.15 However, after multivariable adjustments incorporating CrCl and hsCRP, analytes of arginine methylation pathways alternative to ADMA (eg, SDMA) and the integrated methylation index, ArgMI, still demonstrated independent prognostic value for cardiovascular events. The significant associations between both prevalent significantly obstructive CAD and incident major adverse events and (1) elevations in plasma levels of SDMA (in the absence of a NOS inhibitory effect); (2) reductions in plasma levels of the potent NOS inhibitor MMA; and (3) increased levels of ArgMI, a ratio that serves as an overall gauge for more extensive posttranslational methylation of arginine, suggests that the relationship between arginine methylation pathways and CAD initiation and progression extends well beyond direct NOS inhibition. The lack of prognostic value of plasma methyl-lysine levels argues against the contribution to long-term adverse cardiovascular risks (at least in a high-risk asymptomatic population) by other amino acid (nonarginine) methylation pathways. Taken together, our data demonstrate that not just the endogenous NOS inhibitor ADMA but other methylated arginine metabolites can collectively provide clinically important information relevant in the assessment of prevalent significantly obstructive CAD and long-term cardiovascular risks.
Despite the generally well-accepted prognostic role of ADMA in cardiovascular disease, there has been limited understanding regarding what specific pathophysiologic processes are at play. There has been a long-standing belief that the direct inhibition of NOS is the primary mechanism for the heightened cardiovascular risks associated with elevated ADMA levels. Indeed, animal model studies using either infusion of ADMA or overexpression of DDAH-1 (major catabolic pathway for ADMA) have corroborated a potential role for arginine methylation derivatives and their catabolic pathways in the modulation of NO bioavailability, particularly under conditions where elevated ADMA are present.16–18 Although prior studies in humans have identified the association between SDMA and early renal compromise and the presence of coronary artery disease,19,20 our large prospective study substantially extends these observations by demonstrating that even in a population with normal renal function and after adjustments for traditional CAD risk factors, creatinine clearance, and markers of inflammation (hsCRP), plasma SDMA remains a significant predictor of both prevalent CAD risks and incident risks for MI or stroke, death, and MACE. Moreover, this is the first demonstration to our knowledge that combining SDMA and MMA with ADMA provides added prognostic value. These findings are somewhat unexpected, as SDMA (a stereoisomer of ADMA) has been demonstrated to have no inhibitory effects on NOS.21 However, it is clear that ADMA and SDMA are naturally occurring analogues of L-arginine, the substrate for NO synthesis by nitric oxide synthases (NOSs). Therefore, their structural similarities with L-arginine may suggest a direct inhibition of a common pathway via competition with L-arginine for transport into the cells through the cationic amino acid transporter.21 It is conceivable that direct competition may limit cellular entry of L-arginine, thereby accentuating intracellular L-arginine (substrate) deficiency leading to a decrease in NO production. Direct comparison with substrate estimates of arginine bioavailability (via GABR) indicates that this arginine methylation process may be particularly important in patients with more advanced CAD, as the independent prognostic value of ArgMI were higher in secondary prevention cohort.
Perhaps one of the more remarkable findings in the present study is the inverse relationship between MMA levels and both prevalent significantly obstructive CAD and incident adverse cardiovascular events. This finding argues strongly for a rethinking of our understanding of arginine methylation in cardiovascular risk prediction. Among the methylarginines, MMA is the most potent NOS inhibitor and is widely used as a pharmacological inhibitor of NOSs.21,22 Although present at lower levels in plasma, our study clearly identifies the unexpected inverse relationship between plasma levels of this potent NOS inhibitor and CAD risks (Table 2). As far as we are aware, this study is the first to examine plasma levels of MMA alone or in combinations with ADMA and SDMA in relationship to prevalent and incident CAD risks. Further studies are needed to validate these findings, but the present data clearly illustrate the complexity of arginine methylation metabolic pathways and the importance of understanding globally the “arginine/NO metabolome” rather than examining levels of single specific markers because each is involved in multiple interconnecting metabolic pathways. It is therefore conceivable that the increased risk associated with relatively low plasma level of MMA is a reflection of heightened posttranslational modification of proteins through dimethylation reactions and proteolysis, producing ADMA and SDMA in patients with cardiovascular diseases or those at risk of cardiovascular events. Thus, augmentation of arginine methylation pathways as indicated by the proposed ArgMI may provide a more comprehensive index of cardiovascular risk. The present findings also argue that inflammation and oxidation pathways, which are known to enhance protein arginine residue posttranslational modification by methylation and subsequent proteolysis, may indeed be a dominant mechanism accounting for the established association between ADMA and both prevalent cardiovascular disease and incident adverse events.
Because lysine methylation pathways have no known interactions with the production of NO or its endogenous inhibitors, we expected no relationship between methyl-lysine levels and cardiovascular disease and sought to examine levels of this alternative methylated amino acid as a “control” for generalized protein catabolic activity. Our studies show free methyl-lysine levels demonstrate limited prognostic value regarding either prevalence of significantly obstructive CAD or the prediction of future cardiovascular events in the population examined. By simultaneously measuring both methylarginines and methyl-lysine levels, this “internal control” further validates the important contribution of arginine methylation pathways in the disease progression of long-term cardiovascular disease.
Other studies have shown that systemic levels of arginine alone are a poor prognostic indicator, and several studies have recently reported that GABR, an integrated index of arginine bioavailability, has improved prognostic utility in prediction of major adverse cardiac events in several populations, including studies of subjects with cardiogenic shock,9 subjects with sickle cell disease,12 and most recently, in stable cardiology patients.13 Multilogistic regression analyses with models incorporating both indices, ArgMI and GABR, reveal that both remain significant independent predictors of incident MACE risk over the ensuing 3-year period, strongly supporting the contention that both arginine/NO metabolome related indices retain prognostic utility when evaluated within the same cohort. Moreover, further analyses showed that ArgMI and GABR appear to be sensitive to different aspects of cardiovascular risk. ArgMI showed a superior prognostic utility in secondary prevention subjects compared to primary prevention, suggesting protein arginine methylation, proteolysis, and downstream interference of nitric oxide production are biochemical pathways with greater association to pathophysiological processes relevant to later stages of atherosclerotic plaque progression or vulnerable plaque development. In contrast, the GABR showed improved prognostic value among primary prevention subjects compared to secondary prevention, suggesting that processes linked to plaque initiation may have greater sensitivity to substrate (arginine) bioavailability for nitric oxide production. These analyses illustrate the complexity of different aspects of the NO/Arg metabolome and their potential contributions to different aspects of the pathogenesis of CAD along the spectrum of disease evolution including initiation, progression, and acute complications.
There are several limitations to our study. Despite being one of the largest samples sizes reported to date regarding the role of ADMA and cardiovascular risks, the subject population is heterogeneous and location-specific (patients undergoing elective diagnostic cardiac catheterization), the majority of which with varying degrees of CAD and comorbidities. Also, sampling of methylarginines, arginine and its catabolites, and methyl-lysine was performed only at the time of enrollment, precluding monitoring the impact of cardiovascular therapies on the arginine/NO metabolome. Finally, subjects enrolled were elective cases, did not include subjects with acute coronary syndrome, and end points selected were indicators for long-term cardiovascular events. Therefore, the present results cannot be extrapolated for short-term outcomes or in the setting of acute cardiac events.
Plasma levels of arginine mono- and dimethylation are associated with the presence of cardiovascular disease and incident adverse events. Higher levels of both ADMA and SDMA and lower levels of MMA were predictors of prevalent disease, and elevated SDMA and ADMA both were predictive of long-term risks of major adverse cardiac events (heart attack, stroke, or death), even after adjustments for traditional risk factors, hsCRP levels, renal function, and indices of global arginine bioavailability. Based on our findings, an integrated quantification of arginine methylation in the form of an “arginine methylation index” (ArgMI= [ADMA+SDMA]/MMA) provided the strongest independent risk prediction for incident major adverse cardiac events in stable patients undergoing elective cardiac evaluation, and significantly added to the prognostic utility of traditional risk factors. These results suggest that arginine methylation provides important contributions to disease progression beyond direct NOS inhibition.
Sources of Funding
This research was supported by National Institutes of Health grants P01 HL076491-055328, P01 HL087018-020001, and P50 HL077107-050004 (to S.L.H.), and the Cleveland Clinic Clinical Research Unit of the Cleveland Clinic/Case Western Reserve University CTSA 1UL1RR024989 (to S.L.H. and W.H.T.). Supplies and funding for performance of fasting lipid profiles, blood glucose, creatinine, and hsCRP were provided by Abbott Laboratories Inc.
W.H.W.T. reports having received research grant support from Abbott Laboratories Inc. L.C. reports that she has received honorarium for teaching and speaking from Medtronic Inc and honorarium for speaking from AstraZeneca. S.L.H. is named as coinventor on pending and issued patents filed by the Cleveland Clinic that relate the use of biomarkers in inflammatory and cardiovascular disease. S.L.H. reports he is the scientific founder of PrognostiX Inc; has received speaking honoraria from Pfizer, AstraZeneca, Merck, Merck Schering Plough, BioSite, Lilly, Wyeth, and Abbott; has received research grant support from Abbott Laboratories, Pfizer, Merck, and PrognostiX Inc; and has received consulting fees from Abbott Laboratories, Pfizer, PrognostiX Inc, Wyeth, BioPhysical, and AstraZeneca. All other authors have no disclosures to report.
Received August 27, 2008; revision accepted June 4, 2009.
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