Simultaneous determination of nitroglycerin and dinitrate metabolites in metabolism studies using liquid chromatography–mass spectrometry with electrospray ionization

Miyayama, Takashi; Tsou, Pei Suen; Fung, Sun Mi; Fung, Ho‐Leung

doi:10.1016/j.jchromb.2006.02.059

Cited by 12 publications

(7 citation statements)

References 14 publications

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“…The method was validated using quality control samples, which were processed similarly as the samples. As described previously [31], the accuracy of the assay for the three nitrates, over the range of 1.5–8 ng/ml, were between 92–117% of the theoretical concentration with a CV<9%.…”

Section: Methodsmentioning

confidence: 67%

“…Samples from the incubation media were collected everyday following NTG incubation as described in section 2.2 for NTG, 1,2-GDN, and 1,3-GDN analysis, stored at −80° C until analysis, and analyzed using our published LC-MS/MS method [31]. The samples were diluted with equal volume of solution of 50% methanol and water containing the internal standard, 1,2,4-butanetriol-1,4-dinitrate, centrifuged at 16,000 × g for 20 minutes at 4° C. Samples for constructing standard curves were prepared with the identical proportion of RPMI medium.…”

Section: Methodsmentioning

confidence: 99%

“…The samples were diluted with equal volume of solution of 50% methanol and water containing the internal standard, 1,2,4-butanetriol-1,4-dinitrate, centrifuged at 16,000 × g for 20 minutes at 4° C. Samples for constructing standard curves were prepared with the identical proportion of RPMI medium. Concentrations of NTG and its metabolites 1,2-GDN and 1,3-GDN in rat plasma were determined via the LC-MS/MS method [31], with the following minor modifications. Sirocco protein precipitation plate was placed on the collection plate, and 150 µl of acetonitrile was added.…”

Section: Methodsmentioning

confidence: 99%

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Nitroglycerin alters matrix remodeling proteins in THP-1 human macrophages and plasma metalloproteinase activity in rats

Krishnatry¹,

Fung²,

Brazeau³

et al. 2011

Nitric Oxide

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Several studies suggested that long-term nitrate therapy may produce negative outcomes in patient mortality and morbidity. A possible mechanism may involve nitrate-mediated activation of various extracellular matrix (ECM) proteases, particularly matrix metalloproteinase-9 (MMP-9), and adhesion molecules in human macrophages, leading to the destabilization of atherosclerotic plaques. We examined the gene and protein regulating effects on THP-1 human macrophages by repeated exposure to therapeutically relevant concentrations of nitroglycerin (NTG) and possible involvement of nuclear factor (NF)-κB signaling mechanism in mediating some of these observed effects. THP-1 human macrophages repeatedly exposed to NTG (at 10 nM, added on days 1, 4 and 7) exhibited extensive alterations in the expression of multiple genes encoding ECM proteases and adhesion molecules. These effects were dissimilar to those produced by a direct nitric oxide donor, diethylenetriamine NONOate. NTG exposure significantly up-regulated NF-κB DNA nuclear binding activity and MMP-9 protein expression, and reduced tissue inhibitor of metalloproteinase-1 (TIMP-1) expression; these effects were abrogated in the presence of the NF-κB inhibitor parthenolide (a chemical inhibitor derived from the feverfew plant). Further, we examined whether our in vitro findings (an elevated MMP-9/TIMP-1 ratio and gelatinase activity) can be translated to in vivo effects, in a rat model. Sprague-Dawley rats exposed continuously to NTG subcutaneously for 8 days via mini-osmotic pumps showed significant induction of plasma MMP-9 dimer concentrations and the expression of a complex of MMP-9 with lipocalin-2 or neutrophil gelatinase associated lipocalin (NGAL). Plasma gelatinase activity was significantly increased by NTG over the entire study period, attaining peak elevation at day 6. Plasma TIMP-1 protein was down-regulated significantly by day 2 and days 4 to 7 in the NTG-treated rats. Pharmacokinetic monitoring of NTG and its dinitrate metabolites indicated that concentrations were well within therapeutic levels observed in humans. Our studies indicate that clinically relevant concentrations of NTG not only altered ECM matrix by changing the expression of multiple genes that govern cellular integrity, affecting cellular MMP-9/TIMP-1 balance in THP-1 human macrophages possibly via NF-κB activation, but also led to systemic changes in MMP-9/ TIMP-1 expression and gelatinase activity in rats. These effects may contribute to extracellular matrix degradation and possible atherosclerotic plaque destabilization.

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Section: Methodsmentioning

confidence: 67%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Nitroglycerin alters matrix remodeling proteins in THP-1 human macrophages and plasma metalloproteinase activity in rats

Krishnatry¹,

Fung²,

Brazeau³

et al. 2011

Nitric Oxide

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“…Denitration of NTG was measured using liquid chromatography tandem mass spectrometry with electrospray ionization as previously described by Miyayama et al [14]. Standard solutions containing NTG, 1, 2-GDN, 1, 3-GDN and the internal standard BTDN were dissolved in 1: 1 v/v PBS (50 mM, pH 7.4): 100% methanol, with final concentrations of the three nitrates ranging from 0.375 to 15 ng/ml and BTDN at 2.5 ng/ml.…”

Section: Methodsmentioning

confidence: 99%

In vitro organic nitrate bioactivation to nitric oxide by recombinant aldehyde dehydrogenase 3A1

et al. 2013

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Organic nitrates (ORNs) are commonly used anti-ischemic and anti-anginal agents, which serve as an exogenous source of the potent vasodilator nitric oxide (NO). Recently, both mitochondrial aldehyde dehydrogenase-2 (ALDH2) and cytosolic aldehyde dehydrogenase-1a1 (ALDH1A1) have been shown to exhibit the ability to selectively bioactivate various ORNs in vitro. The objective of the present research was to examine the potential role of ALDH3A1, another major cytosolic isoform of ALDH, in the in vitro bioactivation of various ORNs, and to estimate the enzyme kinetic parameters toward ORNs through mechanistic modeling. The extent of bioactivation was assayed by exposing recombinant ALDH3A1 to various concentrations of ORNs, and measuring the concentration-time profiles of released NO via a NO-specific electrode. Metabolite formation kinetics was monitored for nitroglycerin (NTG) using LC/MS/MS. Our results showed that ALDH3A1 mRNA and protein were highly expressed in C57BL/6 mouse aortic, cardiac, and hepatic tissues, and it was able to release NO from several ORNs, including NTG, isosorbide dinitrate (ISDN), isosorbide-2-mononitrate (IS-2-MN), and nicorandil with similar Vmax (0.175 – 0.503 nmol/min/mg of ALDH3A1), and Km values of 4.01, 46.5, 818 and 5.75 × 103 μM respectively. However, activation of isosorbide-5-mononitrate (IS-5-MN) by ALDH3A1 was undetectable in vitro. ALDH3A1 was also shown to denitrate NTG, producing primarily glyceryl 1, 2-dinitrate (1, 2-GDN) in preference to glyceryl 1, 3-dinitrate (1, 3-GDN). Therefore, ALDH3A1 may contribute to the bioactivation of ORNs in vivo.

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“…Nitroglycerin (glyceryltrinitrate,G TN, CAS 55-63-0)h as been used in the treatmentof anginapectorisformore thanac entury [1].Following oraladministration,G TN ismetabolized asafirst step totwog lyceryld initrate metabolitesbyanonspecificenzymaticprocess [2], thus forthe pharmacokineticstudyof oralGTN, its main two stable metabolites(half-lives:30 -60 min);1,2-dinitroglycerin (1,2-GDN, CAS 621-65-8) and 1,3-dinitroglycerin (1,3-GDN, CAS 623-87-0)should bed etermined in plasma [ 3].Thesetwom etabolitesexert pharmacologicale ffects qualitativelyt he same ast hoseo fthe parent drug [4].GTN isalsorapidlybroken downat3 7°Cin humanblood witht 1/2 of 6.2min [5].Asingle orald ose of 13mg nitroglycerin hasbeen administered tohealthy volunteers without anynotable side effect [6].Miyayama etal. quantified GTN and its activem etabolites in cell culturem ediumusing aliquid chromatographic-mass spectrometricmethod [7].Blumenthal etal. [8] measured lowplasmac oncentrationsof nitroglycerin (0.1 to0.2ng/mL)f ollowing oraladministration of 6.5 mg asasustained-releasecapsule.…”

Section: Introductionmentioning

confidence: 99%

Determination of glyceryl trinitrate and its two main metabolites in human plasma using a new sensitive gas chromatography method

Ghandforoush-Sattari

Zakeri–Milani

Mashayekhi³

et al. 2011

Arzneimittelforschung

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The aim of the present study was to determine the concentrations of nitroglycerin (glyceryl trinitrate, GTN, CAS 55-63-0) and its two main stable metabolites; 1,2-dinitroglycerin (1,2-glyceryl dinitrate, GDN, CAS 621-65-8) and 1,3-dinitroglycerin (1,3-GDN, CAS 623-87-0) in human plasma using a capillary gas chromatography method with an electron capture detection. Using the GC conditions, linear calibrations were obtained for 1,3-GDN from 0.14 to 3 ng/mL, for 1,2-GDN from 0.06 to 6 ng/mL, and for GTN from 0.01 to 0.3 ng/mL in plasma samples by the following calibration curve equations: [y = 0.1924x - 0.0088 (r = 0.999)], [y = 0.2273x + 0.0164 (r = 0.995)], [y = 17.434x - 0.0751] for 1,3-GDN, 1,2-GDN, and GTN respectively. The calculated limits of quantification values for GTN, 1,2-GDN, and 1,3-GDN were 0.03 ng/mL, 0.2 ng/mL, and 0.15 ng/mL respectively. This method was verified with a bioequivalence study of an Iranian brand of oral sustained release nitroglycerin with an innovator formulation.

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Simultaneous determination of nitroglycerin and dinitrate metabolites in metabolism studies using liquid chromatography–mass spectrometry with electrospray ionization

Cited by 12 publications

References 14 publications

Nitroglycerin alters matrix remodeling proteins in THP-1 human macrophages and plasma metalloproteinase activity in rats

Nitroglycerin alters matrix remodeling proteins in THP-1 human macrophages and plasma metalloproteinase activity in rats

In vitro organic nitrate bioactivation to nitric oxide by recombinant aldehyde dehydrogenase 3A1

Determination of glyceryl trinitrate and its two main metabolites in human plasma using a new sensitive gas chromatography method

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