Simultaneous Measurement of [<sup>15</sup>N]Nitrate and [<sup>15</sup>N]Nitrite Enrichment and Concentration in Urine by Gas Chromatography Mass Spectrometry as Pentafluorobenzyl Derivatives

Tsikas, Dimitrios; Schwarz, Alexandra; Stichtenoth, Dirk O.

doi:10.1021/ac902970m

Cited by 41 publications

(24 citation statements)

References 6 publications

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“…213 Electrochemical properties of UMEs with a tip diameter less than 25 μm substantially differ from bigger electrodes. 103,106,364,[368][369][370] In a solution with a supporting electrolyte, mass transport in the vicinity of the UME surface is mainly governed by diffusion, reaching steady-state and time-independent conditions within a fraction of a second. Usually, current values measured with UMEs are in the fem-toA to nanoA range; therefore, resistive drops of the current in the electrolyte solution are negligible.…”

Section: G87mentioning

confidence: 99%

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

Gulaboski

Mirčeski

Kappl

et al. 2019

J. Electrochem. Soc.

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Reactive oxygen species (ROS) exhibit different spatial and temporal distributions as well as concentrations in-and outside the cell, thereby functioning as signaling or pathogen-destroying molecules. Especially the ROS H 2 O 2 is important for the patho/physiological status of an organism. Electrochemistry (EM) and electron spin resonance (ESR)-based techniques allow quantification of H 2 O 2 in artificial and living systems, coping a concentration range from low nM up to mM. Working electrodes for EM are optimized by diverse modifications and, additionally, redox mediators are used. Ultramicroelectrodes allow scanning of single cells to spatially resolve and quantify extracellular H 2 O 2 in real-time. With ESR spectroscopy, • O 2¯, but not H 2 O 2 , can be directly determined by spin probes in-and outside of cells in suspensions. Monitoring H 2 O 2 requires formation of intermediate radicals, detectable with spin probes. Low μM [H 2 O 2 ] can thus be assessed specifically. Using suitable spin traps, in-vivo ESR and immuno-spin trapping can visualize different radicals at their respective production sites in small animals, organs and tissues. Here, the redox reaction cascades may interfere with cell metabolism. Optimization of all methods established for H 2 O 2 determination would be favorable to finally combine them for mutual validation. Thus, a deeper insight into cellular ROS metabolism can be obtained.

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Section: G87mentioning

confidence: 99%

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

Gulaboski

Mirčeski

Kappl

et al. 2019

J. Electrochem. Soc.

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“…The higher excretion rate of nitrite in DMD compared to healthy controls suggests an impaired reabsorption of nitrite in the kidneys, finally resulting in loss of NO bioavailability mainly in the form of nitrite. In humans, renal carbonic anhydrases are involved in the reabsorption of nitrite in the proximal tubule of the nephron (Tsikas et al 2010b;Chobanyan-Jürgens et al 2012b). The urinary nitrate-tonitrite molar ratio U NOx R is a useful measure of nitritedependent carbonic anhydrase activity .…”

Section: Adma and No Synthesis In Dmdmentioning

confidence: 99%

The l-arginine/NO pathway and homoarginine are altered in Duchenne muscular dystrophy and improved by glucocorticoids

et al. 2015

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The L-arginine/nitric oxide (L-Arg/NO) pathway regulates endothelial function and may play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). Yet, this pathway is poorly investigated in children suffering from DMD. Endothelial dysfunction can affect the perfusion of contracting muscles, thus leading to ischemia and hypoxia. In the present study, we tested the hypothesis that reduced NO production due to elevated synthesis of N (G),N (G)-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of NO synthesis, is a possible pathophysiological mechanism for progressive intramuscular muscle ischemia and disturbed endothelial function in children with DMD. Given the possible antagonistic action of homoarginine (hArg) on ADMA, we also analyzed this amino acid. We investigated 55 male patients with DMD and 54 healthy male controls (HC; aged 11.9 ± 4.8 vs. 11.1 ± 4.9 years, mean ± SD). Urinary creatinine and metabolites of the L-Arg/NO pathway were measured in plasma and urine by GC-MS or GC-MS/MS. Urine levels of ADMA and its major urinary metabolite dimethylamine (DMA), nitrite and nitrate (P < 0.001 for all) and hArg (P = 0.002) were significantly higher in DMD patients compared to HC, while the urinary DMA/ADMA molar ratio was lower (P = 0.002). In plasma, nitrate (P < 0.001), hArg (P = 0.002) and the hArg/ADMA ratio (P < 0.001) were lower in DMD than in HC. In plasma, ADMA (631 ± 119 vs. 595 ± 129 nM, P = 0.149), arginine and nitrite did not differ between DMD and HC. In DMD, positive correlations between ADMA, DMA or nitrate excretion and the stage of disease (according to Vignos and Thompson) were found. In DMD patients on steroid medication, lower concentrations of ADMA in plasma, and of DMA, ADMA, nitrate and hArg in urine were observed compared to non-treated patients. The L-Arg/NO pathway is impaired in DMD patients, with the disease progression being clinically negatively correlated with the extent of impairment. One of the underlying mechanisms in DMD may involve insufficient antagonism of ADMA by hArg. Steroids, but not creatine supplementation, seems to improve the L-Arg/NO pathway in DMD.

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“…With this approach, background correction is not necessary, and assay sensitivity can be greatly improved. 15 N-nitrite and 15 N-nitrate can be measured by GC-MS as their pentafluorobenzyl derivatives [11], or by a liquid chromatography tandem mass spectrometric (LC-MS/MS) assay as a fluorescent 2,3-diaminonaphthalene (DAN) derivative [12]. These methods are anticipated to be superior because of their higher specificity and sensitivity in biological samples.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of an LC–MS/MS assay for 15N-nitrite for cellular studies of l-arginine action

Shin

Fung

2011

Journal of Pharmaceutical and Biomedical Analysis

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The utility of an LC-MS/MS assay for nitrite determination in studying L-arginine (ARG) cellular action was examined in vitro. EA.hy926 human endothelial cells or cellular fractions (membrane and cytosol) were exposed to 0 – 500 μM of 15N4-ARG for 2 hours. 14N-Nitrite and 15N-nitrite in the cell lysate and in the incubation medium were derivatized with 2,3-diaminonaphthalene (DAN) to form 14N- and 15N-naphthotriazole (i.e., 14N-NAT and 15N-NAT). Peak responses of 14N-NAT and 15N-NAT were analyzed by LC-MS/MS with 1H-naphth[2,3-d]imidazole as an internal standard. The calibration curves of DAN-derivatized 14N-NAT and 15N-NAT from 14N-nitrite and 15N-nitrite were linear. Intra- and inter-day variability of the quantification was below 14.2 % in quality control samples. Following incubation of EA.hy926 cells with 15N4-ARG, saturable increases of 15N-nitrite accumulation with increasing 15N4-ARG exposure were observed clearly. This increase however could not be detected by the classical fluorescence method, nor were changes in 14N-nitrite level observed. When cellular fractions were exposed to 15N4-ARG, 15N-nitrite formation was only observed in the membrane fragments. The sensitive and selective LC-MS/MS method reported here can be applied to quantify accumulated nitrite levels in human endothelial cells. The selectivity of this stable-isotope labeled LC-MS/MS method offers an advantage over other traditional methods for elucidating cellular ARG action when its stable isotope is employed as a substrate.

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Simultaneous Measurement of [¹⁵N]Nitrate and [¹⁵N]Nitrite Enrichment and Concentration in Urine by Gas Chromatography Mass Spectrometry as Pentafluorobenzyl Derivatives

Cited by 41 publications

References 6 publications

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

The l-arginine/NO pathway and homoarginine are altered in Duchenne muscular dystrophy and improved by glucocorticoids

Evaluation of an LC–MS/MS assay for 15N-nitrite for cellular studies of l-arginine action

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Simultaneous Measurement of [15N]Nitrate and [15N]Nitrite Enrichment and Concentration in Urine by Gas Chromatography Mass Spectrometry as Pentafluorobenzyl Derivatives

Cited by 41 publications

References 6 publications

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

The l-arginine/NO pathway and homoarginine are altered in Duchenne muscular dystrophy and improved by glucocorticoids

Evaluation of an LC–MS/MS assay for 15N-nitrite for cellular studies of l-arginine action

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Simultaneous Measurement of [¹⁵N]Nitrate and [¹⁵N]Nitrite Enrichment and Concentration in Urine by Gas Chromatography Mass Spectrometry as Pentafluorobenzyl Derivatives