Designing modulators of dimethylarginine dimethylaminohydrolase (DDAH): A focus on selectivity over arginase

DDAH inhibition presents a novel promising pharmaceutical strategy to lower NO formation. To date, several potent DDAH inhibitors have been published, most of them representing analogues of l-arginine. While inhibitory effects on NOSs have already been considered, selectivity over arginase has been neglected so far. In our view, the latter selectivity is more important since an additional inhibition of arginase decreases the desired effects on NO levels. Thus, we particularly focus on selectivity over arginase. We present a comprehensive selectivity profile of known DDAH inhibitors by covering their inhibitory potency on arginase. Among the studied compounds, N^ω-(2-methoxyethyl)-l-arginine (2a, L-257) that is already selective over NOSs also only modestly affected arginase activity and is thus far the most suitable DDAH inhibitor for pharmacological studies.     

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

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

Determination of asymmetric and symmetric dimethylarginines in plasma of hyperhomocysteinemic subjects

Summary. The aim of this study was to investigate the possible relationship among dimethylarginines (asymmetric, ADMA; symmetric, SDMA) and homocysteine (Hcy) levels in subjects affected by chronic, mild to intermediate, hyperhomocysteinemia.ADMA and SDMA were assayed by an optimised HPLC method in 75 patients (Hcy = 20.8 μmol/L, 17.1–30.2; median and percentile range) and, for comparison, in 85 healthy subjects (Hcy = 8.0 μmol/L, 7.0–9.1). In controls, the cut-off values were set at 0.61 μmol/L for ADMA and 0.56 or 0.48 μmol/L for male and female SDMA, respectively. In patients, ADMA and SDMA levels were increased (p<0.001) with respect to controls, but no correlation with Hcy was observed. Hyperhomocysteinemic subjects showed a different behaviour in respect to ADMA and SDMA levels and this allowed their stratification in 3 subgroups characterized by ADMA and SDMA in the normal range, only SDMA, or both ADMA and SDMA over the cut-off values. A lack of correlation with Hcy was again observed, thus minimizing the direct role of Hcy on ADMA and SDMA metabolism and suggesting the need for further studies on this issue.     

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

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

早发型重度子痫前期患者血清ADMA、INH-A与胎儿生长受限的关系研究

摘要 目的：探讨早发型重度子痫前期（SPE）患者血清非对称性二甲基精氨酸（ADMA）、抑制素A（INH-A）与胎儿生长受限（FGR）的关系。方法：选取2021年1月～2023年1月于海南医学院第一附属医院分娩的110例早发型SPE患者为观察组,另选取同期健康孕妇100例为对照组,根据是否合并FGR将早发型SPE患者分为FGR组46例和非FGR组64例。比较观察组与对照组血清ADMA、INH-A水平。采用多因素Logistic回归分析早发型SPE患者合并FGR的影响因素,受试者工作特征曲线（ROC）分析血清ADMA、INH-A水平对早发型SPE患者合并FGR的预测效能。结果：与对照组比较,观察组血清ADMA、INH-A水平升高（P＜0.05）。与非FGR组比较,FGR组ADMA、INH-A、收缩压、舒张压、血肌酐、24 h尿蛋白定量、脐动脉血流收缩期末流速/舒张期末流速比值（S/D）水平升高,而孕前体质指数（BMI）降低（P＜0.05）。多因素Logistic回归分析显示,24 h尿蛋白定量、动脉血流S/D、ADMA和INH-A升高为早发型SPE患者合并FGR的独立危险因素（P＜0.05）。ROC分析显示,血清ADMA、INH-A水平联合预测早发型SPE患者合并FGR的曲线下面积大于单独预测。结论：早发型SPE患者血清ADMA、INH-A水平异常升高,且参与FGR的发生、发展,此外,血清ADMA、INH-A水平联合对早发型SPE患者合并FGR的预测效能较高。     

MicroRNA-21 介导非对称性二甲基精氨酸诱导的内皮细胞衰老

目的:观察非对称性二甲基精氨酸(ADMA)对内皮细胞中microRNA-21(miR-21)表达的影响,探讨microRNA-21在ADMA诱导的内皮细胞衰老中的作用。方法:人脐静脉内皮细胞(HUVEC)与10 uM的ADMA孵育48小时后收集细胞提取总RNA及蛋白,荧光定量实时RT-PCR检测miR-21表达,Western blot检测超氧化物歧化酶2(SOD2)表达,衰老相关半乳糖苷酶(SA-β-gal)染色鉴定衰老的内皮细胞;然后HUVEC与miR-21抑制剂转染6小时后继续与10 uM的ADMA孵育48小时留取细胞按上述方法检测相关指标。结果:HUVEC与ADMA孵育后miR-21表达量明显增加(P<0.01),同时衰老的内皮细胞数量增多(P<0.05),而SOD2表达减少(P<0.01);MiR-21抑制剂转染HUVEC后ADMA诱导的miR-21表达明显减少,同时衰老的内皮细胞减少,而SOD2表达明显增加(所有P<0.01)。结论:ADMA诱导了HUVEC中miR-21表达及细胞衰老,miR-21介导了ADMA诱导的内皮细胞衰老作用,其机制可能与其抑制SOD2表达有关。     

Impact of advanced glycation end products (AGEs) signaling in coronary artery disease

Coronary artery disease remains the leading cause of mortality in adult diabetic population with however, a high predominance also in non-diabetic subjects. In search of common molecular mechanisms and metabolic by-products with potential pathogenic role, increased advanced glycation end products (AGEs) present a critical biomarker for CAD development in both cases. Interaction of AGEs with their transmembrane cell receptor, RAGE in endothelial and smooth muscle cells as well as in platelets, activates intracellular signaling that leads to endothelial injury, modulation of vascular smooth muscle cell function and altered platelet activity. Furthermore, tissue accumulation of AGEs affects current treatment approaches being involved in stent restenosis. The present review provides an update of AGE-induced molecular mechanisms involved in CAD pathophysiology while it discusses emerging therapeutic interventions targeting AGE reduction and AGE-RAGE signaling with beneficial clinical outcome.     

Unique Features of Human Protein Arginine Methyltransferase 9 (PRMT9) and Its Substrate RNA Splicing Factor SF3B2

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.