Asymmetrical dimethylarginine plasma clearance persists after acute total nephrectomy in rats

Elevated plasma concentrations of symmetrical dimethylarginine (SDMA) and asymmetrical dimethylarginine (ADMA) are repeatedly associated with kidney failure. Both ADMA and SDMA can be excreted in urine. We tested whether renal excretion is necessary for acute, short-term maintenance of plasma ADMA and SDMA. Sprague-Dawley rats underwent sham operation, bilateral nephrectomy (NPX), ureteral ligation, or ureteral section under isoflurane anesthesia. Tail-snip blood samples (250 microl) were taken before and at 6- or 12-h intervals for 72 h after operation. Plasma clearance was assessed in intact and NPX rats. High-performance liquid chromatography determined SDMA and ADMA concentrations. Sodium, potassium, creatinine, blood urea nitrogen (BUN), and body weight were also measured. Forty-eight hours after NPX, SDMA increased 25 times (0.23 +/- 0.03 to 5.68 +/- 0.30 microM), whereas ADMA decreased (1.17 +/- 0.08 to 0.73 +/- 0.08 microM) by 38%. Creatinine and BUN increased, paralleling SDMA. Sham-operated animals showed no significant changes. Increased SDMA confirms continuous systemic production of SDMA and its obligatory renal excretion, much like creatinine. In contrast, decreased plasma ADMA suggests that acute total NPX either reduced systemic ADMA formation and/or systemic hydrolysis of ADMA increased 48-h post-NPX. However, plasma clearance of ADMA appeared unchanged 48 h after NPX. We conclude that renal excretory function is needed for SDMA elimination but not needed for acute, short-term ADMA elimination in that systemic hydrolysis is fully capable of clearing plasma ADMA.     

Accumulation of symmetric dimethylarginine in hepatorenal syndrome

In patients with cirrhosis, nitric oxide (NO), asymmetric dimethylarginine (ADMA), and possibly symmetric dimethylarginine (SDMA) have been linked to the severity of the disease. We investigated whether plasma levels of dimethylarginines and NO are elevated in patients with hepatorenal syndrome (HRS), compared with patients with cirrhosis without renal failure (no-HRS). Plasma levels of NO, ADMA, SDMA, and l-arginine were measured in 11 patients with HRS, seven patients with no-HRS, and six healthy volunteers. SDMA concentration in HRS was higher than in no-HRS and healthy subjects (1.47 +/- 0.25 vs. 0.38 +/- 0.06 and 0.29 +/- 0.04 microM, respectively; P < 0.05). ADMA and NOx concentrations were higher in HRS and no-HRS patients than in healthy subjects (ADMA, 1.20 +/- 0.26, 1.11 +/- 0.1, and 0.53 +/- 0.06 microM, respectively; P < 0.05; NOx, 94 +/- 9.1, 95.5 +/- 9.54, and 37.67 +/- 4.62 microM, respectively; P < 0.05). In patients with HRS there was a positive correlation between serum creatinine and plasma SDMA (r2 =0.765, P < 0.001) but not between serum creatinine and ADMA or NOx. The results suggest that renal dysfunction is a main determinant of elevated SDMA concentration in HRS. Accumulation of ADMA as a result of impaired hepatic removal may be the causative factor initiating renal vasoconstriction and SDMA retention in the kidney.     

Asymmetric dimethylarginine, oxidative stress, and vascular nitric oxide synthase in essential hypertension

We reported impaired endothelium-derived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis that the patients in this study have increased circulating levels of the cNOS inhibitor, asymmetric dimethylarginine (ADMA), or the lipid peroxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (HODE), which is a marker of reactive oxygen species. Patients had significantly (P < 0.001) elevated (means +/- SD) plasma levels of ADMA (P(ADMA), 766 +/- 217 vs. 393 +/- 57 nmol/l) and symmetric dimethylarginine (P(SDMA): 644 +/- 140 vs. 399 +/- 70 nmol/l) but similar levels of L-arginine accompanied by significantly (P < 0.015) increased rates of renal ADMA excretion (21 +/- 9 vs. 14 +/- 5 nmol/mumol creatinine) and decreased rates of renal ADMA clearance (18 +/- 3 vs. 28 +/- 5 ml/min). They had significantly increased plasma levels of HODE (P(HODE): 309 +/- 30 vs. 226 +/- 24 nmol/l) and renal HODE excretion (433 +/- 93 vs. 299 +/- 67 nmol/micromol creatinine). For the combined group of normal and hypertensive subjects, the individual values for plasma levels of ADMA and HODE were both significantly (P < 0.001) and inversely correlated with microvascular EDRF/NO and positively correlated with mean blood pressure. In conclusion, elevated levels of ADMA and oxidative stress in a group of hypertensive patients could contribute to the associated microvascular endothelial dysfunction and elevated blood pressure.     

High-throughput liquid chromatographic-tandem mass spectrometric determination of arginine and dimethylated arginine derivatives in human and mouse plasma

The balance between nitric oxide (NO) and vasoconstrictors like endothelin is essential for vascular tone and endothelial function. L-Arginine is converted to NO and L-citrulline by NO synthase (NOS). Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. ADMA is degraded by dimethylamino dimethylhydrolases (DDAHs), while SDMA is exclusively eliminated by the kidney. In the present article we report a LC-tandem MS method for the simultaneous determination of arginine, ADMA, and SDMA in plasma. This method is designed for high sample throughput of only 20-mul aliquots of human or mouse plasma. The analysis time is reduced to 1.6 min by LC-tandem MS electrospray ionisation (ESI) in the positive mode. The mean plasma levels of l-arginine, ADMA, and SDMA were 74+/-19 (SD), 0.46+/-0.09, and 0.37+/-0.07 microM in healthy humans (n=85), respectively, and 44+/-14, 0.72+/-0.23, and 0.19+/-0.06 microM in C57BL/6 mice. Also, the molar ratios of arginine to ADMA were different in man and mice, i.e. 166+/-50 and 85+/-22, respectively.     

High-performance liquid chromatographic assay of asymmetric dimethylarginine,symmetric dimethylarginine,and arginine in human plasma by derivatization with naphthalene-2,3-dicarboxaldehyde

Asymmetric dimethylarginine (ADMA) is an emerging cardiovascular risk factor. Its increased levels have been hypothesized to be a cause of endothelial dysfunction in pathological conditions such as hypertension, dyslipidemia, renal failure, hyperglycemia, and hyperhomocysteinemia. It acts as a potent competitive inhibitor of nitric oxide synthase. Methods using ortho-phthaldialdehyde (OPA) as derivatization reagent are widely performed in HPLC determination of ADMA, but they produce derivatives whose fluorescence rapidly decreases during time. Moreover, these methods do not allow a clear separation of ADMA from its stereoisomer symmetric dimethylarginine (SDMA). Our work describes a new method to determine ADMA, SDMA, and arginine that uses, as derivatizing reagent, naphthalene-2,3-dicarboxaldehyde (NDA). Chromatograms with low background, showing a complete separation of ADMA and SDMA, are obtained. NDA derivatives are considerably more stable than the OPA derivatives. The calibration curves of ADMA and SDMA are linear within the range of 0.01-16.0 microM. Coefficients of variation are less than 1.7% for within day and less then 2.3% for day to day. Absolute mean recoveries from supplemented samples are between 100 and 104%. These characteristics make this method reliable and easily manageable for large routine analyses.     

Asymmetrical (ADMA) and symmetrical dimethylarginine (SDMA) as potential risk factors for cardiovascular and renal outcome in chronic kidney disease – possible candidates for paradoxical epidemiology?

Summary. Background: Asymmetrical dimethylarginine (ADMA) is an inhibitor of nitric-oxide synthase. It has been linked to atherosclerotic risk in the general population as well as in end-stage renal disease patients (ESRD), whereas symmetrical dimethylarginine (SDMA) is thought to be biological inactive. Prospective data concerning the role of both dimethylarginines are rare in patients with chronic kidney disease. Methods: 200 patients with chronic kidney disease (mean age 57.6 ± 13.0 years, 69 female, 131 male); 82 with chronic renal failure (CRF), 81 on maintenance haemodialysis (HD) and 37 renal transplant recipients (RTR) were prospectively followed for 24 months. ADMA and SDMA were measured by HPLC. The relation of plasma levels of ADMA and SDMA together with conventional risk factors for the cardiovascular and renal outcome was investigated with Cox proportional hazards model. Results: Mean serum levels of SDMA were significantly increased in all groups compared to the control group (P ≤ 0.0005), ADMA was increased only in HD and RTR (P ≤ 0.004). Forty-seven cardiovascular events (CVE) occurred during follow-up, 35 patients died, and 39 patients reached ESRD. Multivariate analysis showed diabetes (RR 3.072, P = 0.01), ESRD (RR 11.915, P < 0.0005), elevated CRP levels (RR 3.916, P < 0.0005) and surprisingly a lower ADMA level (RR 0.271, P = 0.008) as independent risk factors for CVE. Serum creatinine (RR 11.378, P = 0.001), haemoglobin (RR 0.710, P = 0.038 for an increment of 1 mmol/l), and SDMA levels (RR 1.633, P = 0.006, per 1 μmol/l increment) were predictors for the progression to ESRD. Conclusions: Data from a heterogeneous group of patients with chronic kidney disease provide evidence that conventional risk factors seem to play a more important role than elevated serum levels of ADMA or SDMA for cardiovascular events. Increasing serum SDMA concentration seems to play an additive role for the renal outcome besides serum creatinine and haemoglobin levels. Whether ADMA might possibly be a candidate for the phenomenon of “paradoxical epidemiology” in chronic kidney disease needs further investigation.     

Determination of arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other biological samples by high-performance liquid chromatography

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), may be related to reduced biosynthesis of nitric oxide in diseases associated with accelerated atherosclerosis. The closely related compound symmetric dimethylarginine (SDMA) does not inhibit NOS, but may compete with arginine for cellular uptake, thereby limiting substrate availability for NOS. We report on a method for the simultaneous measurement of arginine, ADMA, and SDMA as a tool to gain insight in the role of these compounds in the regulation of NOS activity. Sample cleanup was performed by solid-phase extraction on polymeric cation-exchange columns using monomethylarginine as internal standard. After derivatization with ortho-phthaldialdehyde reagent containing 3-mercaptopropionic acid, analytes were separated by isocratic reversed-phase HPLC with fluorescence detection. The stable derivatives were separated with near baseline resolution. Using a sample volume of 0.2 ml, linear calibration curves were obtained with limits of quantification of 0.08 microM for arginine and 0.01 microM for ADMA and SDMA. Analytical recovery was 98-102%, and interassay CV was better than 3%. Plasma from healthy volunteers (n = 53) contained 94 +/- 26 microM arginine, 0.42 +/- 0.06 microM ADMA, and 0.47 +/- 0.08 microM SDMA. Due to its high precision and sensitivity this method is a valuable tool in research on the metabolism of dimethylated arginines and their role in the regulation of NOS activity.     

Asymmetric and Symmetric Dimethylarginine as Risk Markers for Total Mortality and Cardiovascular Outcomes: A Systematic Review and Meta-Analysis of Prospective Studies

BackgroundA growing number of studies linked elevated concentrations of circulating asymmetric (ADMA) and symmetric (SDMA) dimethylarginine to mortality and cardiovascular disease (CVD) events. To summarize the evidence, we conducted a systematic review and quantified associations of ADMA and SDMA with the risks of all-cause mortality and incident CVD in meta-analyses accounting for different populations and methodological approaches of the studies.MethodsRelevant studies were identified in PubMed until February 2015. We used random effect models to obtain summary relative risks (RR) and 95% confidence intervals (95%CIs), comparing top versus bottom tertiles. Dose-response relations were assessed by restricted cubic spline regression models and potential non-linearity was evaluated using a likelihood ratio test. Heterogeneity between subgroups was assessed by meta-regression analysis.ResultsFor ADMA, 34 studies (total n = 32,428) investigating associations with all-cause mortality (events = 5,035) and 30 studies (total n = 30,624) investigating the association with incident CVD (events = 3,396) were included. The summary RRs (95%CI) for all-cause mortality were 1.52 (1.37–1.68) and for CVD 1.33 (1.22–1.45), comparing high versus low ADMA concentrations. Slight differences were observed across study populations and methodological approaches, with the strongest association of ADMA being reported with all-cause mortality in critically ill patients. For SDMA, 17 studies (total n = 18,163) were included for all-cause mortality (events = 2,903), and 13 studies (total n = 16,807) for CVD (events = 1,534). High vs. low levels of SDMA, were associated with increased risk of all-cause mortality [summary RR (95%CI): 1.31 (1.18–1.46)] and CVD [summary RR (95%CI): 1.36 (1.10–1.68) Strongest associations were observed in general population samples.ConclusionsThe dimethylarginines ADMA and SDMA are independent risk markers for all-cause mortality and CVD across different populations and methodological approaches.     

HPLC analysis of asymmetric dimethylarginine,symmetric dimethylarginine,homoarginine and arginine in small plasma volumes using a Gemini-NX column at high pH

There is increasing recognition of the clinical importance of endogenous nitric oxide synthase inhibitors in critical illness. This has highlighted the need for an accurate high performance liquid chromatography (HPLC) method for detection of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in small volumes of blood. Here, the validation of an accurate, precise HPLC method for the determination of ADMA, SDMA, homoarginine and arginine concentrations in plasma is described. Solid phase extraction is followed by derivatisation with AccQ-Fluor™ and reversed phase separation on a Gemini-NX column at pH 9. Simultaneous detection by both UV–vis and fluorescence detectors affords extra validation. This solid phase extraction method gives absolute recoveries of more than 85% for ADMA and SDMA and relative recoveries of 102% for ADMA and 101% for SDMA. The intra-assay relative standard deviations are 2.1% and 2.3% for ADMA and SDMA, respectively, with inter-assay relative standard deviations of 2.7% and 3.1%, respectively. Advantages of this method include improved recovery of all analytes using isopropanol in the solid phase extraction; sharp, well-resolved chromatographic peaks using a high pH mobile phase; a non-endogenous internal standard, n-propyl l-arginine; and accurate and precise determination of methylated arginine concentrations from only 100 μL of plasma.     

Direct analysis of un-derivatized asymmetric dimethylarginine (ADMA) and L-arginine from plasma using mixed-mode ion-exchange liquid chromatography-tandem mass spectrometry

A high-throughput analytical method was developed for the measurement of asymmetric dimethylarginine (ADMA) and L-arginine (ARG) from plasma using LC/MS/MS. The sample preparation was simple and only required microfiltration prior to analysis. ADMA and ARG were assayed using mixed-mode ion-exchange chromatography which allowed for the retention of the un-derivatized compounds. The need for chromatographic separation of ADMA from symmetric dimethylarginine (SDMA) was avoided by using an ADMA specific product ion. As a result, the analytical method only required a total run time of 2 min. The method was validated by linearity, with r2>or=0.995 for both compounds, and accuracy, with no more than 7% deviation from the theoretical value. The estimated limit of detection and limit of quantification were suitable for clinical evaluations. The mean values of plasma ADMA and ARG taken from healthy volunteers (n=15) were 0.66+/-0.12 and 87+/-35 microM, respectively; the mean molar ratio of ARG to ADMA was 142+/-81.     

Circulating Levels of Dimethylarginines,Chronic Kidney Disease and Long-Term Clinical Outcome in Non-ST-Elevation Myocardial Infarction

Background

Mechanisms linking chronic kidney disease (CKD) and adverse outcomes in acute coronary syndromes (ACS) are not fully understood. Among potential key players, reduced nitric oxide (NO) synthesis due to its endogenous inhibitors, asymmetric (ADMA) and symmetric (SDMA) dimethylarginine could be involved. We measured plasma concentration of arginine, ADMA and SDMA and investigated their relationship with CKD and long-term outcome in non-ST-elevation myocardial infarction (NSTEMI).

Methodology/Principal Findings

We prospectively measured arginine, ADMA, and SDMA at hospital admission in 104 NSTEMI patients. CKD was defined as an estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m². We considered a primary end point of combined cardiac death and re-infarction at a median follow-up of 21 months. In CKD (n = 33) and no-CKD (n = 71) patients, arginine and ADMA were similar, whereas SDMA was significantly higher in CKD patients (0.65±0.23 vs. 0.42±0.12 µmol/L; P<0.0001). Twenty-four (23%) patients had an adverse cardiac event during follow-up: 12 (36%) were CKD and 12 (17%) no-CKD patients (P = 0.02). When study population was stratified according to arginine, ADMA and SDMA median values, only SDMA (median 0.46 µmol/L) was associated with the primary end-point (P = 0.0016). In models adjusted for age, hemoglobin and left ventricular ejection fraction, the hazard ratio (HR) for CKD and SDMA were high (HR 2.93, interquartile range [IQR] 1.15–7.53; P = 0.02 and HR 6.80, IQR 2.09–22.2; P = 0.001, respectively) but, after mutual adjustment, only SDMA remained significantly associated with the primary end point (HR 5.73, IQR 1.55–21.2; P = 0.009).

Conclusions/Significance

In NSTEMI patients, elevated SDMA plasma levels are associated with CKD and worse long-term prognosis.     

Nitric Oxide Synthesis Metabolites—As Potential Markers in Chronic Kidney Disease in Children

Nitric oxide (NO) is an important signaling molecule for many physiological and pathological processes. Diseases associated with abnormal NO synthesis include cardiovascular diseases, insulin-dependent diabetes, or chronic kidney disease (CKD). The aim of the paper was to evaluate NO synthesis metabolites, i.e., asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), dimethylamine (DMA), arginine, citrulline in plasma of patients with different severity of CKD and to seek possible links between these parameters and the development of this disease. Forty-eight CKD children and thirty-three age-matched controls were examined. Patients were divided into groups depending on the CKD stages (Group II-stage II, Group III-stage III, Group IV-stage IV, and Group RRT children on dialysis). To determine the concentrations of the above-mentioned metabolites in plasma liquid chromatography-mass spectrometry was used. There were significant differences observed in levels of ADMA, SDMA, DMA, and citrulline between control vis CKD groups (p values ranging from <0.001 to 0.029). Plasma arginine concentration was also higher in CKD patients compared to the control group but statistically insignificant. ADMA levels in CKD children were statistically significantly higher in relation to particular stages of CKD (RRT vis II stage of CKD: p = 0.01; RRT vis III-IV stages of CKD: p < 0.046). Citrulline levels in CKD children were statistically significantly higher in RRT group vis control (p < 0.001). Children with CKD develop disturbances in most metabolites of NO synthesis. Dialysis children treated show the greatest disturbances of plasma ADMA and citrulline levels. ADMA seems to be a good indicator of the gradual progression of the CKD, which is proved by the negative correlation with eGFR.     

Dimethylarginine Levels in Cerebrospinal Fluid of Hyperacute Ischemic Stroke Patients are Associated with Stroke Severity

We hypothesise that asymmetric and symmetric dimethylarginine (ADMA, SDMA) are released in cerebrospinal fluid (CSF) due to ischemia-induced proteolysis and that CSF dimethylarginines are related to stroke severity. ADMA and SDMA were measured in CSF of 88 patients with ischemic stroke or TIA within 24 h after stroke onset (mean 8.6 h) and in 24 controls. Stroke severity was assessed by the National Institutes of Health Stroke Scale (NIHSS) score at admission. Outcome was evaluated by institutionalization due to stroke and the modified Rankin scale. Dimethylarginine levels were higher in patients with stroke than in TIA patients, who had higher levels than controls and correlated with the NIHSS. Logistic regression analysis confirmed that dimethylarginines were independently associated with stroke severity. The SDMA/ADMA ratio did not differ significantly between controls and stroke patients. CSF dimethylarginine levels are increased in hyperacute ischemic stroke and are associated with stroke severity. R. Brouns is a research assistant of the Fund for Scientific research Flanders (FWO-Vlaanderen).     

Hypothermic renal perfusion during aortic surgery reduces the presence of lipocalin-2 and preserves renal extraction of dimethylarginines in rats

Cold perfusion through the renal arteries during renal ischemia has been suggested to diminish postoperative renal damage after juxtarenal aortic aneurysm repair. As the kidneys play a key role in dimethylarginine metabolism, which in turn is associated with renal hemodynamics, we hypothesized that the protective effect of cold perfusion is associated with a preserved renal extraction of dimethylarginines. Renal ischemia was induced in three groups of anesthetized Wistar rats (n = 7/group), which underwent suprarenal aortic clamping (45 min) with no perfusion (group 1), renal perfusion with 37°C saline (group 2), or renal perfusion with 4°C saline (group 3), respectively, followed by 90 min of renal reperfusion in all groups. The sham group had no clamping. In group 3 (renal ischemia with cold perfusion), postoperative serum creatinine levels as well as the presence of luminal lipocalin-2 and its associated brush-border damage were lower compared with groups 1 and 2 (P < 0.05). Also, renal extraction of asymmetrical (ADMA) and symmetrical (SDMA) dimethylarginine as well as the arginine/ADMA ratio, which defines the bioavailability of nitric oxide, remained intact in group 3 only (P < 0.04). The arginine/ADMA ratio correlated with cortical flow, lipocalin-2, and creatinine rises. Warm and cold renal perfusion (groups 2 and 3) during ischemia were similarly effective in lowering protein nitrosylation levels, renal leukocyte accumulation, neutrophil gelatinase-associated lipocalin (NGAL) expression in distal tubules, and urine NGAL (P < 0.05). These data support the use of cold renal perfusion during renal ischemia in situations where renal ischemia is inevitable, as it reduces tubular damage and preserves renal extraction of dimethylarginines. Renal perfusion with saline per se during renal ischemia is effective in diminishing renal leukocyte accumulation and oxidative stress.     

Increased plasma level of asymmetric dimethylarginine in hypertensive rats facilitates platelet aggregation: role of plasma tissue factor

This study was designed to explore the role of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthesis, in platelet aggregation in hypertension and its possible mechanisms. Spontaneously hypertensive rats (SHR) and L-NAME-induced hypertensive rats were orally administered with L-arginine (1 g/(kg·day) for 14 days. Systolic blood pressure, platelet aggregation, and plasma tissue factor (TF) level and activity were measured. The plasma concentration of ADMA in SHR was determined. In vitro, platelet-rich plasma isolated from Wistar rats was prepared in order to observe the effect of exogenous ADMA on platelet aggregation and TF level and (or) activity in platelet-rich plasma. In both types of hypertensive rats, systolic blood pressure, platelet aggregation, and the level and activity of plasma TF were elevated compared with corresponding control animals. Plasma ADMA level was also increased in SHR. Treatment with L-arginine, a competitor of ADMA, lowered blood pressure and inhibited platelet aggregation concomitantly with a decrease in plasma TF level and activity in both types of hypertensive rats. We also found that exogenous ADMA promoted platelet aggregation and increased TF level and (or) activity in platelet-rich plasma, an effect that was inhibited by pretreatment with L-arginine. Importantly, the enhanced platelet aggregation induced by exogenous ADMA was reduced by pretreatment with anti-TF antibody. The results suggest that endogenous ADMA may be involved in platelet hyperaggregation status in hypertension, and the facilitation of platelet aggregation by ADMA is related to upregulation of the level and activity of plasma TF.     

Free fatty acids do not acutely increase asymmetrical dimethylarginine concentrations.

Concentrations of asymmetrical dimethylarginine (ADMA) and free fatty acids (FFAs) are elevated in insulin resistance which is associated with impaired vascular function. We hypothesized that FFAs could alter vascular tone by affecting ADMA concentrations. Plasma FFA levels were increased in seventeen healthy male volunteers by Intralipid/heparin infusion; hemodynamic and biochemical parameters were measured after 90 minutes. Plasma collected before and during Intralipid/heparin or equivalent synthetic FFAs was incubated with human umbilical vein endothelial cells (HUVECs) in vitro. Intralipid/heparin infusion resulted in an approximately seven-fold increase in plasma FFA levels to 1861 +/- 139 micromol/l, which was paralleled by increased systemic blood pressure and forearm blood flow. Intralipid/heparin did not affect ADMA (baseline mean 0.59 [95 % confidence interval [CI]: 0.54; 0.64] and 0.56 [CI: 0.51; 0.59] after 90 minutes), but slightly decreased SDMA (from 0.76, [CI: 0.70; 0.83] to 0.71 [CI: 0.64; 0.74], p < 0.05), and had no effect on ADMA/SDMA ratio. There was no correlation between ADMA and FFA concentrations or forearm blood flow. Incubation of HUVECs with FFA-rich plasma or synthetic FFAs induced an ADMA release after 24 hours, but not after 90 minutes. Acutely increased FFA levels caused hemodynamic effects but did not affect ADMA. Prolonged elevation of FFA levels might influence vascular function by increasing ADMA levels.     

High clinical accuracy of asymmetric dimethylarginine and symmetric dimethylarginine in patients with ischemic heart disease

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) were found in various clinical settings including coronary heart disease. To assess ADMA and SDMA diagnostic validity in patients with different stages of ischemic heart disease, we studied these markers in patients having stable angina pectoris (SAP), unstable angina (USAP), and acute myocardial infarction (AMI). The results were compared with the values of healthy individuals. Plasma ADMA and SDMA levels were measured by high-performance liquid chromatography. In all patient groups both markers were significantly elevated in comparison with control ones (p?<?0.001). In SAP patients, the median ADMA value was 0.75 (0.31–2.73)?μmol/L, and SDMA 1.11 (0.69–0.1.42)?μmol/L, in USAP patients, the marker values were 0.94 (0.34–3.13)?μmol/L and 1.23 (0.88–4.72)?μmol/L, and in AMI patients, 0.98 (0.48–2.01)?μmol/L and 1.26 (0.75–2.93)?μmol/L, while in healthy subjects they were 0.31 (0.17–0.87)?μmol/L and 0.29 (0.20–0.83)?μmol/L, respectively. SDMA was found significantly different in SAP and AMI patients (p?<?0.05). Diagnostic accuracy was determined by receiver operating characteristic (ROC) curve analysis. The highest area under the ROC (AUC) for ADMA was obtained in AMI patients (0.976), while for SDMA in USAP patients (1.000). There was no significant difference between the AUCs. The greatest sensitivity and specificity were found in the USAP group (95.65 and 96.30?% for ADMA, and 100?% for each characteristic of SDMA). Considering these results, SDMA showed better clinical accuracy in assessing ischemic disease, where it could be used as a valid marker and a therapeutic target.     

The role of asymmetric dimethylarginine in the regulation of nitric oxide level in rats with acute renal injury

Nitric oxide (NO) is synthesized from arginine (ARG) by NO synthase (NOS). Asymmetric dimethylarginine (ADMA), a competitive inhibitor of NOS, participates in the endogenous regulation of NO synthesis. The main amount of ADMA is enzymatically degraded by dimethylarginine dimethylaminohydrolase (DDAH) widely expressed in renal tissue. The aim of our study was to compare the changes in DDAH activity and ARG synthesis in kidneys, ADMA and ARG concentration in plasma and their urinary excretion under physiological conditions and in acute renal injury (ARI) induced by glycerol in rats. Urinary nitrite/nitrate excretion (NOx) was estimated as an indicator of whole-body NO synthesis. DDAH activity was decreased, ADMA excretion was increased and plasma ADMA did not change in ARI. Plasma ARG concentration, renal ARG synthesis and urinary NOx excretion were decreased. In conclusion, the diminished enzymatic hydrolysis of the NOS inhibitor ADMA and the reduced synthesis of the NOS substrate ARG might affect NO production in ARI.     

Chromatographic-mass spectrometric methods for the quantification of L-arginine and its methylated metabolites in biological fluids

L-Arginine (Arg) and its methylated metabolites play a major role in the synthesis of the cell signaling molecule nitric oxide (NO). Arg serves as a substrate for the enzyme NO synthase (NOS), which produces NO, whereas monomethylarginine (L-NMMA) and asymmetric dimethylarginine (ADMA) act as competitive inhibitors of NOS. Symmetric dimethylarginine (SDMA) has virtually no inhibitory effect on NOS activity, but shares the pathway for cell entry and transport with Arg and ADMA. Accurate and reliable quantification of these substances in various biological fluids is essential for scientific research in this field. In this review, chromatographic-mass spectrometric methods for Arg and its methylated metabolites ADMA and SDMA are discussed. Mass spectrometric detection provides an intrinsic higher selectivity than detection by means of UV absorbance or fluorescence. Taking advantage of the high selectivity, approaches involving mass spectrometric detection require less laborious sample preparation and produce reliable results. A consensus emerges that the concentration values in plasma of young healthy volunteers are about 65 microM for Arg, 0.4 microM for ADMA and 0.5 microM for SDMA.     

Aortic stenosis is associated with elevated plasma levels of asymmetric dimethylarginine (ADMA).

OBJECTIVES: We tested the hypothesis that the presence of aortic stenosis (AS) is associated with elevation of plasma levels of asymmetric dimethylarginine (ADMA), a physiological inhibitor of nitric oxide synthase, a mediator and marker of endothelial dysfunction and an indicator of incremental cardiovascular risk. BACKGROUND: The presence of aortic sclerosis (ASC), the precursor of AS is independently associated both with endothelial dysfunction, and with incremental coronary event risk. It remains uncertain whether elevations of ADMA levels might mediate endothelial dysfunction in these conditions. METHODS: Forty two consecutive patients referred for echocardiography for evaluation of AS, who had calculated aortic valve areas of <1.4 cm(2) (AS group) were evaluated together with 42 consecutive age-matched referred patients (non-AS group). Plasma ADMA levels were measured by high-performance liquid chromatography (HPLC). Determinants of elevation of plasma ADMA levels were identified via stepwise multiple linear regression analysis. RESULTS: Plasma ADMA levels were not statistically different between the AS and non-AS group (median 0.59 vs 0.54 micromol/L, p=0.13, Mann-Whitney test) on univariate analysis. However, in backward stepwise multiple linear regression, the presence of AS was a significant predictor of elevated ADMA levels (p=0.04, 95% CI=0.001, 0.072). In addition, elevated plasma ADMA levels were also associated with history of atrial fibrillation (p=0.009, 95% CI=0.015, 0.100), and negatively associated with creatinine clearance (p=0.01, 95% CI=-0.002, 0.000), and the use of statin therapy (p=0.01, 95% CI=-0.081, -0.011). CONCLUSIONS: AS is independently associated with elevation of ADMA levels, beyond that implied by "conventional" risk factors for endothelial dysfunction. The clinical status of AS as an incremental marker of cardiovascular risk may reflect ADMA-mediated endothelial dysfunction.