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Analytical Biochemistry Jan 2016The L-arginine metabolites methylated at the guanidino moiety, such as N(G)-monomethyl-L-arginine (LNMMA), asymmetric N(G),N(G)-dimethyl-L-arginine (ADMA), and symmetric...
The L-arginine metabolites methylated at the guanidino moiety, such as N(G)-monomethyl-L-arginine (LNMMA), asymmetric N(G),N(G)-dimethyl-L-arginine (ADMA), and symmetric N(G),N(G')-dimethyl-L-arginine (SDMA), are long known to be present in human plasma. Far less is known about the structural isomer of LNMMA, N(δ)-monomethyl-L-arginine (δ-MMA). In prior work, it has been detected in yeast proteins, but it has not been investigated in mammalian plasma or cells. In this work, we present a method for the simultaneous and unambiguous quantification of LNMMA and δ-MMA in human plasma that is capable of detecting δ-MMA separately from LNMMA. The method comprises a simple protein precipitation sample preparation, hydrophilic interaction liquid chromatography (HILIC) gradient elution on an unmodified silica column, and triple stage mass spectrometric detection. Stable isotope-labeled D6-SDMA was used as internal standard. The calibration ranges were 25-1000 nmol/L for LNMMA and 5-350 nmol/L for δ-MMA. The intra- and inter-batch precision determinations resulted in relative standard deviations of less than 12% for both compounds with accuracies of less than 6% deviation from the expected values. In a pilot study enrolling 10 healthy volunteers, mean concentrations of 48.0 ± 7.4 nmol/L for LNMMA and 27.4 ± 7.7 nmol/L for δ-MMA were found.
Topics: Adult; Chromatography, High Pressure Liquid; Humans; Isomerism; Male; Mass Spectrometry; Middle Aged; Pilot Projects; Reproducibility of Results; Young Adult; omega-N-Methylarginine
PubMed: 26464121
DOI: 10.1016/j.ab.2015.10.001 -
International Journal of Cardiology Jan 2016Pathways of oxidative stress, nitric oxide bioavailability and L-arginine derivatives are hypothesized to be related to atrial fibrillation (AF). Circulating methylated...
BACKGROUND
Pathways of oxidative stress, nitric oxide bioavailability and L-arginine derivatives are hypothesized to be related to atrial fibrillation (AF). Circulating methylated L-arginine metabolites can be assessed in the general population and may show an association with AF.
METHODS
We determined L-arginine and its metabolites asymmetric dimethylarginine (ADMA), L-N(ω)-monomethylarginine (NMMA) and symmetric dimethylarginine (SDMA) in the population-based Gutenberg Health Study (n=5000), mean age 55 ± 11 years, 51% men, in association with clinical variables of AF such as electrocardiographic and echocardiographic measures and manifest AF.
RESULTS
Individuals with AF (N=161), 71% men, were older, mean age 64.9 ± 8.3 years. In Bonferroni-corrected multivariable-adjusted regression analyses we observed moderate inverse associations for L-arginine, SDMA, and L-arginine/ADMA ratio with ventricular heart rate, and for L-arginine and L-arginine/ADMA ratio with QTc interval. L-arginine was correlated with QRS duration. In echocardiographic analyses, SDMA was related to left atrial diameter and deceleration time, ADMA and NMMA were correlated with left ventricular mass. ADMA (odds ratio [OR] 1.21, 95% confidence interval [CI] 1.11-1-32; p=0.013) and NMMA (OR 1.17, 95% CI 1.09-1.26, p=0.014) were related to prevalent AF. L-arginine/ADMA ratio was inversely associated (OR 0.8, 95% CI 0.71-0.90, p=0.0082). Results were similar after adjustment for creatinine.
CONCLUSIONS
In our large, population-based cohort, we observed moderate associations of l-arginine metabolites and intermediate electrocardiographic and echocardiographic variables and AF. Our findings support further investigations to define the role of L-arginine derivatives in AF and their clinical utility.
Topics: Adult; Aged; Arginine; Atrial Fibrillation; Biomarkers; Chromatography, Liquid; Echocardiography; Electrocardiography; Female; Germany; Humans; Male; Middle Aged; Morbidity; Oxidative Stress; Population Surveillance; Tandem Mass Spectrometry; omega-N-Methylarginine
PubMed: 26580348
DOI: 10.1016/j.ijcard.2015.05.102 -
Vascular Pharmacology Feb 2016
Review
Topics: Animals; Biopterins; Endothelium, Vascular; Enzyme Inhibitors; Humans; Hypotension; Nitric Oxide Synthase; Sepsis; omega-N-Methylarginine
PubMed: 26556765
DOI: 10.1016/j.vph.2015.10.008 -
Journal of Bacteriology Apr 2017Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central...
Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including and , the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or factors regulating DDAH expression in PAO1. First, results revealed that can utilize , -dimethyl-l-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the gene was observed to be induced in the presence of methylarginines, including -monomethyl-l-arginine (l-NMMA) and ADMA. Third, induction of the gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative sigma factor RpoN. Both PA1196 and RpoN were essential for the expression of the gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for imethylarginine imethylminohydrolase egulator. Interestingly, DdaR and its target gene are conserved only among strains, suggesting that this particular species has evolved to utilize methylarginines from its environment. Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the sigma factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among strains.
Topics: Amidohydrolases; Arginine; Bacterial Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Pseudomonas aeruginosa; RNA Polymerase Sigma 54; omega-N-Methylarginine
PubMed: 28167521
DOI: 10.1128/JB.00001-17 -
Cellular and Molecular Biology... May 2017Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We...
Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We examined interactions of PMN and intestinal epithelial cell-like CaCo-2 cells to elucidate their regulation of inflammatory signalling and the impact of cyclooxygenase (COX), nitric oxide (NO) and platelet-activating factor (PAF). Human PMN and CaCo-2 cells, separately and in co-incubation, were stimulated with the calcium ionophore A23187 or with N-Formyl-methionyl-leucyl-phenylalanin (fMLP) that activates PMN only. Human neutrophil elastase (HNE) and respiratory Burst were measured. To evaluate the modulation of inflammatory crosstalk we applied inhibitors of COX (acetyl salicylic acid; ASA), NO-synthase (N-monomethyl-L-arginin; L-NMMA), and the PAF-receptor (WEB2086). Unstimulated, co-incubation of CaCo-2 cells and PMN led to significantly reduced Burst and elevated HNE as compared to PMN. After stimulation with A23187, co-incubation resulted in an inhibition of Burst and HNE. Using fMLP co-incubation failed to modulate Burst but increased HNE. Without stimulation, all three inhibitors abolished the effect of co-incubation on Burst but did not change HNE. ASA partly prevented modulation of Burst L-NMMA and WEB2086 did not change Burst but abolished mitigation of HNE. Without stimulation, co-incubation reduced Burst and elevated HNE. Activation of PMN and CaCo-2 cells by fMLP as compared to A23187 resulted in a completely different pattern of Burst and HNE, possibly due to single vs. dual cell activation. Anti-inflammatory effect of co-incubation might in part be due to due to COX-signalling governing Burst whereas NO- and PAF-dependent signalling seemed to control HNE release.
Topics: Aspirin; Azepines; Caco-2 Cells; Calcimycin; Humans; Inflammation; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Respiratory Burst; Triazoles; omega-N-Methylarginine
PubMed: 28719356
DOI: 10.14715/cmb/2017.63.5.22 -
Journal of Applied Physiology... Dec 2017Broxterman RM, Trinity JD, Gifford JR, Kwon OS, Kithas AC, Hydren JR, Nelson AD, Morgan DE, Jessop JE, Bledsoe AD, Richardson RS. Single passive leg movement assessment...
Broxterman RM, Trinity JD, Gifford JR, Kwon OS, Kithas AC, Hydren JR, Nelson AD, Morgan DE, Jessop JE, Bledsoe AD, Richardson RS. Single passive leg movement assessment of vascular function: contribution of nitric oxide. J Appl Physiol 123: 1468-1476, 2017. First published August 31, 2017; doi:10.1152/japplphysiol.00533.2017.-The assessment of passive leg movement (PLM)-induced leg blood flow (LBF) and vascular conductance (LVC) is a novel approach to assess vascular function that has recently been simplified to only a single PLM (sPLM), thereby increasing the clinical utility of this technique. As the physiological mechanisms mediating the robust increase in LBF and LVC with sPLM are unknown, we tested the hypothesis that nitric oxide (NO) is a major contributor to the sPLM-induced LBF and LVC response. In nine healthy men, sPLM was performed with and without NO synthase inhibition by intra-arterial infusion of N-monomethyl-l-arginine (l-NMMA). Doppler ultrasound and femoral arterial pressure were used to determine LBF and LVC, which were characterized by the peak change (ΔLBF and ΔLVC) and area under the curve (LBF and LVC). l-NMMA significantly attenuated ΔLBF [492 ± 153 (l-NMMA) vs. 719 ± 238 (control) ml/min], LBF [57 ± 34 (l NMMA) vs. 147 ± 63 (control) ml], ΔLVC [4.7 ± 1.1 (l-NMMA) vs. 8.0 ± 3.0 (control) ml·min·mmHg], and LVC [0.5 ± 0.3 (l-NMMA) vs. 1.6 ± 0.9 (control) ml/mmHg]. The magnitude of the NO contribution to LBF and LVC was significantly correlated with the magnitude of the control responses ( r = 0.94 for ΔLBF, r = 0.85 for LBF, r = 0.94 for ΔLVC, and r = 0.95 for LVC). These data establish that the sPLM-induced hyperemic and vasodilatory response is predominantly (~65%) NO-mediated. As such, sPLM appears to be a promising, simple, in vivo assessment of NO-mediated vascular function and NO bioavailability. NEW & NOTEWORTHY Passive leg movement (PLM), a novel assessment of vascular function, has been simplified to a single PLM (sPLM), thereby increasing the clinical utility of this technique. However, the role of nitric oxide (NO) in mediating the robust sPLM hemodynamic responses is unknown. This study revealed that sPLM induces a hyperemic and vasodilatory response that is predominantly NO-mediated and, as such, appears to be a promising simple, in vivo, clinical assessment of NO-mediated vascular function and, therefore, NO bioavailability.
Topics: Adult; Arterial Pressure; Enzyme Inhibitors; Hemodynamics; Humans; Hyperemia; Leg; Male; Movement; Nitric Oxide; Regional Blood Flow; Vasodilation; Young Adult; omega-N-Methylarginine
PubMed: 28860173
DOI: 10.1152/japplphysiol.00533.2017 -
American Journal of Physiology. Heart... Mar 2016We tested the hypothesis that women exhibit greater vasodilator responses to β-adrenoceptor stimulation compared with men. We further hypothesized women exhibit a...
We tested the hypothesis that women exhibit greater vasodilator responses to β-adrenoceptor stimulation compared with men. We further hypothesized women exhibit a greater contribution of nitric oxide synthase and cyclooxygenase to β-adrenergic-mediated vasodilation compared with men. Forearm blood flow (Doppler ultrasound) was measured in young men (n = 29, 26 ± 1 yr) and women (n = 33, 25 ± 1 yr) during intra-arterial infusion of isoproterenol (β-adrenergic agonist). In subset of subjects, isoproterenol responses were examined before and after local inhibition of nitric oxide synthase [N(G)-monomethyl-l-arginine (l-NMMA); 6 male/10 female] and/or cyclooxygenase (ketorolac; 5 male/5 female). Vascular conductance (blood flow ÷ mean arterial pressure) was calculated to assess vasodilation. Vascular conductance increased with isoproterenol infusion (P < 0.01), and this effect was not different between men and women (P = 0.41). l-NMMA infusion had no effect on isoproterenol-mediated dilation in men (P > 0.99) or women (P = 0.21). In contrast, ketorolac infusion markedly increased isoproterenol-mediated responses in both men (P < 0.01) and women (P = 0.04) and this rise was lost with subsequent l-NMMA infusion (men, P < 0.01; women, P < 0.05). β-Adrenergic vasodilation is not different between men and women and sex differences in the independent contribution of nitric oxide synthase and cyclooxygenase to β-mediated vasodilation are not present. However, these data are the first to demonstrate β-adrenoceptor activation of cyclooxygenase suppresses nitric oxide synthase signaling in human forearm microcirculation and may have important implications for neurovascular control in both health and disease.
Topics: Adrenergic beta-Agonists; Adult; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Female; Forearm; Humans; Infusions, Intra-Arterial; Isoproterenol; Ketorolac; Male; Microcirculation; Nitric Oxide Synthase; Prostaglandin-Endoperoxide Synthases; Sex Factors; Ultrasonography, Doppler; Vasodilation; omega-N-Methylarginine
PubMed: 26747505
DOI: 10.1152/ajpheart.00886.2015 -
American Journal of Physiology. Heart... Jan 2022Central adiposity is associated with greater sympathetic support of blood pressure. β-adrenergic receptors (β-AR) buffer sympathetically mediated vasoconstriction and...
Central adiposity is associated with greater sympathetic support of blood pressure. β-adrenergic receptors (β-AR) buffer sympathetically mediated vasoconstriction and β-AR-mediated vasodilation is attenuated in preclinical models of obesity. With this information, we hypothesized β-AR vasodilation would be lower in obese compared with normal weight adults. Because β-AR vasodilation in normal weight adults is limited by cyclooxygenase (COX) restraint of nitric oxide synthase (NOS), we further explored the contributions of COX and NOS to β-AR vasodilation in this cohort. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured and forearm vascular conductance (FVC) was calculated (FVC = FBF/MAP). The rise in FVC from baseline (ΔFVC) was quantified during graded brachial artery infusion of isoproterenol (Iso, 1-12 ng/100 g/min) in normal weight ( = 36) and adults with obesity ( = 22) (18-40 yr old). In a subset of participants, Iso-mediated vasodilation was examined before and during inhibition of NOS [-monomethyl-l-arginine (l-NMMA)], COX (ketorolac), and NOS + COX (l-NMMA + ketorolac). Iso-mediated increases in FVC did not differ between groups ( = 0.57). l-NMMA attenuated Iso-mediated ΔFVC in normal weight ( = 0.03) but not adults with obesity ( = 0.27). In normal weight adults, ketorolac increased Iso-mediated ΔFVC ( < 0.01) and this response was lost with concurrent l-NMMA ( = 0.67). In contrast, neither ketorolac ( = 0.81) nor ketorolac + l-NMMA ( = 0.40) altered Iso-mediated ΔFVC in adults with obesity. Despite shifts in COX and NOS, β-AR vasodilation is preserved in young adults with obesity. These data highlight the presence of a compensatory shift in microvascular control mechanisms in younger humans with obesity. We examined β-adrenergic receptor-mediated vasodilation in skeletal muscle of humans with obesity and normal weight. Results show that despite shifts in the contribution of cyclooxygenase and nitric oxide synthase, β-adrenergic-mediated vasodilation is relatively preserved in young, otherwise healthy adults with obesity. These data highlight the presence of subclinical changes in microvascular control mechanisms early in the obesity process and suggest duration of obesity and/or the addition of primary aging may be necessary for overt dysfunction.
Topics: Adrenergic beta-Agonists; Adult; Blood Vessels; Cyclooxygenase Inhibitors; Female; Humans; Isoproterenol; Ketorolac; Male; Muscle, Skeletal; Nitric Oxide Synthase Type III; Obesity; Prostaglandin-Endoperoxide Synthases; Receptors, Adrenergic, beta; Vasodilation; omega-N-Methylarginine
PubMed: 34738833
DOI: 10.1152/ajpheart.00449.2021 -
Journal of the American Society For... Jul 2019Arginine methylation is a common protein post-translational modification (PTM) that plays a key role in eukaryotic cells. Three distinct types of this modification are...
Arginine methylation is a common protein post-translational modification (PTM) that plays a key role in eukaryotic cells. Three distinct types of this modification are found in mammals: asymmetric NN-dimethylarginine (aDMA), symmetric NN-dimethylarginine (sDMA), and an intermediate N-monomethylarginine (MMA). Elucidation of regulatory mechanisms of arginine methylation in living organisms requires precise information on both the type of the modified residues and their location inside the protein amino acid sequences. Despite mass spectrometry (MS) being the method of choice for analysis of multiple protein PTMs, unambiguous characterization of protein arginine methylation may not be always straightforward. Indeed, frequent internal basic residues of Arg methylated tryptic peptides hamper their sequencing under positive ion mode collision-induced dissociation (CID), the standardly used tandem mass spectrometry method, while the relative stability of the aDMA and sDMA side chains under alternative non-ergodic electron-based fragmentation techniques, electron-capture and electron transfer dissociations (ECD and ETD), may impede differentiation between the isobaric residues. Here, for the first time, we demonstrate the potential of the negative ion mode collision-induced dissociation MS for analysis of protein arginine methylation and present data revealing that the negative polarity approach can deliver both an unambiguous identification of the arginine methylation type and extensive information on the modified peptide sequences.
Topics: Amino Acid Sequence; Arginine; Peptides; Protein Processing, Post-Translational; Proteins; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; omega-N-Methylarginine
PubMed: 30915654
DOI: 10.1007/s13361-019-02176-9 -
Journal of Separation Science Aug 2014We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for...
Simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine, and L-arginine using monolithic silica disk-packed spin columns and a monolithic silica column.
We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for the simultaneous determination of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G')-dimethyl-L-arginine in human plasma. For solid-phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and then the derivatives are separated on a monolithic silica column. L-arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2-1.0 μM for methylated arginines and 40-200 μM for L-arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r(2)). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3-4.8% (intraday) and 3.0-6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, N(G),N(G')-dimethyl-L-arginine and L-arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 μM (n = 6), respectively.
Topics: Arginine; Calibration; Chromatography, High Pressure Liquid; Fluorescent Dyes; Healthy Volunteers; Humans; Linear Models; Reproducibility of Results; Silicon Dioxide; Solid Phase Extraction; omega-N-Methylarginine
PubMed: 24895248
DOI: 10.1002/jssc.201400240