Asymmetric Dimethylarginine In Plasma Research Articles

Корекція функції ендотелію - нова ціль профілактики і лікування артеріальної гіпертензії в пацієнтів із серцево-судинною патологією

Purpose - to determine the dynamics of endothelial progenitor cells (EPCs) and asymmetric dimethylarginine (ADMA) in pregnant women with hypertension as markers of the effectiveness of endothelial dysfunction correction on the background of L-arginine therapy (Tivortin, manufactured by Yuria-Pharm). Materials and methods. The study pregnant women were divided into two groups: women with hypertension at 12-15 weeks of gestation (n=29) and women with congenital heart disease at 12-14 weeks of gestation (n=21). The control group consisted of 40 healthy pregnant women. The examination was conducted before the start of oral L-arginine (Tivortin aspartate) and 7-8 weeks after the start of therapy. Tivortin aspartate was administered orally 1 measuring spoon 5 times a day for 6 weeks. The study of the level of EPCs (CD45+/CD34+ phenotype of peripheral blood) was performed by flow cytometry using reagents for the determination of CD34, CD45 differentiation clusters produced by Beckman Coulter Inc. The results are presented in % of ERC from the total number of leukocytes, as well as in absolute values - the number of cells per 1 ml of blood in absolute values. The level of ADMA in plasma was determined by an indirect enzyme-linked immunosorbent assay using the ADMA ELISA test system (manufactured by Immunodiagnostik AG, Germany). The reference values for pregnant women are 0.26-0.60 μmol/l. Results. After treatment with L-arginine (Tivortin), the ADMA index decreased compared to baseline data in the studied groups of pregnant women. Thus, in pregnant women with hypertension, ADMA decreased by almost 15%, which is a marker of improved endothelial function. The same dynamics was observed in the group of women with congenital heart disease. In both study groups of pregnant women, the number of EPCs increased by almost 10% after the addition of L-arginine (Tivortin) to the treatment complex. There was a positive correlation between a decrease in mean systolic blood pressure, an increase in EPCs and a decrease in ADMA after complex treatment. Studies have shown a direct correlation (r=0.75) between blood pressure and the number of ЕРСs and an inverse correlation (r=-0.68) between blood pressure and ADMA. The reduction in blood pressure during treatment was accompanied by an improvement in endothelial function, namely, an increase in the number of ЕРСs, mainly due to the vasodilation potential of the endothelium. There was also a decrease in ADMA against the background of a decrease in systolic blood pressure during treatment. Conclusions. The clinical relevance of asymmetric dimethylarginine as a specific marker in patients with hypertension has been established: a significant increase in its level is observed in patients with cardiovascular disease, in particular, with hypertension, which confirms the role of endothelial dysfunction as a leading pathogenetic factor in the development of hypertension in the absence of anatomical and pathological changes in the vessels. The concept of maintenance therapy in patients with cardiovascular disease aims to restore adequate NO bioavailability by administering exogenous L-arginine. Based on the results of the study of the effect of L-arginine (Tivortin) on the index of ЕРСs, which are actively involved in the repair of damaged endothelium, it can be concluded that this drug has a positive effect on the prognosis of life in patients with hypertension, restoring endothelial function. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

<i>NOS2</i>, <i>NOS3</i>, <i>SONE</i> gene transcripts levels in peripheral blood leukocytes and their relationship with markers of endothelial dysfunction in hypertension

Objective. The aim of the study was to evaluate the level of expression of the NOS2, NOS3, SONE genes in peripheral blood leukocytes (PBL) of patients with hypertension (HTN) and to study the relationship between the level of transcripts of these genes and the content of nitric oxide metabolites and markers of endothelial dysfunction.Design and methods. The study included healthy people (25 people) and patients with HTN (stages I–II) before prescribing antihypertensive drugs (15 people) and taking cardioselective β-adrenergic receptor blockers for more than a year (metoprolol (25 mg per day) or bisoprolol (5–10 mg per day)) (20 people). The level of gene transcripts was assessed by real-time polymerase chain reaction (PCR). The level of nitric oxide metabolites was determined by the colorimetric method using the Griess reagent. The content of asymmetric dimethylarginine (ADMA), soluble forms of vascular cell adhesion molecule (sVCAM), and intercellular adhesion molecule (sICAM) in blood plasma was determined by ELISA. The content of malondialdehyde (MDA) in blood plasma was determined spectrophotometrically by color reaction with thiobarbituric acid. Statistical processing of the results was carried out using the Statgraphics Centurion XVI software package (version 16.1.11).Results. The level of nitric oxide metabolites in the blood plasma of HTN patients without antihypertensive therapy was 2,1 times higher than in healthy individuals (p = 0,001) and 1,7 times higher than in patients with HTN taking metoprolol or bisoprolol (p = 0,002). The relative content of mRNA of the NOS3 gene in PBL of individuals included in the study did not differ (p > 0,05). The level of NOS2 gene transcripts in PBL of HTN patients before the prescription of antihypertensive drugs exceeded that in healthy individuals (p = 0,0009) and in HTN patients taking metoprolol or bisoprolol (p = 0,0002). The number of SONE transcripts in the PBL of HTN patients was higher than in people with normal blood pressure (p < 0,00001 when comparing patients before the prescription of antihypertensive therapy and individuals from the control group; p = 0,04 when comparing patients with HTN taking antihypertensive drugs and normotensive subjects). The content of MDA, ADMA, sVCAM was higher in the plasma of HTN patients without antihypertensive therapy compared with people from the control group (p = 0,005, 0,003, 0,039, respectively) and patients taking metoprolol or bisoprolol (p = 0,0006, 0,019, 0,016, respectively). The content of nitric oxide metabolites positively correlated with NOS2, SONE, VCAM1 mRNA level in PBL, the content of MDA and ADMA in blood plasma (p < 0,05). A positive correlation was found between the concentration of MDA and ADMA in plasma (p = 0,03).Conclusions. An increase in the level of nitric oxide metabolites in HTN is associated with an increase in the transcriptional activity of the NOS2 gene, a disturbance of the redox balance of the body, and the development of endothelial dysfunction. The SONE gene is probably involved in the modulation of nitric oxide levels in HTN not only as an antisense transcript that destabilizes the mRNA of the NOS3 gene in vascular endothelial cells, but also indirectly, namely, through the regulation of homeostasis of immune system cells through autophagy.

Overexpression of alanine-glyoxylate aminotransferase 2 protects from asymmetric dimethylarginine-induced endothelial dysfunction and aortic remodeling

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Abstract 13928: Asymmetric Dimethylarginine (ADMA) Mediates the Effect of miRNA-762 on All-Cause Mortality in Patients With Coronary Artery Disease

Introduction: Plasma concentrations of the endogenous L-arginine homologue asymmetric dimethylarginine (ADMA) independently predict cardiovascular and all-cause mortality. We sought to identify miRNAs that mediate the effects of ADMA on mortality or influence mortality through regulation of ADMA concentrations. Methods: In 993 participants of the Ludwigshafen Risk and Cardiovascular Health study (age 63.0±10.5 years, 31.4% females and 74.7% with coronary artery disease (CAD)) we measured ADMA and miRNAs concentrations in citrate plasma and investigated associations between them and with all-cause mortality. Results: miRNA-762 was positively and significantly correlated with ADMA concentrations (p=0.0013) and with all-cause mortality after adjusting for age and sex (HR (95%CI: 1.51(1.26-1.81); p=7.37E-06). The association remained significant after additionally adjusting for body mass index, CAD, arterial hypertension, diabetes, smoking, lipids, C-reactive protein, fibrinogen, renal function, homocysteine and medication (p=0.04). Mediation analyses showed that ADMA mediated 67% of the effect of miRNA-762 on mortality while miRNA-762 only mediated 8% of the effect of ADMA. Conclusions: miRNA-762 is an independent predictor of all-cause mortality in patients with CAD. The effects of miRNA-762 on mortality might be at least partially mediated by ADMA. miRNA-762 has been recently shown to regulate apoptosis in cardiomyocytes in myocardial infarction. We are currently investigating the effects of miRNA-762 on ADMA metabolism and stress responses in cultured cardiomyocytes and endothelial cells.

134 Placental dimethylarginine dimethylaminohydrolase and maternal and cord concentrations of asymmetric dimethylarginine in preeclampsia

Asymmetric dimethylarginine (ADMA) concentrations are reportedly increased in women with preeclampsia (preE). Dimethylarginine dimethylaminohydrolase (DDAH) degrades ADMA, but its placental expression and relationship with ADMA has not been well studied. The objective of this study was to compare ADMA concentrations at delivery in maternal plasma and DDAH protein expression in placenta in women with preE compared to matched controls. The secondary objective was to compare cord blood ADMA concentrations at delivery between pregnancies complicated by preeclampsia and controls. Placenta, cord plasma, and maternal plasma at delivery were obtained from women diagnosed with preE and controls matched by gestational age, race, and BMI enrolled in the Indiana University Building Blocks of Pregnancy Biobank. Placenta tissue was homogenized and DDAH-1 in placental homogenate and ADMA in plasma were measured by ELISA. Differences in DDAH and ADMA concentrations between women with preE and controls were assessed using t-tests. Stepwise logistic regression was used to evaluate covariates. 25 women with preE and 23 matched controls were studied. There was no difference in DDAH-1 between women with preE (1.60±0.79 ng/mL) compared to controls (1.75±0.75 ng/mL, p=0.5). Multivariable analysis did not show a difference in concentrations with gestational age, BMI, race, or age. Hispanic women had lower DDAH-1 than non-Hispanic women (1.25±0.39 ng/mL vs 1.77±0.80 ng/mL, respectively, p=0.009). Maternal ADMA concentrations did not differ between cases (0.092±0.049 μmol/L) and controls (0.082±0.049 μmol/L, p = 0.64). There was no difference in cord concentrations of ADMA in 16 infants born from moms with preE (0.80±0.17 ng/mL) compared to 16 controls (0.73±0.23 ng/mL, p = 0.34). No significant correlation was observed between maternal ADMA, cord ADMA and DDAH. There is no statistical difference in plasma ADMA, placental DDAH-1, and neonatal ADMA between women with preE and controls in a small retrospective study. ADMA through pregnancy and activity of DDAH-1 needs further study.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Sequence Variation in the DDAH1 Gene Predisposes for Delayed Cerebral Ischemia in Subarachnoidal Hemorrhage.

Delayed cerebral ischemia (DCI) often causes poor long-term neurological outcome after subarachnoidal hemorrhage (SAH). Asymmetric dimethylarginine (ADMA) inhibits nitric oxide synthase (NOS) and is associated with DCI after SAH. We studied single nucleotide polymorphisms (SNPs) in the NOS3, DDAH1, DDAH2, PRMT1, and AGXT2 genes that are part of the L-arginine–ADMA–NO pathway, and their association with DCI. We measured L-arginine, ADMA and symmetric dimethylarginine (SDMA) in plasma and cerebrospinal fluid (CSF) of 51 SAH patients at admission; follow-up was until 30 days post-discharge. The primary outcome was the incidence of DCI, defined as new infarctions on cranial computed tomography, which occurred in 18 of 51 patients. Clinical scores did not significantly differ in patients with or without DCI. However, DCI patients had higher plasma ADMA and SDMA levels and higher CSF SDMA levels at admission. DDAH1 SNPs were associated with plasma ADMA, whilst AGXT2 SNPs were associated with plasma SDMA. Carriers of the minor allele of DDAH1 rs233112 had a significantly increased relative risk of DCI (Relative Risk = 2.61 (1.25–5.43), p = 0.002). We conclude that the DDAH1 gene is associated with ADMA concentration and the incidence of DCI in SAH patients, suggesting a pathophysiological link between gene, biomarker, and clinical outcome in patients with SAH.

Melatonin rescued methotrexate-induced spatial deficit and hyperhomocysteinemia and increased asymmetric dimethylarginine in plasma and dorsal hippocampus in developing rats

AimsWith the improvement of the survival rates in children acute lymphoblastic leukemia (ALL), some children ALL survivors show impaired cognitive function. Methotrexate (MTX), an essential component in ALL treatment, has been reported to be related to neurologic sequelae and to increased oxidative stress through its interactions with enzymes in the folate pathway. Asymmetric dimethylarginine (ADMA) is the main endogenous inhibitor of nitric oxide synthase, and increased ADMA may result from increased oxidants. Melatonin is an antioxidant; however, its role in MTX neuropathy is not well studied. We developed a rat model mimicking child ALL treatment to explore peripheral and central homocysteine and ADMA regulation after MTX and found potential treatment choice. Main methodsPreweaning male Sprague-Dawley rats were used in this study. Experiment 1 evaluated spatial performance in rats with intrathecal (IT) MTX, intraperitoneal (IP) MTX, or combined IT and IP MTX, protocols mimicking ALL treatment in children. Experiment 2 focused on rats with combined IT and IP MTX, evaluating spatial performance and plasma and dorsal hippocampal homocysteine and ADMA levels, their regulation, and the protective effect of melatonin. Key findingsCombined IT and IP MTX treatment caused in spatial deficits in developing rats, and melatonin restored the spatial performance. Alterations in peripheral and central homocysteine and ADMA concentrations and their regulation were found and could be alleviated by melatonin treatment. SignificancesCombined IP and IT MTX treatment caused spatial deficits in developing rats. Melatonin could restore spatial performance through alleviating the effects on the imbalance of oxidative stress.

Pathogenetic justification and clinical significance of using arginine for prevention of critical ischemia of lower extremities and fatal manifestations of multifocal atherosclerosis

The aim — to investigate the results of treatment with L‑arginine of patients with intermittent claudication for prevention of critical stages of limb ischemia.Materials and methods. 86 patients with intermittent claudication were randomly divided into two groups of 43 patients each. Patients of the first group received 8 g of L‑arginine (Tivortin) twice a day, patients of the second group — 40 mg of prostaglandin E1 (PGE1) twice a day.Results and discussion. Administration of L‑arginine contributed to an increase in the distance of painless walk by 230 ± 63 %, the absolute walk distance — by 155 ± 48 % (p < 0.05), the administration of PGE1 — by 209 ± 63 % and 144 ± 28 %, respectively (p < 0.05). Treatment with L‑arginine increased the value of the coefficient of L‑arginine/asymmetric dimethylarginine in plasma and the excretion of nitrates and cGMP in the urine, which indicated the normalization of the biosynthesis of endogenous NO. PGE1 therapy did not significantly affect endothelium‑dependent vasodilation parameters. The coefficient of quality of life, estimated in an analog scale, increased from 3.51 ± 0.18 to 8.3 ± 0.4 in the arginine group and to 7.0 ± 0.5 in the PGE1 group (all p < 0.05).Conclusions. Arginine is a drug of choice to prevention of severe forms of lower limbs ischemia and other manifestations of multifocal atherosclerosis.

The role of asymmetric dimethylarginine in the development of arterial&#x0D; hypertension.

The results of numerous studies of recent decades confirm the crucial role of vascular endothelium in regulating vascular homeostasis. A plethora of recent studies have shed light on the clinical significance of endothelial dysfunction in essential hypertension. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. At present, it is considered as a generally recognized marker of endothelial dysfunction by most researchers. In vitro experiments showed that ADMA inhibits endothelium-dependent arterial relaxation, increases the level of indicators characterizing the degree of oxidative stress in endothelial cells, enhances the synthesis of the superoxide anion radical by endothelial cells. The molecular mechanisms described above, activated with an increase in the concentration of ADMA, cause various disturbances in the function of the cardiovascular system, which gave grounds to consider the level of ADMA as a criterion and risk factor for the development of cardiovascular diseases. Thus, ADMA plays a key role in the development and progression of CVD associated with a spectrum of diseases and pathological conditions characterized by a disturbance in NO production. Despite clinical and experimental confirmation of the relationship between the increase in ADMA in plasma and the development of cardiovascular events, the unambiguous etiopathogenetic role of ADMA in CVD requires further research. In order to accurately answer the question of whether ADMA is an etiological factor or a biological marker of CVD, additional analysis is needed to study the biochemical, genetic and pharmacological aspects of ADMA metabolism, the results of which are presented in this article.

Comparison of Three Sample Preparation Procedures for the Quantification of L-Arginine, Asymmetric Dimethylarginine, and Symmetric Dimethylarginine in Human Plasma Using HPLC-FLD

Increased asymmetric dimethylarginine (ADMA) in human plasma has been associated with reduced generation of nitric oxide, leading to atherosclerotic diseases. ADMA may therefore be an important biomarker for cardiovascular disease. In the present study, three sample preparation techniques were compared regarding the quantification of L-arginine and ADMA in human plasma: (A) protein precipitation (PP) based on aqueous trichloroacetic acid (TCA), (B) PP using a mixture of ammonia and acetonitrile, and (C) solid-phase extraction (SPE). The samples were analysed by using high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The analytical performance of (A) was comparable with that of (C), demonstrating recoveries of >90%, coefficient of variations (CVs, %) of <8, and a resolution (Rs) between ADMA and symmetric dimethylarginine (SDMA) of 1.2. (B) was disregarded due to recoveries below 75%. (A) was validated with good results regarding linearity (>0.994), precision (<5%), and sensitivity (lower limit of quantification (LLOQ)) of 0.14 µM and 12 nM for L-arginine and ADMA, respectively. Due to the simplicity and speed of procedure (A), this approach may serve as preferred sample preparation of human plasma samples before HPLC-FLD in providing important information regarding elevated ADMA concentrations.

Simultaneous determination of arginine, citrulline, and asymmetric dimethylarginine in plasma by reverse-phase high-performance liquid chromatography.

BACKGROUND:Arginine, citrulline and asymmetric dimethylarginine (ADMA) are three molecules in the nitric oxide (NO) pathway which provide useful information about vascular endothelial function. ADMA accumulates with patients with chronic kidney disease (CKD) and inhibits NO synthesis. We describe the modification of a previously established method for the measurement of amino acids analysis for simultaneous detection of arginine, citrulline, and ADMA in plasma and to validate its performance in patients with CKD.MATERIALS AND METHODS:Arginine, citrulline, and ADMA were simultaneously separated by reverse-phase high-performance liquid chromatography by precolumn derivatization with O-phthalaldehyde using the modified method. It was then applied for analysis in thirty patients with CKD and thirty healthy controls so as to cover the entire measuring range, i.e., normal and uremic range.RESULTS:The method showed a good performance in terms of linearity, precision, and recovery. The detection limit of the assay for ADMA was found to be 0.05 μmol/L at a signal-to-noise ratio of 3:1. The average within run coefficient of variation for ADMA using this method was 4.7% in the normal range and 1.9% in the uremic range, while the average between-day precision in the normal and uremic range was 6.5% and 5.2%, respectively. Patients with CKD were found to have higher concentration of ADMA compared to controls.CONCLUSION:This method can be useful in assessing the baseline cardiovascular risk in an individual as well as in the follow-up of the patients who are receiving L-arginine, and thus, assess the response to treatment by simultaneous measurement of arginine and ADMA.

Asymmetric Dimethylarginine correlates significantly with tumour necrosis alfa but not with brachial ankle pulse wave velocity in the T2DM-METS

Background: Endothelial dysfunction is an important phenomenon in the pathogenesis of atherosclerosis and is related to the derangements of nitric oxide (NO) synthase in the vessel wall. Asymmetric Dimethylarginine (ADMA) is an endogenous, competitive inhibitor of nitric oxide synthase and is induced by inflammatory cytokines of tumour necrosis factor (TNF)-a in vitro. Increased ADMA levels are associated with reduced NO synthesis as assessed by impaired endothelium-dependent vasodilatation. There is cause and effect relationship between endothelial dysfunction and vascular stiffening. Measurement of brachial ankle pulse wave velocity (baPWV) is simple and applicable for cardiovascular risk screening and as a marker for the severity of atherosclerotic vascular damage. Aim: To investigate the correlation between ADMA level with TNF-a and baPWV in the type-2 diabetes mellitus (T2DM)-Metabolic Syndrome (Mets) patients. Method: This is a cross sectional study with T2DM-Mets patients who came to the outpatient clinic of Soetomo Hospital Surabaya during January 2010 to December 2012. Subjects met the inclusion and exclusion criteria were measured their ADMA, TNF-a, and blood glucose levels in plasma. Brachial ankle pulse wave velocity (ba-PWV) was determined by using the V Serra-1000. The study was approved by the local Research Ethics Committee and subjects gave written informed consent. Results: Thirty-seven T2DM-Mets patients consisted of 15 (40.5%) males and 22 (59.5%) females who met inclusion and exclusion criteria were enrolled in this study. Their mean of age was 51 ± 5.2 years old, duration of illness was 16.49 ± 23.4 months, A1C level was 8.5 ± 0.9%, BMI was 26.7 ± 4.5 kg/m2, ADMA level was 0.572 ± 0.2 μmol/L, TNF-a level was 10.0 ± 16.5 pg/mL, and ba-PWV was 1,624.5 ± 295.5 cm/s. Spearman’s correlation analysis showed that ADMA level was significantly correlated with TNF-a level (p = 0.026; r = 0.366). However, no significant correlation found with ba-PWV (p = 0.134; r = 0.251). Conclusion: ADMA level is correlated with TNF-a level in this T2DM-MetS population.

Modulating DDAH/NOS Pathway to Discover Vasoprotective Insulin Sensitizers.

Insulin resistance syndrome (IRS) is a configuration of cardiovascular risk factors involved in the development of metabolic disorders including type 2 diabetes mellitus. In addition to diet, age, socioeconomic, and environmental factors, genetic factors that impair insulin signaling are centrally involved in the development and exacerbation of IRS. Genetic and pharmacological studies have demonstrated that the nitric oxide (NO) synthase (NOS) genes are critically involved in the regulation of insulin-mediated glucose disposal. The generation of NO by the NOS enzymes is known to contribute to vascular homeostasis including insulin-mediated skeletal muscle vasodilation and insulin sensitivity. By contrast, excessive inhibition of NOS enzymes by exogenous or endogenous factors is associated with insulin resistance (IR). Asymmetric dimethylarginine (ADMA) is an endogenous molecule that competitively inhibits all the NOS enzymes and contributes to metabolic perturbations including IR. The concentration of ADMA in plasma and tissue is enzymatically regulated by dimethylarginine dimethylaminohydrolase (DDAH), a widely expressed enzyme in the cardiovascular system. In preclinical studies, overexpression of DDAH has been shown to reduce ADMA levels, improve vascular compliance, and increase insulin sensitivity. This review discusses the feasibility of the NOS/DDAH pathway as a novel target to develop vasoprotective insulin sensitizers.

Plasma homoarginine, arginine, asymmetric dimethylarginine and total homocysteine interrelationships in rheumatoid arthritis, coronary artery disease and peripheral artery occlusion disease.

Elevated circulating concentrations of total L-homocysteine (thCys) and free asymmetric dimethylarginine (ADMA) are long-established cardiovascular risk factors. Low circulating L-homoarginine (hArg) concentrations were recently found to be associated with increased cardiovascular morbidity and mortality. The biochemical pathways of these amino acids overlap and share the same cofactor S-adenosylmethionine (SAM). In the present study, we investigated potential associations between hArg, L-arginine (Arg), ADMA and thCys in plasma of patients suffering from rheumatoid arthritis (RA), coronary artery disease (CAD) or peripheral artery occlusive disease (PAOD). In RA, we did not find any correlation between ADMA or hArg and thCys at baseline (n = 100) and after (n = 83) combined add-on supplementation of omega-3 fatty acids, vitamin E, vitamin A, copper, and selenium, or placebo (soy oil). ADMA correlated with Arg at baseline (r = 0.446, P < 0.001) and after treatment (r = 0.246, P = 0.03). hArg did not correlate with ADMA, but correlated with Arg before (r = 0.240, P = 0.02) and after treatment (r = 0.233, P = 0.03). These results suggest that hArg, ADMA and Arg are biochemically familiar with each other, but unrelated to hCys in RA. In PAOD and CAD, ADMA and thCys did not correlate.

Asymmetric dimethylarginine, a biomarker of cardiovascular complications in diabetes mellitus.

Cardiovascular (CV) complications are an essential causal element of prospect in diabetes mellitus (DM), with carotid atherosclerosis being a common risk factor for prospective crisis of coronary artery diseases and/or cerebral infarction in DM subjects. From another point of view, asymmetric dimethylarginine (ADMA) has been established as an inhibitor of endogenous nitric oxide synthesis and the relationship between ADMA and arteriosclerosis has been reported. In our study with 87 type 2 DM (T2DM) patients, we have examined whether ADMA and other CV risk factors are the useful predictors of DMCV complications. After the measurement of the respective CV risk factors, we have followed the enrolled T2DM patients for 5 years. We have finally analyzed 77 patients. DMCV complications developed in 15 cases newly within 5 years, and 4 cases recurred. The concentrations of ADMA in plasma were markedly more elevated in 19 DM patients with CV complications than in 58 DM patients without CV complications. Urinary albumin (U-Alb), mean intimal-medial thickness (IMT) and ankle brachial index (ABI) were also higher in patients with CV complications. Multiple regression analyses showed that U-Alb had an influence on the high level of ADMA (standardized β = 6.59, P = 0.00014) independently of age, systolic BP, fibrinogen, mean IMT, plaque score, and ABI. The review indicates what is presently known regarding plasma ADMA that might be a new and meaningful biomarker of CV complications in DM subjects.

Correlation between asymmetric dimethylarginine and cerebral vasospasm secondary to subarachnoid hemorrhage

Objective To investigate the correlation between asymmetric dimethylarginine (ADMA) and cerebral vasospasm (CVS) secondary to subarachnoid hemorrhage (SAH).Methods Seventy-eight healthy male adult Sprague-Dawley rats were randomly allocated into control group (n =6),sham group (n =36) and SAH group (n =36).In SAH group,a SAH model was established by double autologous blood injection into cisterna magna.Arterial blood,cerebrospinal fluid (CSF) and basilar artery samples of each group were collected at the preconcerted time.ADMA levels in the plasma and CSF samples were assessed using enzyme-linked immuno sorbent assay (ELISA).Nitrite and nitrate concentrations were used as a marker of nitric oxide production in plasma and CSF samples and determined by using nitrate reductase method.Pathomorphological changes were observed with optical microscope and inner diameter of basilar artery was measured by Micrometer after hematoxylin-eosin staining was performed.Results (1) In SAH group,ADMA levels in CSF samples were significantly increased at 1st,3rd,5th and 7th day (P ＜0.05),and NO levels in CSF samples were significantly decreased at 3rd,5th,and 7th day as compared with sham group (P＜0.01).(2) The ratio of thickness and inner diameter of basilar artery was used to indicate the degree of CVS.The level of ADMA in CSF samples was strongly correlated with the degree of CVS (r =0.641,P ＜0.01).Nitrite levels in CSF samples were negatively correlated with ADMA levels in CSF samples (r =-0.687,P ＜0.01).(3) Plasma ADMA levels in sham group and SAH group were similar and remained essentially unchanged during the several days after SAH.Plasma ADMA levels were neither correlated with NO levels (r =0.019,P ＞ 0.05) nor with the degree of vasospasm (r =-0.046,P ＞ 0.05).Conclusion Asymmetric dimethylarginine reduces the level of nitric oxide in CSF by inhibiting nitric oxide synthase and may be involved in the progression of cerebral vasospasm.The level of ADMA in CSF samples was strongly correlated with the degree of CVS in SAH rats.There is no evident correlation between the level of ADMA in plasma and the degree of CVS. Key words: Subarachnoid hemorrhage; Cerebral vasospasm ; Asymmetric dimethylarginine ; Nitric oxide

A new derivatization method coupled with LC‐MS/MS to enable baseline separation and quantification of dimethylarginines in human plasma from patients to receive on‐pump CABG surgery

Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide synthase and a risk factor for cardiovascular events. We have developed a new derivatization method to enable baseline separation of the regio-isomers, ADMA, and symmetric dimethylarginine (SDMA), within 15 min on a C18 reverse phase column. Reacting naphthalene-2,3-dicarboxaldehyde with ADMA and SDMA in the presence of 2-mercaptoethanol produces corresponding 2,3-dihydro-benzo[f]isoindol-1-ones that are more stable than previously reported ortho-phthaldialdehyde and 2-mercaptoethanol derivatives. LC-MS/MS quantitation of these derivatives can be used to determine ADMA and SDMA concentrations in the plasma of patients to receive on-pump coronary artery bypass grafting (CABG) surgery. The LOD, LOQ and lower LOQ (LLOQ) of this method were determined to be 2.6, 8.7, and 25 nM for ADMA, and 2.5, 8.3, and 25 nM for SDMA, respectively, with consumption of only 50 μL of plasma. The relative standard deviations and relative errors of the intraday and interday determinations, as measurements of reproducibility and accuracy, are all within 15%. The ADMA and SDMA concentrations in patient plasma are 298.1 ± 11.2 nM (mean ± S.E.M., n = 123) and 457.7 ± 19.8 nM (mean ± S.E.M., n = 123), respectively. Upon unblinding of our clinical trial, these predetermined values might explain patient clinical outcomes associated with on-pump CABG surgery, as ADMA is known to inhibit nitric oxide production. Furthermore, this derivatization reaction in conjunction with LC-MS/MS analysis may open a venue to explore alternative chemical labeling modes for LC-MS/MS applications, such as analysis of other amino acids, metabolites, and peptides containing primary amine group(s).

Asymmetric dimethylarginine in adults with cystathionine β-synthase deficiency

In hyperhomocysteinemia (HHcy), an independent risk factor for cardiovascular diseases, endothelial dysfunction due to reduced bioavailability of nitric oxide is a consistent finding. However, the underlying mechanisms remain unknown.Increased levels of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) have been associated with HHcy, and may contribute, at least in part, for the homocysteine-induced endothelial dysfunction, but whether cystathionine β-synthase (CBS) deficiency is associated with increased ADMA has hardly been investigated.To address this question, we measured total homocysteine (tHcy), ADMA and symmetric dimethylarginine (SDMA) in plasma of 22 adult CBS deficient patients, using established HPLC techniques. Results showed that in CBS deficient patients with elevated levels of tHcy (median (total range): 33 (14–237)μmol/L), both ADMA and SDMA levels were normal. Moreover, tHcy and ADMA concentrations were not correlated (rs=0.017, p=0.94). Our results favor the hypothesis that the negative vascular effects of HHcy have an ADMA-independent etiology.

Role of the human erythrocyte in generation and storage of asymmetric dimethylarginine

Proteolytic activity in whole blood may lead to release of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). We investigated the role of the human erythrocyte in storage and generation of ADMA in healthy controls (n = 36) and critically ill patients (n = 38). Both free and total (sum of free and protein-incorporated) ADMA were measured. Upon incubation of intact erythrocytes with extracellular ADMA (0 to 40 μmol/l), equilibrium between intra- and extracellular ADMA was reached within 3 h. Compared with controls, patients had significantly higher basal concentrations of ADMA in plasma (0.88 ± 0.75 vs. 0.41 ± 0.07 μmol/l) and erythrocytes (1.28 ± 0.55 vs. 0.57 ± 0.14 μmol/l). Intracellular and plasma ADMA were significantly correlated in the patient group only (r = 0.834). Upon lysis, followed by incubation at 37°C for 2 h, free ADMA increased sevenfold (to 8.60 ± 3.61 μmol/l in patients and 3.90 ± 0.78 μmol/l in controls). In lysates of controls, free ADMA increased further to 9.85 ± 1.35 μmol/l after 18 h. Total ADMA was 15.43 ± 2.44 μmol/l and did not change during incubation. The increase of free ADMA during incubation corresponded to substantial release of ADMA from the erythrocytic protein-incorporated pool (21.9 ± 4.6% at 2 h and 60.8 ± 7.6% at 18 h). ADMA was released from proteins other than hemoglobin, which only occurred after complete lysis and was blocked by combined inhibition of proteasomal and protease activity. Neither intact nor lysed erythrocytes mediated degradation of free ADMA. We conclude that intact erythrocytes play an important role in storage of ADMA, whereas upon erythrocyte lysis large amounts of free ADMA are generated by proteolysis of methylated proteins, which may affect plasma levels in hemolysis-associated diseases.

Diagnostical significance of dimethylarginine in the development of hepatorenal syndrome in patients with alcoholic liver cirrhosis

Chronic consumption of alcohol during a longer period of time leads to the development of cirrhosis with the reduction in metabolic liver function and disorders in arginine metabolism. Hepatorenal syndrome (HRS) is the most severe complication of alcoholic liver cirrhosis. The aim of the study was to analyze disorders in arginine metabolism by monitoring concentrations of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in patients with liver cirrhosis and HRS. The study included three groups of subjects: a group of patients with cirrhosis and HRS (24 patients), a group of patients with cirrhosis without HRS (18 patients) and a control group composed of 42 healthy voluntary blood donors. Concentrations of ADMA, SDMA and L-arginine in plasma were measured in all groups using the high pressure liquid chromatography (HPLC) method. The concentration of SDMA was significantly higher in the patients with HRS compared to the patients without HRS and it was also higher than the values obtained from the healthy participants 1.76 +/- 0.3 micromol/L; 1.01 +/- 0.32 and 0.520 +/- 0.18 micromol/L, respectively; p < 0.01). The concentrations of ADMA were higher in the cirrhotic patients with HRS than in those without this serious complication of cirrhosis. The concentration of ADMA in all the examined cirrhotic patients was higher than those obtained from healthy volunteers (1.35 +/- 0.27 micromol/L, 1.05 +/- 0.35 micromol/L and 0.76 +/- 0.21 micromol/L, respectively). In the patients with terminal alcoholic liver cirrhosis, the concentrations of ADMA and SDMA correlated with the progress of cirrhosis as well as with the development of cirrhosis complications. In the patients with HRS there was a positive correlation between creatinine and SDMA in plasma (r2 0.0756,p < 0.001) which was not found between creatinine and ADMA. The obtained results demonstrate that the increase in SDMA concentration is proportionate to the progression of chronic damage of the liver and kidneys. Increased ADMA concentration can be a causative agent of renal insufficiency in patients with cirrhosis.