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Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor, which has been associated with total and cardiovascular mortality in various clinical settings. Studies on its structural isomer, symmetric dimethylarginine (SDMA), are scarce. This study aimed to determine the associations of both ADMA and SDMA levels with secondary cardiovascular disease events and all-cause mortality in patients with stable coronary heart disease (CHD). In the observational prospective cohort study KAROLA, 1,148 CHD patients were followed for a median of 8.1 years. ADMA and SDMA were determined by liquid chromatography–tandem mass spectrometry. Baseline ADMA and SDMA levels were categorized in quartiles or standardized by their respective standard deviation, and appropriate hazard ratios and 95 % confidence intervals (HR [95 % CI]) were estimated in Cox proportional hazards models. 150 patients experienced secondary cardiovascular disease events (CVD) and 121 patients died. After adjustment for confounders, ADMA was not associated with the risk of secondary CVD events (HR per standard deviation increase: 1.02 [95 %CI: 0.86–1.21]), whereas an association was suggested for SDMA (HR 1.17 [1.00–1.37]). Higher hazard ratios were observed in all-cause mortality models (ADMA: HR 1.15 [0.95–1.37]; SDMA: HR 1.29 [1.09–1.52]). Our results suggest that especially SDMA might possibly have potential as a risk marker for all-cause mortality and to a lesser extent for secondary cardiovascular events. Future studies are needed to quantify these associations more precisely and should, in particular, further address the possibility of residual confounding by impaired kidney function.

Availability of L-arginine, the exclusive substrate for nitric oxide synthases, plays an important role in kidney ischemia/reperfusion injury. The endogenous L-arginine derivatives asymmetrical dimethylarginine (ADMA) and symmetrical dimethylarginine (SDMA) block cellular L-arginine uptake competitively, thereby inhibiting the production of nitric oxide. ADMA also blocks nitric oxide synthase activity directly. Here, we investigate the pathomechanistic impact of ADMA and SDMA on ischemic acute kidney injury. Rats were subject to bilateral renal ischemia (60 minutes)/reperfusion (24 hours) injury. Impairment of renal function was determined with inulin clearance (glomerular filtration rate) and para-aminohippurate (PAH) clearance (renal plasma flow). L-arginine, ADMA, and SDMA levels were measured by liquid chromatography–tandem mass spectrometry. L-arginine was extracted from renal tissue and analyzed by enzyme-linked immunosorbent assay, and protein and messenger RNA expressions were determined by Western blot and real-time reverse transcription polymerase chain reaction. Renal function deteriorated severely after ischemia/reperfusion injury, as demonstrated by inulin and PAH clearance. Serum ADMA and SDMA increased, but tissue expression of specific ADMA or SDMA synthesizing and metabolizing enzymes (protein arginine methyltransferases and dimethyl arginine dimethylaminohydrolases) did not alter. Serum L-arginine increased as well, whereas intracellular L-arginine concentration diminished. Renal messenger RNA expression of cationic amino acid transporters, which mediate L-arginine uptake, remained unchanged. In serum, the ratio of L-arginine to ADMA did not alter after ischemia/reperfusion injury, whereas the ratios of L-arginine to SDMA and ADMA to SDMA decreased. A marked increase in serum SDMA, especially when accompanied by a diminished L-arginine-to-SDMA ratio, might reflect competitive inhibition of cellular L-arginine uptake by SDMA. As a consequence, a pathologic renal L-arginine deficiency in ischemic acute kidney injury results.

Hypertension is a major risk factor for many cardiovascular diseases and leads to subsequent concomitant pathologies such as left ventricular hypertrophy (LVH). Translational approaches using large animals get more important as they allow the use of standard clinical procedures in an experimental setting. Therefore, the aim of this study was to establish a minimally invasive ovine hypertension model using chronic angiotensin II (ANG II) treatment and to characterize its effects on cardiac remodeling after 8 weeks. Sheep were implanted with osmotic minipumps filled with either vehicle control ( n = 7) or ANG II ( n = 9) for 8 weeks. Mean arterial blood pressure in the ANG II‐treated group increased from 87.4 ± 5.3 to 111.8 ± 6.9 mmHg ( P = 0.00013). Cardiovascular magnetic resonance imaging showed an increase in left ventricular mass from 112 ± 12.6 g to 131 ± 18.7 g after 7 weeks ( P = 0.0017). This was confirmed by postmortem measurement of left ventricular wall thickness which was higher in ANG II‐treated animals compared to the control group (18 ± 4 mm vs. 13 ± 2 mm, respectively, P = 0.002). However, ANG II‐treated sheep did not reveal any signs of fibrosis or inflammatory infiltrates as defined by picrosirius red and H&E staining on myocardial full thickness paraffin sections of both atria and ventricles. Measurements of plasma high‐sensitivity C‐reactive protein and urinary 8‐iso‐prostaglandin F 2 α were inconspicuous in all animals. Furthermore, multielectrode surface mapping of the heart did not show any differences in epicardial conduction velocity and heterogeneity. These data demonstrate that chronic ANG II treatment using osmotic minipumps presents a reliable, minimally invasive approach to establish hypertension and nonfibrotic LVH in sheep.

Background: An investigation was conducted to determine the fate of the iridoid derivative deacetylasperulosidic acid (DAA) after oral application to mice. Methods: DAA was extracted from Morinda citrifolia leaf and purified by preparative HPLC. The identity was verified by MS and NMR spectroscopy. A sample of DAA was radioactively labelled with tritium and applied to mice by gavage. The pharmacokinetic of the radioactivity was investigated in blood, organs, urine and feces. Metabolites were isolated in blood and urine by HPLC and identified by LC-MS. In vitro incubation of DAA with mouse duodenum and liver homogenate and human fecal bacteria was performed and possible metabolites were separated with HPLC. Results: DAA was rapidly absorbed and excreted mainly via the kidneys with a half-life of 30 minutes. Radioactivity was present in all organs with highest concentrations in kidney and liver. Almost 100% of the radioactivity isolated from urine and organs could be identified as unchanged DAA. Additionally, no metabolism could be observed after in vitro incubation of DAA with mouse small intestine or liver homogenate, however, a total breakdown of the molecule was observed after incubation of DAA with human intestinal bacteria. Conclusions: The absorption and excretion of glycosides such as DAA in mammals without hydrolysis is possibly a defense mechanism of animals against the toxicity of these compounds. Key words: Deacetylasperulosidic acid, DAA, tritium label, pharmacokinetic,iridoid, metabolism

International audience; OBJECTIVES: Within the past decade, biochemical markers have emerged as attractive tools to assess pulmonary arterial hypertension (PAH) prognosis, being noninvasive and easily repeatable.The objective of this study was to determine whether biomarkers measured at initial diagnostic right-sided heart catheterization predict 3-year all-cause mortality for incident cases of PAH independently of clinical and hemodynamic parameters. METHODS: Patients with incident PAH were enrolled between December 2003 and April 2006 in six centers from the French Network on Pulmonary Hypertension and followed for 3 years.Venous blood samples were taken during right-sided heart catheterization, and analyses were centralized. RESULTS: Among 110 enrolled patients, 11 underwent lung or heart/lung transplantation, and 27 died during follow-up. The Kaplan-Meier estimates of survival were 91%, 78%, and 75% at 1, 2, and 3 years, respectively. Plasma big endothelin-1 (hazard ratio [HR] per 1-SD increase, 1.48; 95% CI,1.14-1.92), serum troponin T . 0.01 mg/L (HR, 2.35; 95% CI, 1.05-5.29), and urinary F 2 -isoprostanes(15-F2t -isoprostane) (HR per 1-SD increase, 1.76; 95% CI, 1.31-2.36) were associated with increased unadjusted hazard of death. In multivariate analysis adjusting for patient characteristics, the level of urinary F 2 -isoprostanes was the only biomarker that remained independently associated with increased hazard of death (HR per 1-SD increase, 1.82; 95% CI, 1.28-2.60). CONCLUSIONS: This study shows that levels of urinary F 2 -isoprostane, a biomarker of lipid peroxidation,quantified at initial diagnostic right-sided heart catheterization are independently associated with mortality in a cohort of patients with incident PAH.

Hypertension, a major risk factor for cardiovascular disease worldwide, is increasing significantly in urbanised South Africans. Impaired glomerular filtration is a potential contributor to hypertension. Although HIV infection is widespread, little is known regarding its contribution to diminished estimated glomerular filtration rate (eGFR) and, in turn, hypertension in Africans. We compared eGFRs and cardiovascular profiles of newly identified HIV infected African men (N=53) not yet undergoing anti-retroviral therapy, and uninfected African men of similar age and anthropometry. The aim of the study was to determine whether eGFR is diminished in treatment naive HIV infected individuals and whether eGFR is associated with a potential modulator of hypertension, namely serum L-arginine. Cardiovascular risk factor profiles of HIV infected and uninfected men were similar. In men with healthy eGFRs90 ml min(-1) per 1.73 m(2), eGFR was significantly lower with HIV infection (114 (90; 147)) compared with that in uninfected men: (120 (91; 168)), P=0.043. Despite the absence of clinically-diagnosed renal dysfunction, eGFR associated significantly with serum L-arginine only in HIV infected men (R(2)=0.277, β=-0.299, P=0.034), whereas L-arginine did not stay in the model for uninfected men. This difference suggests that the fate of L-arginine as a substrate for nitric oxide generation may be altered in HIV infected individuals. Subsequently this is likely to escalate endothelial dysfunction, contributing to later hypertension and cardiovascular disease. Our findings show that while glomerular filtration rate is not associated with L-arginine in uninfected men, it is diminished and significantly negatively associated with serum L-arginine in HIV infected men.

BACKGROUND Symmetric dimethylarginine (SDMA) is a by-product of protein methylation. Once released from proteins, SDMA is eliminated by the kidneys; consequently, plasma concentration has been suggested as a sensitive marker of renal function. Furthermore, recent work implicates SDMA in the pathogenesis of cardiovascular disease. To date, reference limits for SDMA plasma concentrations in healthy individuals are lacking. METHODS This study defined reference limits for plasma SDMA concentrations in 840 relatively healthy individuals of the Offspring Cohort from Framingham Heart Study (mean age 56 years, 61% women). Plasma SDMA concentrations were determined by LC-MS/MS using a stable isotope dilution assay. RESULTS The median SDMA concentration in the reference sample was 0.37 μmol/L (Q1, Q3:0.32, 0.43 μmol/L) and the reference limits were 0.225 and 0.533 (2.5th and 97.5th percentile). In a multivariable regression model, serum creatinine, age and total homocysteine were positively associated with SDMA (p

Conjugated linoleic acid (CLA) showed a wide range of beneficial biological effects with relevance for cardiovascular health in animal models and humans. Most human studies used olive oil as a reference. This study assessed the effect of CLA as compared with safflower oil on endothelial function and markers of cardiovascular risk in overweight and obese men. Heated safflower oil and olive oil were given for additional descriptive control.Eighty-five overweight men (aged 45-68 years, body mass index 25-35 kg/m(2)) were randomized to receive 4.5 g/d of the CLA isomeric mixture, safflower oil, heated safflower oil, or olive oil in a 4-week double-blind study. Endothelial function was assessed by peripheral arterial tonometry (PAT) index determination in the fasting and postprandial state (i.e., 4 hours after consumption of a fat- and sucrose-rich meal).CLA as compared with safflower oil consumption did not impair fasting or postprandial PAT index but decreased body weight. CLA as compared with safflower oil did not change total, low-density lipoprotein (LDL), or high-density lipoprotein (HDL) cholesterol; triglycerides; insulin sensitivity indices; C-reactive protein; soluble adhesion molecules; oxidized LDL; lipoprotein a (Lp[a]); paraoxonase; or platelet-activating factor acetylhydrolase (PAF-AH) activity, but significantly reduced arylesterase activity and increased concentrations of the F(2)-isoprostane 8-iso-prostaglandin F (PGF)(2α).CLA did not impair endothelial function. Other parameters associated with metabolic syndrome and oxidative stress were not changed or were slightly improved. Results suggest that CLA does not increase cardiovascular risk. Increased F(2)-isoprostane concentrations in this context may not indicate increased oxidative stress.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthases. By inhibiting nitric oxide formation, ADMA causes endothelial dysfunction, vasoconstriction, elevation of blood pressure, and aggravation of experimental atherosclerosis. Levels of ADMA and its isomer symmetric dimethylarginine (SDMA), which does not inhibit nitric oxide synthesis, are both elevated in patients with kidney disease. Currently available data from prospective clinical trials in patients with chronic kidney disease suggest that ADMA is an independent marker of progression of renal dysfunction, vascular complications and death. High SDMA levels also negatively affect survival in populations at increased cardiovascular risk, but the mechanisms underlying this effect are currently only partly understood. Beyond glomerular filtration, other factors influence the plasma concentrations of ADMA and SDMA. Elevated plasma concentrations of these dimethylarginines might also indirectly influence the activity of nitric oxide synthases by inhibiting the uptake of cellular L-arginine. Other mechanisms may exist by which SDMA exerts its biological activity. The biochemical pathways that regulate ADMA and SDMA, and the pathways that transduce their biological function, could be targeted to treat renal disease in the future.

Methylarginines have been shown to interfere with NO (nitric oxide) formation by inhibiting NOS (NO synthase)–ADMA (asymmetric dimethylarginine) and cellular L-arginine uptake into the cell [ADMA and SDMA (symmetric dimethylarginine)]. In a recent study, elevation of SDMA was related to long-term mortality in patients recruited 30 days after a stroke event. In the present study, we aimed at investigating the association of SDMA and adverse clinical outcome in the early phase (first 30 days) after acute ischaemic stroke. A total of 137 patients were recruited immediately upon admission to the emergency unit with an acute ischaemic stroke. Plasma levels of methylarginines were determined by a validated LC–MS/MS (liquid chromatography–tandem MS) method. Patients were prospectively followed for 30 days. A total of 25 patients (18.2%) experienced the primary composite endpoint [death, recurrent stroke, MI (myocardial infarction) and rehospitalization]. SDMA plasma levels were significantly higher in stroke patients compared with patients without event (0.89±0.80 compared with 0.51±0.24 μmol/l; P

Antidiabetic thiazolidinediones (TZDs) improve endothelial function in patients with or without type 2 diabetes. The present randomised, placebo-controlled, double-blind study examined the time course of a single dose of rosiglitazone on flow-mediated endothelium-dependent vasodilation (FMD), metabolic parameters, and its effect on inflammatory markers in non-diabetic men. Forty non-obese, healthy men with normal glucose tolerance were randomised to a single dose of rosiglitazone (8 mg) or placebo, and FMD was assessed at baseline as well as after 6 h and 24 h. Rosiglitazone did not significantly affect blood glucose and insulin levels or lipid parameters after 6 and 24 h compared with placebo. Treatment with rosiglitazone significantly increased FMD after 6 h from 4.3% (3.3; 4.9) to 7.6% (5.6; 9.2) ( p

Background and Purpose— Asymmetrical dimethylarginine (ADMA), an inhibitor of endothelial nitric oxide synthase, is a marker of endothelial dysfunction. Elevated circulating ADMA concentrations have been associated with systemic and carotid atherosclerosis, an elevated risk of developing stroke, and magnetic resonance imaging white-matter hyperintensities (WMHs). The relation of plasma ADMA to subclinical vascular brain injury has not been previously studied in a middle-aged, community-based sample. Methods— In 2013 stroke-free Framingham offspring (mean±SD age, 58±9.5 years; 53% women), we related baseline plasma ADMA levels (1995–1998) to subsequent brain magnetic resonance imaging measures (1999–2004) of subclinical vascular injury: presence of silent brain infarcts (SBIs) and large white-matter hyperintensity volumes (LWMHs; defined as >1 SD above the age-specific mean). Results— Prevalences of SBIs and LWMHs were 10.7% and 12.6%, respectively. In multivariable analyses adjusting for age, sex and traditional stroke risk factors, higher ADMA levels were associated with an increased risk of prevalent SBIs (odds ratio [OR] per 1-SD increase in ADMA=1.16; 95% CI, 1.01 to 1.33; P =0.04). We observed that participants in the upper 3 age-specific quartiles (Qs) of plasma ADMA values had an increased prevalence of SBIs (OR for Q2–Q4 vs Q1=1.43; 95% CI, 1.00 to 2.04; P Conclusions— Higher plasma ADMA values were associated with an increased prevalence of SBIs, after adjustment for traditional stroke risk factors. Thus, ADMA may be a potentially useful new biomarker of subclinical vascular brain injury, which is an important correlate of vascular cognitive impairment and risk of stroke.

Background: Methylated arginines are endogenous analogues of L-arginine, the substrate for nitric oxide (NO) synthase. Asymmetric dimethylarginine (ADMA) interferes with NO formation, causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in humans. It is eliminated primarily by enzymatic activity of dimethylarginine dimethylaminohydrolase (DDAH). Methodology/Principal Findings: We investigated whether human DDAH-1 (hDDAH-1) transgenicity protects from ischemic tissue damage in temporary middle cerebral artery occlusion (tMCAO) in mice. Infarct sizes did not significantly differ between hDDAH-1 transgenic (TG) mice and wild-type littermates (WT). As expected, ADMA plasma concentrations were significantly decreased, cerebral hDDAH expression and protein significantly increased in transgenic animals. Interestingly, neither brain tissue DDAH activity nor ADMA concentrations were different between TG and WT mice. In contrast, muscular DDAH activity was generally lower than in brain but significantly increased in TG mice. Conclusion/Significance: Our study demonstrates that hDDAH-1 transgenic mice are not protected from ischemic cerebral tissue damage in tMCAO. This lack of protection is due to high basal cerebral DDAH activity, which is not further increasable by transgenic overexpression of DDAH.

Background: Accumulating evidence links higher circulating asymmetric dimethylarginine (ADMA) to greater risk of cardiovascular disease (CVD). Relatively small differences in ADMA concentrations between healthy individuals and those with disease underscore the need to formulate reference intervals that may aid risk stratification of individuals.Methods: We formulated reference intervals for plasma ADMA concentrations using a community-based reference sample from the Framingham Offspring Study consisting of 1126 nonsmoking individuals [mean (SD) age 56 (9) years; 60% women] who were free of clinical CVD, hypertension, diabetes, and obesity and who attended a routine examination at which ADMA was assayed. ADMA concentrations were determined using a validated tandem mass spectrometry–liquid chromatography assay.Results: In the study sample, the mean ADMA concentration was 0.52 (0.11) μmol/L, and the reference limits were 0.311 and 0.732 (2.5th and 97.5th percentile). The sex-specific reference limits were 0.310 and 0.745 in men and 0.313 and 0.721 μmol/L in women. In multivariable regression analysis, ADMA plasma concentrations were positively correlated with age and total plasma homocysteine (both P < 0.001).Conclusions: Reference limits calculated for circulating ADMA in our large community-based healthy reference sample confirm the previous observation of a relatively narrow distribution of concentrations. This suggests a tight physiological control of ADMA plasma concentrations, presumably by dimethylarginine dimethylaminohydrolase (DDAH) metabolism of ADMA.

Background and Purpose— Higher plasma concentrations of the endogenous nitric oxides synthase inhibitor asymmetrical dimethylarginine (ADMA) are associated with increased risk of cardiovascular and cerebrovascular events and death, presumably by promoting endothelial dysfunction and subclinical atherosclerosis. We hypothesized that plasma ADMA concentrations are positively related to common carotid artery intimal-media thickness (CCA-IMT) and to internal carotid (ICA)/bulb IMT. Methods— We investigated the cross-sectional relations of plasma ADMA with CCA-IMT and ICA/bulb IMT in 2958 Framingham Heart Study participants (mean age, 58 years; 55% women). Results— In unadjusted analyses, ADMA was positively related to both CCA-IMT (β per SD increment, 0.012; P P P =0.991), but remained significantly and positively related to ICA/bulb IMT (β per SD increment, 0.0246; P =0.002). Conclusions— In our large community-based sample, we observed that higher plasma ADMA concentrations were associated with greater ICA/bulb IMT, but not with CCA-IMT. These data are consistent with the notion that ADMA promotes subclinical atherosclerosis in a site-specific manner, with a greater proatherogenic influence at known vulnerable sites in the arterial tree.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthases. By inhibiting NO formation, ADMA causes endothelial dysfunction, vasoconstriction, elevation of blood pressure, and aggravation of experimental atherosclerosis. Cross-sectional studies in humans have revealed that ADMA plasma concentration is elevated in numerous populations with vascular diseases or at high cardiovascular risk. However, the potential causal relationship between elevated ADMA and cardiovascular events and mortality in humans can only be revealed in prospective clinical studies. This review gives an overview of currently available data from prospective clinical studies in which ADMA has been measured in populations at high, intermediate, or low global vascular risk. Although the analytical methods used to quantify ADMA varied and statistical approaches to assess the association of ADMA with risk differed, these data reveal that ADMA is significantly associated with an increased risk of incident cardiovascular events and total mortality in subjects at a broad range of global risk. Hazard ratios were mostly in a range comparable to that of traditional cardiovascular risk markers even after multivariable adjustments, suggesting that ADMA may be suitable as a diagnostic marker for risk assessment, and that the biochemical pathways that regulate ADMA may be promising therapeutic approaches to treat cardiovascular disease in the future.

Background Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of endothelial nitric oxide synthase (eNOS) that is associated with endothelial dysfunction, and is a risk marker for cardiovascular disease, a significant problem in Type 1 diabetes. The aim of the present study was to measure circulating ADMA, and define its association with endothelial dysfunction and endothelial markers in people with Type 1 diabetes with low likelihood of macrovascular disease. Methods Sixty-one young people with Type 1 diabetes without macrovascular disease or nephropathy and 62 healthy volunteers underwent brachial artery flow-mediated dilatation (FMD) and assay of plasma ADMA and adhesion molecules. Results Age, gender, BMI, lipid profile and renal function were similar in the two groups. People with Type 1 diabetes had impaired FMD compared to healthy controls (5.0 ± 0.4 vs 8.9 ± 0.4%; p < 0.001). Plasma ADMA levels were significantly lower in the people with diabetes compared to healthy controls (0.52 ± 0.12 vs 0.66 ± 0.20 μmol/l, p < 0.001). Plasma ICAM-1, E-selectin and PAI-1 levels were significantly higher in people with diabetes compared to healthy controls (median 201 (IQR 172–226) vs 180 (156–216) μg/l, p = 0.027; 44.2 (32.6–60.9) vs. 33.1 (22.4–51.0) μg/l; p = 0.003 and 70.8 (33.3–85.5) vs 46.3 (23.9–76.8) μg/l, p = 0.035). Plasma ADMA and VCAM-1 levels were positively correlated (r = 0.37, p = 0.003) in people with diabetes. There was no correlation between the plasma ADMA and FMD. Conclusion ADMA levels are not associated with endothelial dysfunction in young adults with Type 1 diabetes without microalbuminuria or known macrovascular disease. This suggests that the impaired endothelial function in these individuals is not a result of eNOS inhibition by ADMA.

Methylarginines have been shown to interfere with nitric oxide (NO) formation by inhibiting NO synthase (asymmetric dimethylarginine, ADMA, and monomethylarginine, NMMA) and the cellular l-arginine uptake system (ADMA, NMMA and symmetric dimethylarginine, SDMA), thereby causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in diverse populations.We investigated whether methylarginines are predictors of mortality in 394 patients after acute ischemic stroke during 7.4 years of follow-up.Patients who died (N=231) were older and more frequently had one of the traditional risk factors for stroke (previous stroke/TIA, atrial fibrillation, prevalent ischemic heart disease, peripheral vascular disease, each p0.05). ADMA (0.52 micromol/l vs. 0.50 micromol/l, p=0.015) and SDMA (0.56 micromol/l vs. 0.43 micromol/l, p0.001) were higher in patients who died. In multivariable-adjusted hazard models, SDMA but not ADMA or NMMA was an independent predictor of all-cause mortality after stroke (SDMA, hazard ratio 2.41 (1.55-3.72), p0.001; ADMA, hazard ratio 1.43 (0.99-2.07), p=0.06). SDMA was significantly associated with atrial fibrillation (0.55 micromol/l vs. 0.50 micromol/l, p=0.03) but there was no significant interaction between SDMA and AF in relation to mortality (p=0.81). SDMA remained significantly associated with mortality after adjusting for eGFR and also additionally adjusting for C-reactive protein, albumin, beta-thromboglobulin, and von Willebrand factor.Our study demonstrates that SDMA is an independent predictor of total mortality after acute stroke irrespective of renal function. SDMA is associated with atrial fibrillation, endothelial and platelet activation, and may therefore play a previously unknown role in the pathophysiology of stroke.

We recently noticed a possible triglyceride-lowering effect during dietary supplementation with L-arginine. The major limitation of prior studies on L-arginine, however, was that triglyceride levels were not the primary end point, and patients were not necessarily hypertriglyceridemic. Therefore, we conducted a 2-arm, randomized, double-blind study in 33 hypertriglyceridemic patients to investigate the hypothesis that oral L-arginine may lower serum triglyceride levels in hypertriglyceridemic patients on and off statins. The study consisted of a 6-week run-in phase, 6 weeks of treatment with L-arginine (n = 22, 1.5 g bid) or placebo (n = 11), and a 6-week extension period where simvastatin (20 mg qd) was added. All patients received dietary advice during each study visit. Routine and lipid laboratory parameters were determined in the local routine clinical laboratory. Treatment with L-arginine alone had no effects on serum lipids compared to placebo. The combination of L-arginine with simvastatin led to a significantly stronger reduction in triglycerides compared to placebo plus simvastatin (-140.5 +/- 149.2 mg/dL vs -56.1 +/- 85.0 mg/dL; P = .048). In addition, we found simvastatin-induced increases in aspartate transaminase and fibrinogen to be attenuated by L-arginine as compared to placebo. We conclude from our data that L-arginine enhances the effects of simvastatin on lipid metabolism, but it has no triglyceride-lowering effects when given alone.

Objectives and methods Methylarginines like asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are formed by post-translation methylation of arginine residues in proteins. ADMA inhibits nitric-oxide synthase and predicts clinical outcomes in various diseases including end stage renal disease (ESRD). SDMA competes with l-arginine for cell entry and is associated with organ failure in patients with severe illness. We investigated the inter-relationships between methylargines, l-arginine and other risk factors and tested the prediction power of methylargines for mortality in a prospective cohort study including 288 ESRD patients. Results ADMA and SDMA exceeded the upper limit of the corresponding normal range in almost all cases (98% and 100%, respectively) and were inter-related (r = 0.30, P

Infusion of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) causes an elevation of blood pressure and depression of cardiac output. Polymorphisms in the promoter region of the ADMA-degrading enzyme dimethylarginine dimethylaminohydrolase 2 (DDAH2) gene have been associated with elevated ADMA concentrations and adverse outcomes in critically ill patients. We hypothesized that two DDAH2 promoter -1151 A/C and -449 G/C polymorphisms (rs805304 and rs805305) will be associated with blood pressure levels, hypertension prevalence and measures of cardiac structure and function in the general population.We genotyped rs805304 and rs805305 in 783 participants of the population-based Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA) Augsburg S3 study. Plasma ADMA concentrations did not differ by rs805304 and rs805305 genotypes. Both polymorphisms were associated with a higher prevalence of hypertension. The odds ratio (adjusted for age, gender and body mass index) for hypertension was 1.70 (95%CI: 1.22-2.36: p=0.002) for those homozygous (n=348) for the -1151A allele and 1.80 (95%CI: 1.29-2.49, p0.001) for individuals homozygous for the -449G allele (n=350). However, both polymorphisms were not related to measures of cardiac structure and function (left ventricular [LV] mass, LV wall thickness, LV end-diastolic diameter, ejection fraction, E/A ratio, isovolumetric relaxation time) in multivariable-adjusted models.The present study indicates that the -1151 A/C and -449 G/C polymorphisms in the DDAH2 promoter region are not related to plasma ADMA levels or measures of cardiac structure and function but are associated with an increased prevalence of hypertension. The mechanisms of this association need further investigation.

Isoprostanes are endogenously formed end products of lipid peroxidation. Furthermore, they are markers of oxidative stress and independent risk markers of coronary heart disease. In patients experiencing coronary heart disease, impaired angiogenesis may exacerbate insufficient blood supply of ischemic myocardium. We therefore hypothesized that isoprostanes may exert detrimental cardiovascular effects by inhibiting angiogenesis. We studied the effect of isoprostanes on vascular endothelial growth factor (VEGF)-induced migration and tube formation of human endothelial cells (ECs), and cardiac angiogenesis in vitro as well as on VEGF-induced angiogenesis in the chorioallantoic membrane assay in vivo. The isoprostanes 8-iso-PGF(2alpha), 8-iso-PGE(2), and 8-iso-PGA(2) inhibited VEGF-induced migration, tube formation of ECs, and cardiac angiogenesis in vitro, as well as VEGF-induced angiogenesis in vivo via activation of the thromboxane A(2) receptor (TBXA2R): the specific TBXA2R antagonists SQ-29548, BM 567, and ICI 192,605 but not the thromboxane A(2) synthase inhibitor ozagrel blocked the effect of isoprostanes. The isoprostane 8-iso-PGA(2) degraded into 2 biologically active derivatives in vitro, which also inhibited EC tube formation via the TBXA2R. Moreover, short hairpin RNA-mediated knockdown of the TBXA2R antagonized isoprostane-induced effects. In addition, Rho kinase inhibitor Y-27632 reversed the inhibitory effect of isoprostanes and the thromboxane A(2) mimetic U-46619 on EC migration and tube formation. Finally, the various isoprostanes exerted a synergistic inhibitory effect on EC tube formation. We demonstrate for the first time that isoprostanes inhibit angiogenesis via activation of the TBXA2R. By this mechanism, isoprostanes may contribute directly to exacerbation of coronary heart disease and to capillary rarefaction in disease states of increased oxidative stress.

Background Effects independent from cholesterol reduction on vascular function are considered to importantly contribute to the beneficial effects of statin therapy in cardiovascular disease. We aimed to evaluate the effect of high versus low dose atorvastatin on endothelial dysfunction in patients with coronary artery disease (CAD) in a setting of comparable cholesterol reduction. Methods and results Fifty-eight patients with CAD were randomly assigned to double-blind treatment for 8 weeks with atorvastatin 80mg per day (A80) or atorvastatin 10mg+ezetimibe 10mg per day (A10E10), respectively. Flow-mediated vasodilation (FMD) of the brachial artery, nitroglycerin-mediated endothelium-independent vasodilation (NMD), lipid, C-reactive protein (CRP) plasma concentrations and urinary 8-iso-prostaglandin F2alpha excretion were measured before and after treatment. Total cholesterol, triglycerides and LDL-cholesterol levels were significantly reduced with no difference between A80 and A10E10. A80 caused significantly stronger improvement of FMD compared to A10E10 (absolute change FMD: A80+2.7±3.0% (post vs. pre p p =0.25), A80 vs. A10E10 p =0.018). NMD was improved by A80 but not by A10E10 (absolute change NMD: A80+2.7±4.6%, A10E10+0.7±3.5%, p =0.12). Both treatment groups caused a comparable reduction of CRP and did not effect urinary 8-iso-prostaglandin F2alpha excretion. There was no correlation between FMD or NMD change and LDL-cholesterol change in either treatment group. Conclusions The present findings clearly suggest that in the presence of comparable LDL-lowering effects of both treatment forms, LDL-cholesterol independent effects of high dose atorvastatin therapy account for the improvement of endothelium-dependent vasodilation in patients with stable CAD.

Background: Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase and may alter NO production during pathological conditions. Concerning Alzheimer’s disease (AD), there are reports on altered cerebral NO metabolism, but only few studies on ADMA concentrations in plasma and cerebrospinal fluid (CSF). Methods: We assessed plasma ADMA in 80 AD patients and 80 age- and gender-matched controls and CSF ADMA in a subgroup of 53 AD patients and 20 controls. Results: ADMA plasma concentrations were increased, while CSF ADMA concentrations were decreased in AD patients. There was a significant association between decreasing CSF ADMA levels and the severity of cognitive impairment. Conclusion: Elevated ADMA in plasma might be a contributing factor for AD through alterations of NO metabolism, for example decreased cerebral microperfusion, while decreased levels of CSF ADMA might lead to a cerebral increase of NO, peroxynitrite production and oxidative protein damage. Our study reveals different mechanisms of plasma and CSF ADMA regulation, both potentially contributing to AD pathology.

Renin-angiotensin system blockade reduces proteinuria and prevents nephropathy progression in patients with type 2 diabetes mellitus (T2D). Experimental evidence demonstrates that angiotensin receptor blockers (ARBs) possess anti-inflammatory potential, which might contribute to reducing proteinuria and providing renoprotection.We conducted a multicentre, double-blind, prospective, parallel-group non-inferiority study of 885 hypertensive [systolic blood pressure/diastolic blood pressure (SBP/DBP)130/80 mmHg] patients with T2D, proteinuria (or =900 mg/24 h) and serum creatinine (or =3.0 mg/dl) who were randomized to once-daily telmisartan 80 mg or valsartan 160 mg; additional antihypertensive therapy was permitted. The primary endpoint was the change from baseline in the 24-h proteinuria after 12 months. Secondary endpoints included changes in 24-h albuminuria, estimated glomerular filtration rate (eGFR) and inflammatory parameters asymmetrical dimethylarginine (ADMA), high-sensitivity C-reactive protein (CRP) and urinary 8-iso-prostaglandin F(2alpha) (8-iso-PGF(2alpha)).Telmisartan and valsartan produced comparable reductions in 24-h urinary protein excretion rates: geometric mean reduction (95% confidence interval) [telmisartan, 33% (27-39%); valsartan, 33% (27-38%)]. No significant differences between treatments were seen in changes from baseline in 24-h urinary albumin excretion rate and eGFR at 12 months. With both treatments, greater renoprotection was seen among patients with better blood pressure control. No significant changes in ADMA or CRP were noted in either group after 12 months, but urinary 8-iso-PGF(2alpha) levels decreased by 14% with telmisartan and by 7% with valsartan (P = 0.040).In patients with T2D, hypertension and overt nephropathy, the renoprotection afforded by telmisartan and valsartan appears similar, and the study was unable to show any effect beyond that due to blood pressure control. At doses used to treat hypertension, there is no evidence of inflammatory parameters being modified by ARBs in patients with more advanced kidney disease due to T2D.

Background: Endothelial function is impaired in hypercholesterolemia and atherosclerosis. Based on mostly indirect evidence, this impairment is attributed to reduced synthesis or impaired biological activity of endothelium-derived nitric oxide (NO). It was the aim of this study to directly estimate and compare whole-body NO production in normo- and hypercholesterolemia by applying a nonradioactive stable isotope dilution technique in vivo. Methods: We enrolled 12 normocholesterolemic and 24 hypercholesterolemic volunteers who were all clinically healthy. To assess whole-body NO synthesis, we intravenously administered l-[guanidino-(15N2)]-arginine and determined the urinary excretion of 15N-labeled nitrate, the specific end product of NO oxidation in humans, by use of gas chromatography-mass spectrometry. In addition, we measured flow-mediated vasodilation (FMD) of the brachial artery, expression of endothelial NOS (eNOS) in platelets, plasma concentration of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), and urinary excretion of 8-isoprostaglandin F2α (8-iso-PGF2α). Results: After infusion of l-[guanidino-(15N2)]-arginine, cumulative excretion of 15N-labeled-nitrate during 48 h was 40% [95% CI 15%–66%] lower in hypercholesterolemic than normocholesterolemic volunteers [mean 9.2 (SE 0.8) μmol vs 15.4 (2.3) μmol/l, P = 0.003]. FMD was on average 36% [4%–67%] lower in hypercholesterolemic than normocholesterolemic volunteers [6.3 (4.0)% vs 9.4 (4.6)%, P = 0.027]. Normalized expression of NOS protein in platelets was also significantly lower in hypercholesterolemic volunteers, whereas there were no significant differences in plasma ADMA concentration or urinary excretion of 8-iso-PGF2α between the 2 groups. Conclusions: This study provides direct evidence for a decreased whole body NO synthesis rate in healthy people with hypercholesterolemia.

What is already known about this subject • Genetic polymorphisms of the P2Y12 ADP receptor on platelets have been shown to contribute to variability in platelet aggregation in healthy humans. • P2Y12 ADP receptor polymorphisms are more frequently present in patients with vascular disease than in healthy people. • The majority of patients with vascular disease receive acetylsalicylic acid as an anti-aggregatory agent, which has also been shown to induce a variable response; however, the role of P2Y12 ADP receptor polymorphisms in the platelet response to acetylsalicylic acid in patients with vascular disease has not yet been studied. What this study adds • The present data show that the platelet response to acetylsalicylic acid is independent of the presence or absence of P2Y12 ADP receptor polymorphisms in patients with stable coronary artery disease who have had their first myocardial infarction. • This is important, as studies in healthy humans had suggested that carriers of P2Y12 ADP receptor polymorphisms may be at increased risk of experiencing cardiovascular events. • However, the observed variability of the platelet response to the cyclooxygenase inhibitor acetylsalicylic acid (in our study) and to the P2Y12 ADP receptor blocker clopidogrel (in a study by Angiolillo et al.[18]) in patients with coronary artery disease is clearly not determined by common P2Y12 ADP receptor polymorphisms. Aims Recently, two genetic polymorphisms of the platelet ADP receptor P2Y12 (haplotypes H2 and 34T) have been implicated in increased platelet aggregation and atherothrombotic risk. It was suggested that these polymorphisms contribute to a diminished response to antiplatelet drugs. Therefore, we investigated the effects of these polymorphisms on platelet aggregation in aspirin-treated patients with coronary artery disease (CAD). Methods Platelet aggregation was studied in platelet-rich plasma from 124 patients with CAD treated with 100 mg aspirin day−1. P2Y12 ADP receptor polymorphisms were determined by PCR-RFLP. The 52G > T polymorphism was used as tag-SNP for the H2 haplotype. Aggregation was induced by 1 mg l−1 collagen. In a subgroup (n = 72), a concentration-response curve to collagen (0.5–10 mg l−1), aggregation at 2 μmol l−1 ADP and 1 mmol l−1 arachidonic acid were determined. Results Whereas arachidonic acid-induced aggregation was inhibited in all patients, collagen and ADP-induced aggregation were highly variable. However, aggregation did not differ significantly between carriers and noncarriers of the 52T-allele (1 mg l−1 collagen: 32.7% (21.9–38.6%) vs. 32.5% (21.2–41.6%); P = 0.77; ADP: 33.1% (29.9–40.9%) vs. 39.1% (31.5–49.7%); P = 0.34), respectively. EC50 values were 1.26 mg l−1 (0.79–2.02) and 1.54 mg l−1 (0.98–2.4) collagen in noncarriers and carriers of the H2 haplotype, respectively (P = 0.56). Moreover, the 34°C > T polymorphism did not significantly affect any of the aggregatory responses. Conclusions Low-dose aspirin inhibits platelet aggregation to the same extent in patients carrying or not carrying the P2Y12 H2 haplotype and/or the 34T allele. Our data do not support the hypothesis that these polymorphisms contribute to an attenuated antiplatelet effect of aspirin.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • L-Arginine is a semiessential amino acid that is converted to nitric oxide (NO) by NO synthase (NOS). • NO improves endothelial function by elevating cyclic guanosine monophosphate. • However, oral L-arginine treatment in humans is hampered by extensive metabolism. WHAT THIS STUDY ADDS • Oral L-citrulline supplementation raises plasma L-arginine concentration and augments NO-dependent signalling in a dose-dependent manner. • L-Citrulline may thus be an alternative to L-arginine in patients with impaired NOS activity. AIMS Oral L-arginine supplementation has been used in several studies to improve endothelium-dependent, nitric oxide (NO)-mediated vasodilation. L-Arginine treatment is hampered by extensive presystemic elimination due to intestinal arginase activity. In contrast, L-citrulline is readily absorbed and at least in part converted to L-arginine. The aim of our study was to assess this metabolic conversion and its subsequent pharmacodynamic effects. METHODS In a double-blind, randomized, placebo-controlled cross-over study, 20 healthy volunteers received six different dosing regimes of placebo, citrulline, and arginine. Pharmacokinetic parameters (Cmax, Tmax, Cmin, AUC) were calculated after 1 week of oral supplementation. The ratio of plasma L-arginine over asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase (arginine/ADMA ratio), urinary cyclic guanosine monophosphate (cGMP) and nitrate excretion rates, and flow-mediated vasodilation (FMD) was measured to assess pharmacodynamic effects. RESULTS L-Citrulline dose-dependently increased AUC and Cmax of plasma L-arginine concentration more effectively than L-arginine (P

This randomized, single-blind, parallel-group study was performed to assess pharmacokinetic interactions potentially occurring during concomitant use of telmisartan and nisoldipine. Patients with essential hypertension (n = 37) were treated with once-daily doses of telmisartan, nisoldipine, or their combination for 6 weeks. The regimen was started at low dose with an increase of dosage after 3 weeks of treatment. AUC(ss) (132%; P < .01) of telmisartan applied in doses of 80 mg was significantly higher after concomitant application with nisoldipine (10 mg), whereas CL/f(ss) (-54%; P < .05) and Vz/f(ss) (-72%; P < .05) were significantly lower. Regarding pharmacokinetic parameters of nisoldipine, significant differences between treatment groups were not detected. In conclusion, the results of this study strongly suggest that concomitant treatment with nisoldipine enhances telmisartan bioavailability in hypertensive individuals. Larger crossover trials will have to establish these observations and investigate whether interaction of both drugs affects telmisartan efficacy and tolerability in clinical use.

What is already known about this subject • Isoprostanes are the product of free radical oxidation of arachidonic acid bound to phospholipids. • Their hydrolysis from phospholipids is presumably catalysed by phospholipases A2. • Atorvastatin reduces protein concentrations of secretory PLA2s and concentrations of LDL, with which PAF-AH (group VII phospholipase) is associated. What this study adds • Atorvastatin affects PAF-AH activity and this effect is strongly associated with its lipid-lowering effect, but it has no effect on groups IIA and V PLA2s' activity. • Thus, PAF-AH is no independent risk factor of cardiovascular diseases. • Moreover, a role of PAF-AH in the liberation of 15-F2t-isoP from phospholipids is excluded. Aims Isoprostanes are the product of free radical oxidation of arachidonic acid, whose hydrolysis from phospholipids is presumably catalysed by phospholipases A2 (PLA2s) such as group IIA or V PLA2s, or group VII PLA2[platelet-activating factor acetylhydrolase (PAF-AH), lipoprotein-associated phospholipase]. Atorvastatin reduces concentrations of low-density lipoprotein (LDL), with which PAF-AH is associated, and PLA2s' protein concentrations. We investigated the effect of atorvastatin on PLA2s and PAF-AH activity and the urinary excretion of 15-F2trans-isoprostane (15-F2t-IsoP, 8-iso-PGF2α, iPF2α-III). Methods Twenty-four hypercholesterolaemic individuals naive to lipid-lowering therapy were randomized to atorvastatin 40 mg or placebo for 6 weeks. The 15-F2t-isoP urinary excretion (gas chromatography/mass spectrometry), PAF-AH and group IIA and V PLA2 activities (photometry) were assessed at baseline and end-point. Results At end-point, 15-F2t-isoP urinary excretion concentrations as well as PLA2s' activity were unchanged under atorvastatin (mean change 0.21 ± 1.79 ng h−1, 95% confidence interval −0.92, 1.35 and 0.33 ± 0.94 nmol min−1 ml−1, −0.27, 0.93) and under placebo (mean change 0.69 ± 1.69 ng h−1, −0.52, 1.90 and 1.29 ± 2.16 nmol min−1 ml−1, −0.25, 2.84). Atorvastatin treatment decreased total (P

Recent studies have shown that in response to vascular damage or ischemia, bone marrow-derived endothelial progenitor cells (EPCs) are recruited into the circulation. To investigate whether antihypertensive treatment has an influence on the number of circulating EPCs, patients with essential hypertension were treated either with the angiotensin receptor antagonist telmisartan, the calcium channel blocker nisoldipine, or their combination for 6 weeks. At baseline and after 3 and 6 weeks of treatment, EPCs were identified and quantified by fluorescence-activated cell sorting (FACS) analysis and by their capacity to generate colony-forming units of the endothelial lineage (CFU-EC) in a methylcellulose-based assay. During treatment, patients in the nisoldipine groups, but not in the telmisartan group, showed a significant mobilization of EPCs, which in part had the capacity to generate large-sized colonies comprising more than 1,000 cells. Moreover, a remarkable correlation between the number of CFU-EC and the number of circulating CD133(+)/CD34(+)/CD146(+) cells was observed, thereby providing strong evidence that cells with this phenotype represent functional EPCs. No correlation was found between the numbers of CFU-EC and the blood pressure levels at any time point during the treatment. Hence, nisoldipine-induced mobilization of EPCs might represent a novel mechanism by which this antihypertensive compound independently of its blood pressure-lowering effect contributes to vasoprotection in patients with essential hypertension.

Elevated circulating ADMA levels have been proposed as the pivotal link between insulin resistance, cardiovascular disease, and endothelial dysfunction in Caucasian population. To evaluate whether there is an association between plasma ADMA concentrations and insulin resistance in Hispanic population, we identified metabolic syndrome NCEP-ATP III criteria and measured ADMA and L-arginine plasma concentrations in 147 Colombian young males consecutively included in a cross-sectional study. In contrast to inflammatory markers, ADMA was not found to be elevated in subjects with metabolic syndrome, furthermore, no significant association between ADMA concentrations and insulin resistance degree was found. In conclusion, our results suggest that at least in our population, ADMA does not seem to be implicated in the pathophysiology of metabolic syndrome. Ethnic-specific or environmental differences in the etiologic mechanisms of metabolic syndrome need to be elucidated in further studies.

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-μl 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 μM in healthy humans ( n = 85), respectively, and 44 ± 14, 0.72 ± 0.23, and 0.19 ± 0.06 μM 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.

We hypothesized that the level of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial nitric oxide (NO) synthase (eNOS), might determine the endothelial effects of statins.Endothelial NO synthase is up-regulated by statins. However, statins failed to improve endothelial function in some studies. Asymmetric dimethylarginine inhibits eNOS by a mechanism that is reversible by L-arginine.Ninety-eight clinically asymptomatic elderly subjects had their plasma ADMA levels screened. Those in the highest (high ADMA, n = 15) and lowest quartiles of the ADMA distribution (low ADMA, n = 13) were eligible to receive, in a randomized order, simvastatin (40 mg/day), L-arginine (3 g/day), or a combination of both, each for 3 weeks. Endothelium-dependent vasodilation (EDD) was assessed by brachial artery ultrasound.Simvastatin had no effect on EDD in subjects with high ADMA (6.2 +/- 1.2% vs. 6.1 +/- 0.9%), whereas simvastatin plus L-arginine significantly improved EDD (9.8 +/- 1.5% vs. 5.3 +/- 0.8%; p0.01). In subjects with low ADMA, simvastatin improved endothelial function when given alone (9.5 +/- 3.2% vs. 6.1 +/- 3.8%; p0.001) or in combination with L-arginine (9.0 +/- 3.1% vs. 6.3 +/- 3.3%; p = 0.001). L-arginine alone improved endothelial function in both groups. Endothelium-independent vasodilation was not affected.Simvastatin does not enhance endothelial function in subjects with elevated ADMA, whereas it does so in patients with low ADMA. Combination of simvastatin with oral L-arginine improves endothelial function in subjects with high ADMA, but has no additional effect in subjects with low ADMA. As NO-mediated effects may play a major role in the therapeutic effects of statins, ADMA concentration is an important factor that influences the "pleiotropic" effects of simvastatin.

Asymmetric dimethylarginine (ADMA) plasma levels have been shown to be elevated in diseases related to endothelial dysfunction such as hypertension, hyperlipidemia, diabetes mellitus, and others. It has been shown that ADMA predicts cardiovascular mortality in patients who have coronary heart disease (CHD). However, the question whether ADMA is an independent risk factor for CHD still remains unresolved.The CARDIAC study is a multicenter case-control study, designed to detect differences in ADMA plasma levels between patients with CHD and controls from the general population. We included in our analysis 131 cases and 131 controls, matched for age, sex, and body mass index.We found that cases had higher ADMA plasma levels than controls (0.70 micromol/L [0.59-0.87 micromol/L] vs 0.60 micromol/L [0.54-0.69 micromol/L], P.001). To evaluate the predictive power of ADMA regarding CHD, we calculated 2 multivariate logistic regression models including laboratory parameters and traditional risk factors. The odds ratio for ADMA in the multivariate model including the laboratory characteristics was 2.59 (1.61-4.17; P.001); the odds ratio for the multivariate model including other risk factors was 6.04 (2.56-14.25; P.001) for the third tertile (0.72 micromol/L) versus the first (0.58 micromol/L).We conclude from the results of our study that ADMA is an independent risk factor for CHD.

The renin angiotensin system has been shown to be involved in the patho genesis of vascular and renal sequelae of diabetes mellitus. In type 2 diabetes mel litus, angiotensin receptor blockers have been shown to exert clinical benefit by reducing the progression of diabetic nephropathy. They also improve endothelium- mediated vascular function. The latter effect is partly due to the reduction of angiotensin II-associated oxidative stress. Moreover, small clinical studies have shown that treatment with angiotensin receptor blockers also reduces the circulating levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase. In the VIVALDI trial, the ability of the angiotensin receptor blocker telmisartan to reduce the progression of diabetic nephropathy (associated with proteinuria) in com parison with valsartan in more than 800 patients with type 2 diabetes during 1 year of treatment is being studied. In order to gain more detailed insight into the poten tial pathomechanisms associated with this effect, further end-points have been defined. Among these are the circulating levels of ADMA and the urinary excretion rate of 8-iso-prostaglandin F2α (8-iso-PGF 2α). The former is an endogenous inhibitor of NO-mediated vascular function(s) and a prospectively determined marker of major cardiovascular events and mortality; the latter is a lipid peroxidation product resulting from the nonenzymatic peroxidation of arachidonic acid, which exerts detrimental vascular effects similar to those of thromboxane A2. Urinary 8-iso-PGF 2α has been shown in clinical studies to be an independent marker of cardiovascular disease. Highlighting the effects of telmisartan on ADMA and 8-iso-PGF levels in such a large cohort of diabetic patients will enhance our understanding of the roles of dys functional NO metabolism and redox mechanisms in the pathogenesis of end-organ damage and its prevention by pharmacotherapy with angiotensin receptor blockers.

Microvascular dysfunction and increased oxidative stress are major hallmarks of the systemic sclerosis disease process. The primary objective of this study was to test whether there is a link between peak postocclusive hyperemia and urinary levels of the F2-isoprostane 15-F2t-IsoP (8-iso-PGF2alpha) in patients suffering from systemic sclerosis. We enrolled 43 patients suffering from systemic sclerosis, 33 patients with primary Raynaud's phenomenon (RP), and 25 healthy volunteers. Microvascular function was assessed using the postocclusive hyperemia monitored by laser Doppler flowmetry. Endothelium-independent response was monitored after 0.4 mg sublingual nitroglycerin. Oxidative stress status was assessed by urinary levels of the F2-isoprostane 15-F2t-IsoP using GC-MS. The peak postocclusive vascular conductance was altered in subjects with systemic sclerosis and primary RP compared to controls (respectively 28 (7-48), 30 (13-48), and 39.9 (13-63) mV/mm Hg, p = 0.01). F2-isoprostanes were increased in the systemic sclerosis group compared to primary Raynaud's phenomenon and healthy controls (respectively 230 (155-387), 182 (101-284), and 207 (109-291) pg/mg, p = 0.006). In patients suffering from systemic sclerosis, there was a significant inverse correlation between F2-isoprostanes and postocclusive hyperemia, expressed as raw data (R = -0.45, p = 0.007) or as an increase over baseline (R = -0.28, p = 0.04). Conversely, no correlation was found with the nitroglycerin response. In conclusion, we provide evidence that there is an inverse correlation between postocclusive hyperemia and urinary F2-isoprostane levels in patients suffering from systemic sclerosis. Whether oxygen free radicals initiate the vascular dysfunction or whether there is an initial trigger that initiates both processes will need to be further clarified in future studies.

The endothelium plays a crucial role in the maintenance of vascular tone and structure by releasing the endothelium-derived vasoactive mediator, nitric oxide (NO). NO is formed in healthy vascular endothelium from the amino acid precursor L-arginine. Endothelial dysfunction is caused by various cardiovascular risk factors, metabolic diseases, and systemic or local inflammation. One mechanism that explains the occurrence of endothelial dysfunction is the presence of elevated blood levels of asymmetric dimethylarginine (ADMA) – an L-arginine analogue that inhibits NO formation and thereby can impair vascular function. Accumulating evidence from prospective clinical studies suggests that elevated plasma or serum levels of ADMA are associated with an increased risk of major adverse cardiovascular events. This article gives an updated overview of the currently available literature on ADMA and cardiovascular disease from prospective clinical trials. Recently, advances have been made in the development of analytical methods that are reliable and fast enough to allow determination of ADMA in clinical routine.

BACKGROUND Asymmetric dimethylarginine (ADMA) acts as an endogenous inhibitor of NO-synthase. In the last years ADMA has emerged as a cardiovascular risk factor. The aim of this study was to determine a reference value for ADMA. METHODS Plasma samples of 500 healthy subjects in the 19-75 year age group were analyzed. Exclusion criteria from this study were smoking, any known significant disease, body-mass-index (BMI) above 30 kg m(-2), elevated plasma lipid levels, impaired renal function, hypertension, and intake of any medication. The ADMA levels were determined by ELISA, (DLD Diagnostics, Hamburg, Germany). RESULTS Mean ADMA plasma concentration of the total population was 0.69 micromol L(-1) (SD 0.20) and 95% of the measured values were in the range from 0.36 micromol L(-1) to 1.17 micromol L(-1). Women below 50 years of age had lower ADMA levels than men below 50 years of age [0.62 (0.17) micromol L(-1) vs. 0.69 (0.19) micromol L(-1); P = 0.001] and woman above 50 years of age had higher ADMA levels than men above 50 years of age [0.80 (0.22) micromol L(-1) vs. 0.73 (0.20) micromol L(-1); P = 0.036]. A regression analysis of ADMA levels and age was performed for each sex. The regression factor was r = 0.444 for women in a squared regression model (P < 0.001) and r = 0.212 for men in a linear regression model (P < 0.001). CONCLUSION The study was able to define a reference value for ADMA plasma levels with 0.36-1.17 micromol L(-1) and found sex dependent correlations between ADMA and age. Women showed a significant increase in ADMA plasma levels with onset of menopause.

Objective To investigate the association of the endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) and coronary endothelial function. Methods and results In 289 patients with coronary artery disease we assessed coronary endothelium-dependent and -independent vascular responses to intracoronary infusion of acetylcholine, adenosine, and nitroglycerin, respectively, and determined plasma ADMA and l-arginine concentrations by HPLC. After 6 months of treatment with either cerivastatin, nifedipine, cerivastatin+nifedipine, or placebo, coronary vascular function testing as well as ADMA and l-arginine determinations were repeated. We observed no correlation of plasma ADMA or l-arginine concentration and coronary response to acetylcholine, adenosine or nitroglycerin baseline, and no correlation of changes of ADMA or l-arginine plasma concentration with changes in coronary function (all r and ρ p >0.05). Conclusion At physiological plasma concentrations ADMA appears to have only little impact on coronary endothelial function.

Coronary artery disease (CAD) and erectile dysfunction (ED) of vascular origin are closely related and share common risk factors. The endogenous NO synthase inhibitor asymmetrical dimethylarginine (ADMA) has recently been identified as an independent risk marker for cardiovascular disease and it was the purpose of the present study to investigate the role ADMA in ED with and without underlying CAD.We determined plasma ADMA levels in 132 men with ED. Patients were divided into a group of 56 men with underlying CAD (ED-CAD) and a group of 76 men without clinical evidence for underlying CAD (ED-No-CAD). Diagnosis of ED was based on the International Index of Erectile Function Score (IIEF-5). Plasma ADMA concentrations in the ED-CAD group were elevated as compared to the ED-No-CAD group, median (IQR): 0.76 (0.65-0.91) micromol/l ADMA vs. 0.49 (0.36-0.71) micromol/l, p0.001. In a multiple logistic regression analysis adjusting for hypertension, hypercholesterolemia, low HDL cholesterol and diabetes or fasting glucoseor =6.1 mmol/l, ADMA remained a strong and independent predictor for presence of CAD. Odds ratios for second and third tertiles as compared to lowest tertile of plasma ADMA were 3.3 (95%CI, 1.1-10.3; p = 0.041) and 8.7 (95%CI, 2.8-27.2; p0.001), respectively.As elevation of ADMA has been found to be associated with many risk factors for both CAD and ED, our data provide further strong evidence for the close interrelation of CAD and ED. Determination of ADMA may help to identify underlying cardiovascular disease in men with ED.

Nitric oxide (NO) is essential in numerous physiologic processes and may be involved in related pathologic processes. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of isoforms of NO synthase in humans (1). ADMA originates from protein arginine methylation by protein arginine methyltransferases after protein hydrolysis (2). Enzymatic hydrolysis by dimethylarginine dimethylaminohydrolases to dimethylamine and citrulline is the major pathway for elimination of ADMA (3). Circulating ADMA is altered in patients with cardiovascular and neurologic diseases, erectile dysfunction, and many other disorders (4)(5)(6), and increased circulating ADMA independently predicts future cardiovascular events and mortality (7)(8). Short-time infusion of ADMA affects hemodynamics and cardiac function in humans (3)(9). Analytical methods for the measurement of ADMA include HPLC, capillary electrophoresis, ELISA, and mass spectrometry (MS). The commonly used HPLC methods with fluorescence detection (10) measure o -phthaldialdehyde derivatives of ADMA. These derivatives are not stable and must be analyzed on-line. Moreover, ADMA must be separated by chromatographic means from its biologically inactive isomer, symmetric dimethylamine (SDMA). Thus, these HPLC methods have long analysis times of up to 45 min. Alternatively, ADMA and SDMA can be analyzed by gas chromatography–mass spectrometry (11)(12), which shows different fragmentation patterns for the respective ions. These methods include several extraction and derivatization steps and require considerable analysis times. Recently, Kirchherr and Kuhn-Velten (13) developed a mass spectrometric method that omits derivatization procedures; however, chromatographic separation of ADMA from SDMA is still required for this method. Here we report a simple and rapid liquid chromatography–tandem MS (LC-MS/MS)-based method with 1 derivatization step and sample pretreatment consisting of protein precipitation. This is the first LC-MS–based method that separates methylated arginine derivatives by specific fragmentation patterns instead of by chromatographic separation. Analyses were performed on a Varian 1200L Triple Quadrupole …