Emilie Destandau

Pierre Leroy

Thierry Oster

Dr Emilie Dear

Destandau

Elke Richling

Dear Pr Igor Clarot

Pr Thierry Oster

Pr Alain Isabelle Le Faou

Lartaud

Marie-Lynda Bouressam

Justine Bonetti

Romain Schmitt

Yi Zhou

Hui Ming

Arnaud Pallotta

Eugenia Belcastro…

Isabelle Fries

Philippe Giummelly

Nathalie Degousee

Pascale Carnet

Etude de la biodisponibilité orale du S-

Introduction

Review in Talanta (191 (2018) 491492493494495496497498499500501502503Table 1. Nitrogen 15 labeled compounds and their metabolic pathways or Table 1. Comparison of performances between derivatization of nitrite ion with Griess and 2,3-diaminonaphthalene (DAN) methods followed by LC-ESI-ITMS/MS analysis (CID: Collision Induced Dissociation; HCD: Higher energy Collision Dissociation; LOQ: limit of quantification; S/N: signal-over-noise; S/B: signal-over-blank) .... 73

Experimental paper in Rapid Communications in Mass Spectrometry (under minor revision):

Table 1. Ion composition of the MS/MS spectra of the 14 N-NAT or 15 N-NAT adduct in CID (35eV) and HCD (50eV) depending on the organic modifier composing the chromatographic mobile phase (solvent S). The NAT adduct was infused in a mixture of a 10 mM-acetic acid aqueous solution containing 40% of the solvent S. The relative abundance (%) of each ion is indicated in parenthesis. Nd: not detected ...... 101 Table 2. Inter-day and intra-day variability of quality control samples for 15 N-nitrite S-nitrosoglutathione (GS 15 NO) and 15 N-nitrate analysis with IS. 15 N-nitrite, GS 15 NO and 15 N-nitrate were spiked into HBSS buffer (n = 6), and the concentration was calculated by using related six-point calibration curve (5, 10, 20, 50, 100, 200 nM) of 15 N-nitrite and S-nitrosoglutathione (GS 15 NO), and five-point calibration curve (250,500,1250,2500, 5000 nM) of 15 N-nitrite. Accuracy was determined as the deviation (%) of calculated concentrations from the nominal concentrations. Precision was determined as the relative standard deviation (RSD) of the 6 measurements.102

nd Chapter

Table 1. Comparison of the physiological parameters in different parts of the gut (Bilat et al. 2017) ) and the RSNO form after 4 h of permeability study from the apical compartment (pH 6.4) to the basolateral compartment (pH 7.4). .................... 125

Table 7. Mass balance for all tested treatments (initial amount 50 nmol) after 4 h of permeability from the apical compartment at pH 6.4 to the basolateral compartment at

Experimental paper to be submitted to Drug Metabolism and Disposition:

Table 1. Specific activity of γ -glutamyltransferase (GGT) and thiol content in isolated rat intestine. (ND # : not detected, under limit of detection; -: undone; GCNA:

L-γ-glutamyl-

General conclusions and perspectives

Table 1. Summary of bioanalytical method performances for S-nitrosoglutathione The concentration of RS 15 NOs is obtained after the pretreatment of the metabolite pool with Hg 2+ . The quantification by LC-MS/MS after DAN derivatization of this sample provides the cumulative concentration  of free 15 N-nitrites + GS 15 NO (converted into nitrites, see reaction 2 in figure 1). The concentration of RS 15 NO  is deduced from result  by subtracting the concentration of free 15 N-nitrites ..104 Administered RSNOs are converted to nitrite and nitrate ions, and other RSNOs through transnitrosation, in the body [START_REF] Minamiyama | Effect of thiol status on nitric oxide metabolism in the circulation[END_REF]. All these NO species can be converted back into NO, and their concentrations are closely linked to physiology or pathology states. For example, NO in the range of pico to nanomolar, shows protective properties, while it becomes cytotoxic in the micromolar range [START_REF] Mocellin | Nitric oxide, a double edged sword in cancer biology: Searching for therapeutic opportunities[END_REF]. Still, there are many unknown parts and challenges for these studies. For example, which method able to distinguish endogenous and exogenous NO sources can be used to analyze these NO species with a sufficient high specificity and the required sensitivity? Thus, the first step of our research is to find a selective and sensitive method, using nitrogen 15 Lorsqu'un seul de ces ions fragments (dits produits) est sélectionné pour atteindre l'analyseur, on est en mode de détection MRM (pour Multiple Reaction Monitoring). In conclusion, DAN derivatization of nitrite ions coupled with LC-ITMS/MS in HCD mode shows a lower LOQ value for the quantification of 15 N labeled NO species than with the Griess method. Consequently, preliminary experiments of GSNO permeability in intestinal barrier models (Caco-2 cell monolayer and isolated rat intestine in Ussing chamber) have been carried out using this method. They allowed concluding that GSNO belongs to medium permeability class of drugs according to BCS rules [START_REF] Peng | Applications of a 7-day Caco-2 cell model in drug discovery and development[END_REF]. Intestinal permeability of RSNOs has been more deeply studied by using both models in the following chapter.

(

nd Chapter: Intestinal permeability studies of S-nitrosothiols using two models

Oral formulations are widely used as they are convenient for drug delivery, especially for CVDs which need chronic treatments. According to the Biopharmaceutical Classification System (BCS) (Amidon et al. 1995), a drug suitable for oral administration should possess high solubility and permeability (Figure 1). The RSNOs such as GSNO and S-nitroso-N-acetyl-D-penicillamine (SNAP), are known to have high solubility, but their intestinal permeability is not yet reported.

Figure 1. Scheme of Biopharmaceutical Classification System.

The main absorption part is small intestine (Figure 2 C). Interestingly, parts of small intestine differ with their physicological characteristics (Table 1) (Bilat et al. 2017). (Bilat et al. 2017).

To study RSNOs intestinal permeability, two models mastered at the EA 3452

Cithefor level have been presently used:

-an in vitro model with a monolayer of the differentiated epithelial cells (Caco-2) (Figure 2 A);

-an ex vivo model using "intact" isolated intestine (ileum) of rat placed in Ussing chamber (Figure 2 B). Previous studies have been realized in the lab with "stripped" isolated rat intestine in Ussing chamber [START_REF] Shah | Synthesis and characterization of S-nitrosoglutathione-oligosaccharide-chitosan as a nitric oxide donor[END_REF]) [START_REF] Shah | Synthesis of S-nitrosoglutathione-alginate for prolonged delivery of nitric oxide in intestines[END_REF]) . As the model offers poor reproducibility, "intact" intestine was preferred. Then, the classification of GSNO permeability was confirmed by using GS 15 NO in the ex vivo model and quantifying the permeated labeled NO species by LC-MS/MS [START_REF] Yu | Comparison between two derivatization methods of nitrite ion labeled with 15 N applied to liquid chromatography-tandem mass spectrometry[END_REF]).

In addition, the roles of denitrosating enzyme γ-glutamyltransferase (GGT; EC

Abstract

Background: S-nitrosoglutathione (GSNO) is a promising nitric oxide (NO) donor in comparison with presently marketed drugs in the field of cardiovascular diseases (CVDs). Oral delivery of GSNO is an ideal route to treat chronic CVDs related to endothelial dysfunction. GSNO is a highly soluble molecule with medium permeability as already demonstrated by using two intestinal barrier models: Caco-2 cell monolayer (Bonetti et al., 2018) and isolated rat intestine [START_REF] Yu | Comparison between two derivatization methods of nitrite ion labeled with 15 N applied to liquid chromatography-tandem mass spectrometry[END_REF]. A passive transportation mechanism was demonstrated in the Caco-2 cell monolayer model (Bonetti et al., 2018). However, little is known about its degradation in the intestine and how the corresponding involved enzymatic and chemical processes can modify its absorption.

Aims:

The present work will focus on GSNO degradation and absorption in isolated rat intestine. The main factors already identified to be involved in the degradation of GSNO and leading to NO release in the aorta, i.e. γ-glutamyltransferase (GGT) and thiols including protein disulfide isomerase (PDI) (Dahboul et al., 2012) (Dahboul et al., 2014) will be studied.

Methods: First, GGT activity and thiol content were measured in isolated rat intestine samples using various substrates. Namely, GSNO and inhibitors (serine-borate complex, SBC) were added for GGT measurement; Ellmanʾs reagents and the thiol blocker (N-ethylmaleimide, NEM) were used for thiol quantification. GGT has to be localized in intestine samples through histological staining. Moreover, mucus, bacteria and epithelium visualization has to be performed by microscopy to understand whether denitrosating processes are localized on epithelial layer and/or on microbiota.

The addition of glycylglycine, a cosubstrate of GGT, and Mg 2+ (glygly-Mg 2+ ) is needed to accelerate the transferase activity. Next, GSNO degradation and absorption in isolated rat intestine were evaluated. For this purpose, GSNO metabolites (S-nitrosothiols (RSNOs), nitrite and nitrate ions) were quantified by spectrophotometry and LC-FL, respectively, in presence or not of glygly-Mg 2+ , SBC and NEM. At last, the absorption of GSNO and its metabolites with or without glygly-Mg 2+ and glygly-Mg 2+ coupled with SBC and NEM was studied with an ex vivo model based on isolated rat intestine placed in an Ussing chamber using LC coupled to Electrospray-Ion Trap tandem Mass Spectrometry (LC-ESI-ITMS/MS). The permeability of labeled GS 15 NO was evaluated by calculating its apparent permeability coefficient (P app ).

Results:

The specific GGT activity measured in isolated rat intestine in presence of glygly-Mg 2+ was 6.8 ± 0.8 nmol of 3-carboxy-4-nitroanilide min -1 .mg -1 of proteins.

Histological studies showed that GGT was localized in epithelial layer/mucus (microbiota) of the intestine (to be done). 26.5 ± 1.2 nmol of thiol .mg -1 of proteins were detected with Ellmanʾs reagents. When incubating GSNO with glygly-Mg 2+ for 60 min, a degradation rate of 0.9 ± 0.1 nmol of GSNO min -1 .mg -1 of proteins was observed. This degradation rate was lowered in presence of SBC and NEM. After incubating GSNO with intestine in absence and presence of activators (glygly-Mg 2+ )

or inhibitors (SBC and NEM) for 60 min, both inside and outside Ussing chamber, less than 1 % of initial GSNO was absorbed in the intestine tissue. Compared to incubating GSNO alone with isolated rat intestine in Ussing chamber, more nitrite ions (NO 2 -) was permeated through intestine (0.7 vs. 0.2 nmol) when GGT activators were added, and more RSNOs were permeated (0.5 vs. 0.1 nmol) when GGT inhibitor and thiol blocker were added. Conventional spectrofluorometry can not be used to quantify permeated NO species through intestine in Ussing chamber, due to its poor limit of

Introduction

In the case of vascular endothelium dysfunction resulting from aging and cardiovascular diseases (CVDs), the activity of the endothelial nitric oxide (NO) synthases decreases and consequently NO is produced at a low level. Meanwhile, inducible NO production increases, and oxidative stress emerges [START_REF] Loo | Enhanced Peroxynitrite Formation Is Associated with Vascular Aging[END_REF].

S-nitrosoglutathione (GSNO) as a physiological NO reservoir shows high potency to

treat CVDs, as it does not present drawbacks of presently marketed NO donors, especially tolerance phenomenon and induction of oxidative stress (Gaucher et al., 2013). Since most of CVDs are chronic, oral administration of GSNO appears to be the most practical route. Noteworthy, a drug suitable for oral administration should possess a good solubility and permeability according to the Biopharmaceutical Classification System (Amidon et al., 1995). Solubility depends on the physico-chemical properties of the drug while permeability is determined by the absorption through the gastrointestinal tract (GIT).

The main drug absorption part in GIT is the small intestine, which consists of duodenum, jejunum and ileum. Jejunum and ileum are often considered for research, because their length is 10-fold more than duodenum (3 vs 0.25-0.3 m) (Kararli, 1995).

Interestingly, ileum shows better absorption than jejunum, due to a longer residence time and a heavier mucus thickness (3-4 vs 2-3 h, 476 vs 123 µm, respectively) (Fallingborg et al., 1989) (Atuma et al., 2001). In addition, mucus is not tightly attached to mucosa. This may contribute to poor reproducibility of related experiments, due to its partial loss. It is situated between intestinal lumen and epithelial layer, where a lot of bacteria coexist.

Apart from transporters that should be taken into consideration, enzymes also play vital roles in drug absorption process. In biological environment, γglutamyltransferase (GGT) is a specific denitrosating enzyme of GSNO. It is primarily found on the cell surface in secretory or absorptive tissues such as kidney, liver, seminal vesicles/prostate, small intestine, and it specially exists in epithelial cells, according to previous immunohistochemical studies (Hanigan and Frierson, 1996).

Actually, GGT is well-known for its anti-oxidant and pro-oxidant activities, because its most abundant physiological substrates are reduced L-glutathione (GSH) and glutathione disulfide (GSSG), which can reflect cell oxidation state by their ratio (Schafer and Buettner, 2001) (Jones, 2002) (Wickham et al., 2011). Besides, GGT is also a predictor of CVDs, due to its higher serum level observed with the progression of atherosclerosis and related complications (Pompella et al., 2004). In addition, it induces GSNO breakdown with first the release of S-nitrosocysteinylglycine, then rapidly liberating NO in the presence of metal ions (Me n+ ), such as Fe 2+ and Cu 2+ (Broniowska et al., 2013) (Figure 1 A). The role of GGT in the release of NO from GSNO and in the vasodilation has already been investigated in an ex vivo model (isolated rat aorta) (Dahboul et al., 2012). Its activity can be reversibly inhibited by serine-borate complex (SBC) (Tate and Meister, 1978).

In a biological environment, GSNO undergoes transnitrosation reaction (Figure 1 B) and decomposes as a consequence of many impacting factors such as thiols (Figure 1 C), reductants and enzymes, resulting in formation of NO and corresponding by-products, e.g. GSSG (Al-Sa'doni et al., 2000). Consequently, SBC to reversibly inhibit GGT and N-ethylmaleimide (NEM) for thiol blockage (Bryan and Grisham, 2007) are usually used to stabilize GSNO during sample pre-treatment steps before analytical measurement. (C) some thiols (e.g. PDI) (glygly:glycylglycine; GSSG: glutathione disulfide; PDI:

protein disulfide isomerase).

To study drug intestinal permeability, numerous models exist, but it is difficult to achieve a simple and relevant model (Bilat et al., 2017). Three kinds of models are commonly used: (i) an in vitro model consisting of two compartments separated by intestinal cells, such as Caco-2 cell monolayer; it is usually for preliminary study; (ii)

an ex vivo model such as isolated animal intestine in Ussing chamber: this is often applied for complementary information, due to a better correlation with in vivo studies than in vitro experiments; and (iii) an in vivo model using rodents for pre-clinical evaluation of drugs, such as pharmacokinetic studies (PK), which is one of the indispensable parts of drug discovery, to explore actively transported drug.

According to the literature, the conversion of nitrate (NO 3 -) to nitrite (NO 2 -) in the GIT through microorganism action is well known (Benjamin et al., 1994); the formation of RSNOs in stomach and their chemical decomposition have also already been reported (Pinheiro et al., 2015). Several reports conclude on the efficiency of GSNO after oral administration in rats: (i) GSNO either as a free molecule (Khan et al., 2011) or formulated [START_REF] Parent | In Situ Microparticles Loaded with S-Nitrosoglutathione Protect from Stroke[END_REF] improves the therapeutic score in a stroke rat model; (ii) Wu et al. used GSNO encapsulated nano-composite particles and showed that the NO storage increased in vessels [START_REF] Wu | Polymer nanocomposites enhance S-nitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta[END_REF]. In addition, GSNO is a highly soluble molecule with medium permeability through both intestinal cell (Caco-2)

monolayer (Bonetti et al., 2018) and isolated rat intestine [START_REF] Yu | Comparison between two derivatization methods of nitrite ion labeled with 15 N applied to liquid chromatography-tandem mass spectrometry[END_REF]. It has passive transportation mechanism in Caco-2 cell monolayer model, based on our previous study (Bonetti et al., 2018). However, little is known about its intestinal absorption, not to mention the involved factors of transferring GSNO from GIT to blood vessel. The present work aims at studying GSNO degradation and absorption in isolated rat intestine.

Methods

Material and Reagents

All reagents were of analytical or chromatographic grade and used without further S-nitrosoglutathione (GSNO) was synthesized according to a method previously described (Parent et al., 2013). Briefly, 20 mM GSH was incubated with 20 mM NaNO 2 in 500 mM HCl at 4°C in the dark for 60 min. The reaction was stopped by neutralization with NaOH (40%) and was further 2-fold diluted with 500 mM phosphate buffer, pH 7.4. The final GSNO concentration was determined by UV spectrophotometry (UV-1800, Shimadzu, Lognes, France) at a wavelength of 334 nm (ε = 922 M -1 ×cm -1 ). GS 15 NO was synthesized as above but using Na 15 NO 2 .

All experiments and assays involving GSNO were conducted under conditions of subdued light.

Isolation of rat intestine

All experiments were performed in accordance with the European Community guidelines (2010/63/EU) for the use of experimental animals in the respect of the 3 Rs'

requirements for Animal Welfare. The protocols and procedures were approved by the advisory regional ethical committee on animal experiments: Comité d'Ethique Lorrain en Matière d'Expérimentation Animale, CELMEA protocol agreement N• 02420.03.

Animals were kept under standard conditions (temperature: 21 ± 1 °C, hygrometry 60 ± 10%, light on from 6 am to 6 pm) and had free access to standard diet (A04, Safe, Villemoisson-sur-Orge, France) and water (reverse osmosis system, Culligan, Brussells, Belgium). Wistar rats (350 g, 8 weeks old, males, Janvier Laboratories, Le Genest Saint Isle, France) were used for the present experiments. Isolated rat intestine (ileum, weight: 100 ± 5 mg) was obtained after sacrifice (anesthetized with sodium pentobarbitone (60 mg.kg -1 , intraperitoneal injection, Sanofi Santé Nutrition Animale, Libourne, France), administrated with heparin (1000 IU.kg -1 heparin Choay, penis vein) and sacrificed by exsanguination). The fat tissue along the mesenteric attachment was removed and the intestine was longitudinally opened, followed by quick rinsing with 1 mL of PBS for 2 times.

Evaluation of GGT activity

γ-Glutamyltransferase (GGT) activity was determined using a specific chromogenic substrate, i.e. GCNA (Schiele et al., 1981). Briefly, isolated rat intestine prepared in section 2.2 was incubated with 1 mM GCNA, 20 mM glygly and 10 mM Protein concentrations were quantified using the bicinchoninic acid (BCA) method (Pierce Protein Assay Kit), with Bovine Serum Albumin as the standard.

Proteins were extracted from isolated rat intestine samples as follows: the rat intestine sample was frozen with liquid nitrogen (N 2 ), next smashed into powder in liquid N 2 with a pestle in a mortar; then, 100 mg of intestine powder was mixed with 400 µL of lysis buffer (0.05 M Tris-HCl, 0.15 M NaCl, 0.1 % sodium dodecyl sulfate (SDS), and 1 % pure Triton X-100), and incubated at 4°C in the dark for 10 min; next, the supernatants were collected after centrifugation at 15,000 x g at 4°C for 15 min, and proteins were precipitated by adding 3.3 mL of acetone and centrifugated at 3000  g at 4°C for 10 min; at last, 200 μL of lysis buffer were used to resuspend the proteins in the pellet before storage at -80 °C and quantification. GGT specific activity was expressed in nmol of obtained product per min per mg of proteins.

Quantification of thiols

The thiol content was measured using the DTNB (Ellman's reagent) method.

Briefly, 750 µL of 1 mM DTNB in Tris buffer (0.1 M, pH 7.4) were incubated with intestine at room temperature (r.t.) in the dark under rotation (35 rpm, CAT Roller RM5-40) for 10 min. Then, 200 µL of medium were transferred to a 96-well microplate and the absorbance was read at 405 nm (EL800 Microplate Reader, BioTek Instruments). The efficiency of NEM to block all thiols including those of cysteine residues in active sites of redoxins like protein disulfide isomerase (PDI) was also assessed. First, intestine was pre-incubated with 5 mL of 10 mM NEM in Tris buffer (0.1 M, pH = 7.4) at 37 °C under rotation for 10 min. Then, intestine was rinsed with 5 mL of PBS for 3 times, Next, DTNB was added to quantify remaining active thiols with procedures described above.

Degradation of GSNO in isolated rat intestine

50 µM GSNO in 5 mL of Tris buffer (0.1 M, pH 7.4) was incubated with isolated rat intestine placed in a 15-mL polypropylene tube at 37°C under rotation (35 rpm, CAT Roller RM5-40), in the absence and in the presence of i) glygly-Mg 2+ , or ii) 20 mM glygly,10 mM MgCl 2 and 20 mM SBC (glygly-Mg 2+ -SBC), or iii) 20 mM glygly, 10 mM MgCl 2 , 20 mM SBC and 10 mM NEM (glygly-Mg 2+ -SBC-NEM). Every 10 min during 60 min, 25 µL of reaction medium were collected and diluted 25 times in PBS for GSNO quantification using the DAN-Hg 2+ assay (Wu et al., 2015) (measurements in triplicate). In parallel, the stability of GSNO in oxygenated Krebs solution in the absence of intestine was evaluated.

Briefly, 100 µL of each standard (concentration range: 0.1 -2 µM) or sample (in triplicate) were mixed with 20 µL of 0.105 mM DAN solution in 0.6 M HCl. The mixture was shaken at 500 rpm (Heidolph, VIBRAMAX 110) at r.t. for 1 min, followed by incubation at 37 °C in the dark for 10 min. Then, 20 µL of 2.1 M NaOH were added.

The mixture was then shaken for another 5-min period, before measuring fluorescence intensity (spectrofluorometer model: FP-8300iRM, JASCO, Tokyo, Japan) coupled with FMP micro-well plate reader. The excitation and emission wavelengths were 375 and 415 nm, respectively. For RSNOs and (RSNOs + NO

Absorption of GSNO in isolated rat intestine

Method for quantifying (NO 2 -) and RSNOs in rat intestine samples was first validated, and applied to study GSNO absorption inside intestine.

Method validation for quantification of NO species in intestine

Three five-point concentration curves of NaNO

Quantification of nitrite ions and RSNOs in intestine

The nitrite ions (NO 2 -

) and RSNOs concentrations were measured after incubating intestine samples with 5 mL of i) 25 or ii) 50 or iii) 100 μM GSNO in a 15-mL tube at 37°C for 60 min under rotation (35 rpm, CAT Roller RM5-40). Intestine was rinsed with 5 mL of cold PBS for 3 times, and frozen in liquid N 2 . Then, RSNOs in intestine tissue were quantified with the LC-FL method described above.

In parallel, NO 2 -and RSNOs concentrations in isolated rat intestine were also measured after incubating intestine with 5 mL of i) 100 μM GSNO alone or ii) 100 μM GSNO in presence of glygly-Mg 2+ or iii) 100 μM GSNO with glygly-Mg 2+ -SBC-NEM, in a 15-mL tube at 37°C for 60 min under rotation; further steps for quantification were similar to operating conditions described above.

Metabolism of GS15NO in isolated rat intestine in Ussing chamber

Before studying the role of intestinal GGT and thiols in GSNO degradation and permeation through isolated rat intestine in Using chamber, the potential influence of the different additives tested (glygly-Mg 2+ , SBC and NEM) was checked on intestine integrity by measuring transepithelial electrical resistance (TEER) value over 2-h, using an adapted Millicell® ERS ohm-meter (Millipore).

The intestinal permeability of 100 μM GS 15 NO with and without glygly-Mg 2+ , and glygly-Mg 2+ -SBC-NEM in Ussing chamber was carried out with the same procedure described before [START_REF] Yu | Comparison between two derivatization methods of nitrite ion labeled with 15 N applied to liquid chromatography-tandem mass spectrometry[END_REF]. In parallel, the same experiments using Ussing chamber but without isolated rat intestine were carried out to evaluate GSNO stability at 37°C with O 2 -CO 2 (95:5, V/V) mixture bubbling.

Quantification of NO species in donor, intestine and acceptor compartments

After a 2-h incubation, NO species in the donor compartment were quantified as described in 2.5, NO 2 -/RSNOs in isolated rat intestine were quantified as in 2.6.2, and 15 N-NO species in the acceptor compartment were quantified using LC coupled to Electrospray-Ion Trap tandem Mass Spectrometry (LC-ESI-ITMS/MS) as described before [START_REF] Yu | Comparison between two derivatization methods of nitrite ion labeled with 15 N applied to liquid chromatography-tandem mass spectrometry[END_REF]. Briefly, permeated 15 N-NO species were derivatized and injected (20-µL loop) into a LC-ESI-ITMS/MS system (Dionex, Ultimate 3000, coupled with LTQ Velos Pro, Thermo Scientific, San José, CA, USA). An Acclaim 120 C-18 column (2.1 × 150 mm ID, 3 μm, Thermo) was eluted with a gradient mobile phase consisting of acetonitrile (from 40 to 80 %) and 0.01 M acetic acid solution. An ion trap mass spectrometer (LTQ Velos Pro, Thermo Scientific, San Jose, CA, USA) fitted with the HESI-II probe (electrospray) was coupled to monitor transitions of m/z 171 to 156 for 15 N-NAT quantification (HCD fragmentation mode). The apparent permeability coefficient (P app ) values were calculated using the following equation:

Papp = dQ dt × 1 A × C0
dQ/dt (mol.s -1 ) refers to the quantity of permeated NO related species (dQ) in the acceptor compartment at the time of quantification (dt), "A" refers to the membrane diffusion area (0.25 cm 2 ), and C 0 refers to the initial concentration of GS 15 NO in the donor compartment.

Histological studies [Operating conditions to be inserted when developed]

Statistical tests

All results were expressed as mean ± standard deviation (SD) or mean ± standard error of the mean (sem). Values were analyzed with one-way ANOVA, followed by a Bonferroni's post-test using the Graphpad Prism 5 software; p < 0.05 was considered as statistically significant.

Results and discussion

Measurement of GGT activity and thiol content in isolated rat intestine

The specific GGT activity in the presence of glygly-Mg 2+ was 6.8 ± 0.8 nmol of 3-carboxy-4-nitroanilide.min -1 .mg -1 of proteins, which was abolished by adding SBC (values were under the limit of detection) (Table 1). In the literature (Carter et al., 1997), the specific GGT activity was 24 700 nmol of 4-nitroanilide.min -1 .mg -1 of proteins in small intestine of female mice; thus, it was around 3600-times higher than the one observed in our experiment using isolated ileum. This huge difference has two possible explanations: (i) a lot of GGT comes from bacteria in GIT, but most of these bacteria were lost during our washing steps realized for intestine treatment; (ii) there is an inter-species or gender variation of GGT activity between female mice and male (Lash et al., 1998). In addition, the specific GGT activity presently measured in rat ileum is higher than the one in rat aorta (0.3 ± 0.01 nmol of 3-carboxy-4-nitroanilide.min -1 .mg -1 of proteins) (Dahboul et al., 2012) and differentiated Caco-2 cells (1.9 ± 0.3 nmol of 3-carboxy-4-nitroanilide.min -1 .mg -1 of proteins) (data not shown). This may result from the different structures between ileum, aorta and Caco-2 cells, and only ileum contains a lot of bacteria with high GGT expression.

[Histology results to be inserted when related experiments be done]

The intact isolated rat intestine showed around 26.5 ± 1.2 nmol of thiols.mg -1 of proteins, and 93.0 ± 0.7 % of these thiols were blocked by NEM (Table 1).

Table 1. Specific activity of γ -glutamyltransferase (GGT) and thiol content in isolated rat intestine. (ND # : not detected, under limit of detection; -: undone; GCNA:

L-γ-glutamyl-3-carboxy-4-nitroanilide;

CNA: 3-carboxy-4-nitroanilide; SBC: serine-borate complex; DTNB: 5,5′-dithiobis(2-nitrobenzoic acid); NEM:

N-ethylmaleimide))
Incubating with isolated rat intestine (n = 3)

1.9 ± 0.1

(n = 3)

Degradation of GSNO in isolated rat intestine

GSNO in Tris buffer (50 µM) under conditions (fast rotation) providing high oxygenation rate at 37°C was stable in the absence of intestine for at least 60 min. In the presence of intestine, around 40 % of GSNO (250 nmol as starting amount) were degraded within 60 min corresponding to a degradation rate of 0.5 ± 0.1 nmol of GSNO min -1 .mg -1 of proteins. This degradation was stimulated by adding glygly-Mg 2+

(around 80 % of GSNO loss), rising the degradation rate to 0.9 ± 0.1 nmol of GSNO min -1 .mg -1 of proteins. 100 % of GSNO catabolism was inhibited by SBC-NEM (GGT inhibitor and thiol blocker) (Figure 2 A). Most of the initial amount of GSNO was converted to NO 3 -ions (Figure 2 B). Perhaps, GGT does not catalyze GSNO degradation without adding co-substrates (glygly-Mg 2+ ). Or there is other factors protecting GSNO from degradation, and SBC could block this process. In summary, intestine GGT and thiols play a significant role in GSNO metabolism.

Method validation with LC-FL for quantification of NO species in intestine

To quantify NO species in the intestine tissue, the conventional fluorometric assay relying upon DAN derivatization (Wu et al., 2015) did not exhibit enough selectivity. Thus a LC-FL method was presently developed for NO 2 -and RSNOs quantification in the rat intestine tissue has a linear range of 0.3 -2.2 nmol.mg -1 of proteins, with a determination coefficient (r²) higher than 0.995. The accuracy as the deviation (%) of calculated concentrations from the nominal concentrations was in the range of 97.0 -113.3 %. Satisfactory precisions were obtained with intra-and inter-day bias less than 8.2 % and 11.9 %, respectively, over the concentration ranges studied.

These ranges were within the criteria stated in the FDA guidance on bioanalytical method validation (Shah et al., 2000) (Table 2). NO 3 -concentrations were not measured as it was already shown that the intracellular reducing power of cells enables their existence (Parodi et al., 2007).

Absorption of GSNO in isolated rat intestine

A concentration-dependent absorption of RSNOs in intestine was observed, but no significant difference for NO 2 -and RSNOs absorption in intestine between 25 and 100 µM GSNO (initial concentrations used for incubation) was observed (Figure 3 A).

Finally, 100 µM GSNO was chosen as working concentration for further experiment.

Method with a lower limit of quantification (LOQ) should be applied to study GSNO with lower concentrations (i.e. less than 25 µM).

The presence of the different additives (glygly-Mg 2+ , SBC and NEM) showed no significant differences for NO 2 -and RSNOs absorption in intestine between each group (Figure 3

Intestinal permeability of GSNO and implication of denitrosating enzymes

Transepithelial electrical resistance (TEER) values were respectively of 24.3 ± 3.4, 21.5 ± 2.4 and 47.1 ± 5.0 ohm×cm². There is not impact of glygly-Mg 2+ or SBC on intestine integrity, but an increase of TEER due to the presence of NEM.

In absence of intestine, 100 µM GSNO with extra addition (or not) of glygly-Mg 2+

or SBC-NEM was stable in Ussing chamber's operating conditions. However, only 29.4 % of initial GSNO amount were remaining in the donor compartment as the intact form, 12.7 % were converted to NO No matter adding extra GGT activator (glygly-Mg 2+ ) or GGT inhibitor-thiol blocker (SBC-NEM) or not, both the absorbed NO species amount inside intestine and the permeated ones through intestine were less than 1%. Also, the final recovery of NO species was calculated by the sum of amounts in donor, acceptor compartments, and rinsing solutions over the initial GSNO amount; the ratio found is 97 ± 12 %, which validates both model and methods (Table 3). Moreover, adding the activator did not change the permeation rate of NO species from GSNO, but has increased NO 2 permeation rate. Meanwhile, adding the inhibitor has increased RSNOs permeation rate, but did not change the permeation rate of NO species. Consequently, the drug classification of GSNO remains the same (medium permeability class), whatever adding GSNO activator or inhibitor (Table 3).

Conclusions and perspectives

GGT and thiol groups in rat intestine are the main factors involved in GSNO degradation. The GGT activator (glygly-Mg 2+ ) contributes to GSNO degradation and increases NO 2 -permeation through intestine, while GGT inhibitor and thiol blocker (SBC-NEM) protect GSNO from degradation, and leads to a higher RSNOs permeation. Other enzymes are maybe involved in GSNO metabolism, such as redoxin (i.e. Protein Disulfide Isomerase, PDI). Actually, PDI in the intestine was identified by western blot (data not shown). In addition, method using LC-ESI-ITMS/MS is needed to study GSNO with low concentration (e.g. 100 µM).

As NO plays important roles in the intestinal homeostatis, it could be interesting (i)

to investigate GSNO impact on a pathological model, and to explore a possible modification of the drug metabolism; (ii) to adapt the analytical method using liquid chromatography/tandem ion trap mass spectrometry to measure GSNO labeled with

Supplementary study: Comparison between the two intestinal barrier models,

Caco-2 cell monolayer and isolated rat intestine in Ussing chamber

The two intestinal barrier models (Caco-2 cell monolayer and isolated rat intestine in Ussing chamber) appear to give complementary data for RSNOs bioavailability studies. Combining data both from above experimental papers and PhD students in EA 3452 (Schmitt R. and Bonetti J.), a comparison between the two models concerning their validation, RSNOs permeability, and influence of GSNO on integrity of intestinal cells or tissue is presently reported.

For Caco-2 cell monolayer model validation, 50 nmol furosemide or 25 nmol propranolol were deposited in the apical compartment, and after a 1-h incubation, permeated furosemide and propranolol were quantified by LC-FL. For isolated intestine in Ussing chamber, 1000 µM sodium fluorescein or 1000 µM propranolol were deposited in the donor compartment, and after a 2-h incubation, permeated fluorescein and propranolol were quantified by FL and LC-FL, respectively. Related P app values were calculated [START_REF] Peng | Applications of a 7-day Caco-2 cell model in drug discovery and development[END_REF]. Parameters of all related instruments, detailed information about measuring intestinal markers and P app values are available in the article (Bonetti et al. 2018).

The procedure of RSNOs permeability studies is already described for Caco- furosemide. The permeated propranolol concentration is not detectable in the ex vivo model using isolated rat intestine in Ussing chamber (LOQ = 0.5 µM). Perhaps, it is trapped in intestine. In addition, sodium fluorescein belongs to high permeability class (P app = (17.27 ± 1.33) x 10 -6 cm.s -1 ) in the isolated rat intestine model. As a result, permeability values of RSNOs obtanined in both models are consistent.

After GSNO incubation, the TEER values for both models were still above the critical levels, which proved that GSNO showed no influence on the integrity of intestinal cells and tissue. This fact has also been confirmed by permeability of sodium fluorescein (less than 20 % of initial amount). Noteworthy, a much smaller amount of sodium fluorescein was used in the cell model, compared to the intestinal tissue (2.5 vs 25,000 nmol). In conclusion, RSNOs permeability, related transportation mechanism (passive)

and preferred absorption site in GIT (jejunum) were studied in an intestinal cell (Caco-2) monolayer model. Meanwhile, GSNO degradation and absorption in intestine, and potential roles of intestinal denitrosationg enzymes (GGT and PDI), thiols and Me n+ were explored using isolated rat intestine. All these work give the consistent conclusion that GSNO permeability belongs to medium class according to FDA rules. In addition, GGT activity and PDI identification in rat intestine were measured and performed. Apart from GGT, thiols in intestine also contributed to GSNO degradation, but Me n+ showed no significant influence. Less than 1 % GSNO was absorbed and permeated in intestine after a 2-h incubation. Further work focused on protecting GSNO from degradation and improving its absorption after oral administration is of great importance.

General conclusions and perspectives

As mentioned in the general introduction part, the analytical methodologies commonly used in the EA 3452 Cithefor are based on conventional spectroscopic techniques described in the literature. However, their LOQ (defined as the lowest concentration of calibration curve with acceptable RSD, less than 15 %) range between 1 and 0.1 μM; it is is not sufficient for bioavailability study of RSNOs after oral administration. Moreover, in an in vivo PK study, it is necessary to distinguish between the different sources of NO: endogenous production, diet and medicine. (Wang et al. 2018) or solid phase extraction [START_REF] Chao | Urinary nitrite/nitrate ratio measured by isotope-dilution LC-MS/MS as a tool to screen for urinary tract infections[END_REF]) is performed to purify analytes. In addition, our work has opened the perspective of more fundamental works such as the study of the reactivity of the ion [NAT+H] + in HCD tandem mass spectrometry in presence of acetonitrile and the mechanism of formation of the fragment ion m/z 156. In the future, Griess assay under HCD mode is promising to be tested to find out the most sensitive novel transition with low interference due to 13 C (e.g. less than 20 %).

Recently, a paper has described an automated online solid-phase derivatization for quantification of low-molecular-mass RSNOs by LC-MS (Wang et al. 2018).

Noteworthy, labeling RSNOs by both 15 N and 13 C contributes to PK studies of both nitroso group carriers and NO species released from RSNOs. A recent review has explained that RSNOs such as SNACET has unique pharmacological profiles for oral use as NO, H 2 S and GSH suppliers [START_REF] Tsikas | S-Nitroso-N-acetyl-L-cysteine ethyl ester (SNACET) and N-acetyl-L-cysteine ethyl ester (NACET)-Cysteine-based drug candidates with unique pharmacological profiles for oral use as NO, H2S and GSH suppliers and as antioxidants: Results and overview[END_REF]. Compared to nitrite ions, the permeation of the RSNOs species will improve the interest for RSNOs oral administration to provide NO supplementation. In addition, RSNOs have also shown other therapeutic effects, such as anti-cancer [START_REF] Foster | Protein S-nitrosylation in health and disease: a current perspective[END_REF] and anti-inflammatory [START_REF] Khan | S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke[END_REF]. For these reasons, exploring NO donors with higher lipophilicity such as S-nitrosoglutathione monoethyl ester [START_REF] Konorev | The mechanism of cardioprotection by S-nitrosoglutathione monoethyl ester in rat isolated heart during cardioplegic ischaemic arrest[END_REF] or formulations [START_REF] Wu | Polymer nanocomposites enhance S-nitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta[END_REF] to increase RSNOs permeability is in need. In summary, it is vital and eventful to evaluate RSNOs bioavailability more deeply, and the developed and validated analytical method using 15 N labeling and LC-ESI-ITMS/MS in a more complex matrix (such as plasma) should be performed to conduct further PK study.

Measurement of nitrite, nitrate ions and S-nitrosothiols

Reference:

Jobgen WS, Jobgen SC, Li H et al. Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography. J Chromatogr B 2007;851:71-82.

Reagents

All solutions are prepared with fresh cold ultrapure water (> 18.2 MΩ.cm) and

2.1 M NaOH

Dilute 6-fold 50% NaOH solution with ultrapure water by mixing 5 mL of 50%

NaOH solution (Mw = 40 g.mol -1 ; ref Fluka 72064) and 25 mL ultrapure water. in a 50 mL volumetric flask, dissolve and adjust the volume with PBS. Prepare daily.

S-nitrosoglutathione (GSNO)

Solutions are prepared, titrated and stored according to protocol entitled "Batch synthesis of S-nitrosoglutathione".

Lysis buffer

Transfer 2 g Triton X-100 (ref Sigma 9002-93-1) in a 500 mL volumetric flask and adjust the volume with 0.1 M HCl.

Methods

Calibration curves for NO 2 -and RSNOs determination

NaNO 2 /GSNO daughter solution n°1 (10 -4 M): transfer 100 µL of 10 -2 M NaNO 2 /GSNO solution in a 10 mL volumetric flask, adjust the volume with PBS. Mix gently for one time. Fluorescence intensity is measured within 10 min.

Operating conditions for direct spectrofluorometric reader

FP-8300iRM spectrofluorimeter (JASCO, Tokyo, Japan) parameters are fixed as follows:

- -(RSNOs + nitrite) concentration is calculated with RSNOs standard curve equation.

-(Nitrate + nitrite) concentration is calculated with nitrate standard curve equation.

-RSNOs concentration is calculated by subtracting nitrite concentration to (RSNOs + nitrite) concentration.

-Nitrate concentration is calculated by subtracting nitrite concentration to (nitrate + nitrite) concentration.

Typical calibration curves obtained in a validation process are indicated in Table 1. Prepare daily.

S-nitrosoglutathione (GS 15 NO)

Solutions are prepared, titrated and stored according to protocol entitled "Batch synthesis of S-nitrosoglutathione", except that Na 15 NO 2 instead of Na 14 NO 2 is used. Mw = 60.05 g.mol -1 ) with 500 mL with ultrapure water, and filter with 0.45-µm HV membrane (Durapore®).

Rinsing mobile phase

Mix 500 mL acetonitrile and 500 mL of ultrapure water, filter with 0.45-µm HV membrane (Durapore®).

Methods

Calibration curve for pure NAT determination

The pure NAT daughter solution ( The mixture is shaken for 0.1 min by vortex at 20-25 °C, followed by freezing at -80 °C.

7 µL of 0.02 mM IS are added before analysis with LC-MS/MS.

Operating conditions for LC-MS/MS

All reversed phase columns used for the present method have to exhibit a number of theoretical plates per meter equal to at least 15,000 for the anthracene peak.

The operating conditions for LC (DIONEX, Ultimate 3000) are as follows:

-Column: Acclaim 120 C18 3 μm, 2.1 X 150 mm (Thermo Scientific).

-

Calculations

-Nitrite concentration is calculated with nitrite standard calibration curve equation.

-(RSNOs + nitrite) concentration is calculated with RSNOs standard calibration curve equation.

-(Nitrate + nitrite) concentration is calculated with nitrate standard calibration curve equation.

-RSNOs concentration is calculated by subtracting nitrite concentration to (RSNOs + nitrite) concentration.

-Nitrate concentration is calculated by subtracting nitrite concentration to (nitrate + nitrite) concentration.

The signal value obtained in the blank is subtracted to all signal values obtained for standards.

Typical validation data are shown in Table 2. They are checked daily by comparing with quality control.

Quantification of RSNOs after incubation of isolated rat intestine with

GSNO

Rinse the isolated rat intestine (100 ± 5 mg) quickly with 1 mL of cold PBS for 2 times, use tissue paper to dry the intestine each time; incubate intestine with 5 mL of 100 μM GSNO in a 15-mL tube at 37°C for 60 min under rotation (35 rpm, CAT Roller RM5-40, Dordrecht, Netherlands), rinse intestine quickly with 5 mL of cold PBS for 3 times. Then freeze intestine and quantify NaNO2/RSNOs as described above.

For RSNOs or (RSNOs + nitrite) measurement, DAN reagent has to be replaced by DAN-HgCl 2 reagent.

Operating conditions for LC-FL

All reversed phase columns used for the present method have to exhibit a number of theoretical plates per meter equal to at least 15,000 for the anthracene peak.

The operating conditions are as follows:

- and at 40 °C for 10 min.

Calculations

-Nitrite concentration is calculated with nitrite standard calibration curve equation.

-(RSNOs + nitrite) concentration is calculated with RSNOs standard curve equation.

- Typical validation data are shown in Table 1. A 50 μM pure NAT solution has been used to check extraction efficiency of the protocol described above, the recovery is 88 % according to peak area ratio of pure NAT with and without intestine (n = 1).

Recoveries when compared to GSNO standard solutions derivatized in the absence of intestine (injected concentration range: 6.25-50 µM): ca. 85%

Intestinal permeability studies of S-nitrosothiols using a Caco-2 cell monolayer model

Principle

The Caco-2 cell line is a continuous cell of heterogeneous human epithelial colorectal adenocarcinoma. Although derived from a colon (large intestine) carcinoma, when cultured under specific conditions, the cells become differentiated and polarized so that their properties (phenotype, morphology and functions) are similar to those of the enterocytes lining the small intestine. When cultured on a cell culture insert filter (e.g. Transwell®; Figure 1), the cells differentiate to form a polarized epithelial cell monolayer that provides a physical and biochemical barrier to the passage of ions and small molecules. The Caco-2 monolayer is widely used in the pharmaceutical industry as an in vitro model to predict the absorption of orally administered drugs [START_REF] Wu | Polymer nanocomposites enhance S-nitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta[END_REF]) (Bilat et al. 2017).

Figure 1. Representation of Caco-2 cells in a Transwell® plate and its application for permeability evaluation of S-nitrosothiols (RSNOs).

References:

Wu

Treatment of cells

Replace the culture medium in basolateral compartment with 1.5 mL of HBSS, and the one in apical compartment with 0.5 mL of HBSS. Then incubate at 37 °C for 15 to 20 min on an orbital shaker at 500 rpm, followed by TEER measurement.

Next, replace the HBSS in basolateral compartment with 1.5 mL of fresh one and the solution in apical compartment with 0.5 mL of RSNOs solution prepared in HBSS (e.g. 50 µM) or HBSS or HBSS without Ca 2+ /Mg 2+ (tight junctions opening, as a positive control of barrier integrity). Incubate cells at 37 °C for 1 or 4 h on a orbital shaker at 500 rpm, before harvesting the full volume of HBSS in both compartments.

Finally, rinse the cells by the addition of 0.5 mL of fresh HBSS in apical compartment and 1.5 mL of fresh one in basolateral compartment, followed by TEER measurement and sodium fluorescein (5 μM) permeability assessment to evaluate monolayer integrity. Briefly, replace the medium in basolateral compartment with 1.5 mL of HBSS and the one in apical compartment with 0.5 mL of sodium fluorescein or HBSS or HBSS without Ca 2+ /Mg 2+ after TEER measurement; incubate at 37 °C for 2 h, measure the fluorescence intensity of both compartments at λ exc = 460 nm, λ em = 515 nm (Fluorimetric Detector, Biotek) (a permeability value of sodium fluorescein ≤ 20 % indicates a confluent monolayer [START_REF] Wu | Polymer nanocomposites enhance S-nitrosoglutathione intestinal absorption and promote the formation of releasable nitric oxide stores in rat aorta[END_REF], calculated according to a calibration curve with stadards of 0.05, 0.0625, 0.125, 0.25, 0.5, 5 µM).

Quantification of nitrite, nitrate ions and RSNOs in both compartments

See protocol entitled "Measurement of nitrite, nitrate ions and S-nitrosothiols using the 2,3-diaminonaphthalene assay by spectrofluorometry" and "Measurement of nitrite, nitrate ions and S-nitrosothiols using the 2,3-diaminonaphthalene assay by LC coupled with tandem mass spectrometry".

Calculations

Calculate the cumulative amounts of RSNOs permeated through the Caco-2 monolayer by the concentrations measured in the basolateral compartment. Calculate the P app values using the following equation:

P app = dQ dt × 1 A × C 0
dQ/dt (mol•s -1 ) refers to the quantity of permeated NO related species (dQ) in the basolateral compartment at the time of quantification (dt), A refers to membrane diffusion area (1.12 cm 2 ), and C 0 refers to the initial concentration of RSNOs in the apical compartment (from 10 -5 to 10 -4 M).

Permeability studies of S-nitrosothiols in isolated rat intestine using Ussing chamber

Principle

The Ussing chamber was invented by the Danish zoologist and physiologist Hans Henriksen Ussing in 1946. The basic design of a classical Ussing chamber is illustrated in Figure 1. The membrane (cells, tissues or synthetic one) is situated vertically in the middle of the model, and separates it into two compartments (equal volume): a donor compartment (also referred to the apical, mucosal or luminal-side membrane) and an acceptor compartment (also referred to the basolateral, serosal, nutrient, or blood-side membrane). The Ussing chamber is an apparatus for measuring the transport of ions, nutrients or drugs across various epithelial cells, tissues or synthetic membrane. Thus, it is useful to explore drug absorption process and barrier functions of living tissues. It is widely used in the pharmaceutical industry as an ex vivo model to predict the absorption of orally administered drugs (Bilat et al. 2017) ( Neirinckx et al. 2011). We presently use it for studying the permeability of S-nitrosothiols (RSNOs) by quantifying RSNOs and their metabolites (nitrite and nitrate ions).

Reagents

All solutions are prepared with fresh cold ultrapure water (> 18.2 MΩ.cm) and stored at 4°C in the dark for maximum one month unless otherwise specified.

Physiological media

Conservation time: one week at 4°C after preparation.

Solution with substrate and enzyme inhibitor

Prepare 20 mM SBC by mixing 5 mL of 100 mM SBC and 20 mL of 100 mM Tris buffer. Weigh and dissolve 6.6 mg of L-γ-glutamyl-3-carboxy-4-nitroanilide (Mw = 328.3 g.mol -1 , ref Sigma AC98) with 20 mL of 20 mM SBC solution. Protect solution from light. Prepare daily.

Methods

Measurement of purified GGT activity

Equilibrate the temperature of the incubator at 37°C, transfer 5 mL of solution with substrate to a 1.5-mL Eppendorf tube, and incubate at 37°C in the dark for 5 min. Next, dilute GGT (1.1 UI/mL) 1:10 in Tris buffer, and add 25 µL of GGT diluted solution to the EP tube above. The blank with substrate is prepared in the same way but without adding GGT. Read absorbance at 405 nm at different time shown in Table 1.

Measurement of GGT activity in rat intestine

Remove Wistar rat intestine (length around 2 cm, weight around 100 mg) after sacrifice (anesthetized with sodium pentobarbitone (60 mg.kg -1 , intraperitoneal injection, Sanofi Santé Nutrition Animale, Libourne, France); administrated with heparin (1000 IU.kg -1 heparin Choay, penis vein and sacrificed by exsanguination)).

Rinse quickly with 1 mL cold PBS for 2 times, use tissue paper to dry the intestine each time. Next, place the intestine tissue in a 5-mL tube and add 5 mL of solution with/without substrate in the dark. The blank with substrate is prepared in the same manner but without intestine. Incubate all the tubes in the dark at 37°C for 10/20/30 min, transfer 1 mL of the solution at different time shown in the above table to Eppendorf tube, centrifuge at 14,000 rpm for 30 s, collect the supernatant to the plastic cuvette, and read absorbance at 405 nm shown in Table 2. For manipulations with SBC (GGT inhibitor): the procedure is the same, except solution with substrate is replaced with solution with substrate and enzyme inhibitor.

Calculation

Calculate the concentration of produced 3-carboxy-4-nitroanilide by using the Beer-Lambert law (A = ԑ x l x c, ԑ = 9,500 L.mol -1 .cm -1 ). Take into consideration of the optical length (l = 1 cm in plastic cuvette and 0.15 cm on microplate) and the possible dilution factor. Next, define the enzymatic activity = Amonut of 3-carboxy-4-nitroanilide/incubation time (nmol.min -1 ).

Specific enzyme activity (nmol.min -1 .mg -1 of proteins) is calculated by dividing enzyme activity by the quantity of proteins (proteins are measured by the BCA Protein Assay kit).

-Specific purified GGT activity: 3200 nmol.min -1 .mg -1 of proteins -Specific rat intestine GGT activity: 6.83 nmol.min -1 .mg -1 of proteins

Western blot for protein disulfide isomerase identification in tissues

Principle

Protein disulfide isomerase (PDI) is an enzyme located in the endoplasmic reticulum or on the plasmic membrane surface of eukaryotes, catalyzing the formation and breakage of disulfide bonds between cysteine residues within proteins during their folding. Its denitrosating action has been identified in isolated aorta (Dahboul et al. 2014).

PDI is presently extracted from the rat intestine, separated on a denaturing gel under electric field and identified using specific anti-bodies [START_REF] Mahmood | Western blot: technique, theory, and trouble shooting[END_REF].

and proteins samples (rat intestine homogenate) (8 μL marker and 50 μL samples) on the gel in the following order (from left to right); put the gel in the tank with the electrophoresis buffer, perform migration with following conditions:

-25 min at 50 V -90 min to 150 V -20 min to 180 V

Transfer of intestinal proteins to nitrocellulose membrane

Activate the nitrocellulose membrane in methanol for 1 min, and in transfer buffer for 5 min.

The proteins after migration, are subjected to an electro-transfer on nitrocellulose membrane (Whatman Protran) in Transfer buffer (pH = 8.3) for 1 h at constant voltage of 100 V.

After the transfer, wash the membrane in 20 mL of TBST for 5 min and blocking the membrane in blocking buffer (20 mL, TBST-milk 5 %) for 2 h at r.t. or overnight at 4 ° C.

Labeling of intestinal proteins

Dilute the primary antibody (anti-PDI, Santa cruz Biotechnology) for 1000-fold in the TBST-Milk 5 % buffer (final volume = 20 mL) and use it to cover the membrane. stirrer, Schwabach, Germany). Wash the membrane in TBST for 4 times (5 min for each wash).

Analysis of intestinal proteins

Enhanced chemiluminescence (ECL) revelation: Tap the membrane on filter paper and place it in a crystallizer. Mix 1 mL of solution 1 and 1 mL of ECL solution 2

(

  bio)chemical conversion giving rise to related species. ................................... 31 Table 2. Probes used for derivatization of NO related species labeled with 15N and corresponding chromatographic methods coupled with MS. ............................ 34 Table 3. Literature overview of 2,3-naphthotriazole adduct analysis resulting from derivatization of NO species with 2,3-diaminonaphthalene by using RPLC-MS/MS. ......................................................................................................................... 36

  ...................................................................................................... 114 1. Experimental paper in Biochemical Pharmacology (155 (2018) 21-31):

  pH 7.4. ............................................................................................................ 126

Figure 1 .Figure 2 .Figure 3 .Figure 2 .Figure 3 . 2 .Figure 1 . 2 )Figure 2 .

 123232122 Figures

Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 1 .Figure 2 .Fig. 2 .Fig. 3 .Fig. 4 .

 345612234 Figure 3. Representative LC-ESI-ITMS/MS chromatogram (conditions given in the experimental section) of the 2,3-naphthotriazole (NAT) derivative and the internal standard (IS, 2-naphthylamine (2-NA)). .......................................................... 105 Figure 4. Fragmentation of the protonated molecular ion of NAT diluted at 2 µM in the chromatographic mobile phase. A) CID at 35 eV of [ 14 N-NAT+H] + (m/z 170) using acetonitrile (ACN), B) HCD at 50 eV of [ 14 N-NAT+H] + using ACN, C) HCD at 50 eV of [ 14 N-NAT+H] + using deuterated acetonitrile (d 3 -ACN) and D) HCD at 50 eV of [ 15 N-NAT+H] + (m/z 171) using ACN. See also Table 1 for complementary information. ....................................................................................................................... 106 Figure 5. Representative ion chromatograms under multiple reaction monitoring (MRM) transition of m/z 171 → 115, corresponding to derivatization of 5 nM 15 N-nitrite in HBSS buffer, blank of the in vitro study and freshly prepared HBSS buffer.107 Figure 6. Permeation of 10, 25, 50 or 100 µM of 15 N labeled S-nitrosoglutathione (GS 15 NO) through an in vitro model of intestinal barrier (differentiated Caco-2 cell monolayer) during 1 h at 37 C. GS 15 NO metabolites (nitric oxide (NO) species concentrations) were quantified in both compartments, n = 3. Results are expressed as mean ± SD. ................................................................................................ 108 Figure S1. Simplified scheme of the LTQ Velos Pro showing where the fragmentation occurs in the 2 different fragmentation modes evaluated in this publication. In CID (Collisionally Induced Dissociation), the parent ion is isolated and fragmented in the LTQ (Linear Trap Quadrupole or Linear Ion Trap). The fragments are then analyzed by the LTQ. In HCD (Higher-energy Collision Dissociation), the parent ion is first isolated in the LTQ then ejected back to the Q 00 region where its fragmentation

Fig. 7 .Fig. 8 .Fig. 9 . 2 .Figure 1 .Figure 2 .Figure 3 .Figure

 7892123 Fig. 7. Basolateral to apical permeability -Quantification in the basolateral compartment of remaining (A) RSNO, (B) NO 2 -and (C) NO 3 after 1 h and 4 h of exposure to 150 nmol of each treatment. Results are shown as mean ± SD of four independent experiments done in duplicate and are compared using one-way ANOVA (excluding NaNO 2 ). *vs. GSNO; # vs. SNAP at the same time; p < 0.05 (Bonferroni post-test)......................................................................................................... 124 Fig. 8. Apical pH 6.4 to Basolateral permeability -Quantification in the basolateral compartment of remaining (A) RSNO, (B) NO 2 -and (C) NO 3 -after 1 h and 4 h of exposure to 50 nmol of each treatment. Results are shown as mean ± SD of three independent experiments done in duplicate and are compared using two-way ANOVA (p treatment (GSNO, NACNO, SNAP; excluding NaNO 2 ), p time (1 h, 4 h) and p interaction ). *vs. GSNO; # vs. SNAP at the same time; p < 0.05 (Bonferroni's multiple comparisons test). ............................................................................................................... 125 Fig. 9. Apical to basolateral compartment -Quantification in the apical compartment of remaining (A) RSNO, (B) NO 2 -and (C) NO 3 -after 1 h and 4 h of exposure to 50 nmol of each treatment. Results are shown as mean ± SD of three independent experiments done in duplicate and are compared using one-way ANOVA (excluding NaNO 2 ). *vs. GSNO; # vs. SNAP at the same time; p < 0.05 (Bonferroni post-test). ....................................................................................................................... 126

-

  la méthode spectrophotocolorimétrique de Griess dans laquelle les ions nitrites réagissent successivement avec la sulfanilamide et la naphtylamine pour produire un adduit azoïque coloré ; -une méthode spectrofluorimétrique dans laquelle les ions nitrites réagissent en milieu acide avec le 2,3-diaminonaphtalène (DAN) pour donner un adduit (2,3-naphtotriazole ; NAT) fluorescent en milieu alcalin. Ces deux méthodes ont en commun l'avantage de permettre en plus de la mesure des ions nitrites, celle des ions nitrates (après réduction chimique ou enzymatique en ions nitrites) et des RSNOs (après clivage de la liaison S-NO par action des ions mercuriques). Les trois espèces (ions nitrates, nitrites et RSNOs) seront donc analysées par la même méthode finale. La première partie de cette étude a concerné le choix entre ces deux méthodes de dérivation ; le principal critère de choix a été la LOQ atteinte par chacune des deux méthodes en examinant avec attention son mode de calcul. En effet, le calcul de la LOQ s'effectue classiquement par le rapport signal-sur-bruit (signal-over-noise ratio : S/N) ; mais il n'est pas approprié si on considère l'analyte « nitrite ». En effet, au cours des manipulations des échantillons et des réactifs au contact de l'air, il survient une contamination de toutes les solutions par les oxydes d'azote présents dans l'atmosphère ainsi qu'une réduction des ions nitrates en nitrites par des bactéries contaminant les solutions. C'est pourquoi il lui sera préféré le rapport signal-sur-blanc (signal-over-blank ratio : S/B) pour calculer la LOQ. Ces travaux ont fait l'objet d'une short communication intitulée « Comparison between two derivatization methods of nitrite ion labeled with 15 N applied to liquid chromatography-tandem mass spectrometry» soumise à Analytical Methods (10 (2018) 3830-3836). La seconde partie de ce chapitre a consisté en un développement plus approfondi de la méthode reposant sur la dérivation des ions nitrites par le DAN sélectionnée en raison de la LOQ la plus faible qu'elle offre. Une optimisation des conditions opératoires a été suivie d'une validation complète de la méthode et d'une application de celle-ci à l'étude de la perméabilité intestinale de GSNO sur un modèle cellulaire. Ces travaux ont fait l'objet d'un article intitulé «Higher-energy Collision Dissociation for the quantification by liquid chromatography/in an in vitro model of intestinal barrier » soumis à Rapid Communications in Mass Spectrometry et actuellement en cours de révision. Le second chapitre est consacré aux études de perméabilité intestinale menées sur le GSNO pris comme modèle de RSNOs. Un rapide rappel des nombreux modèles de barrière intestinale couramment utilisés pour évaluer la biodisponibilité orale des médicaments est réalisé en début de chapitre. En effet une revue très complète traitant ce sujet a été récemment publiée (Bilat et al. 2017). L'approche expérimentale s'est axée sur l'utilisation des deux modèles maîtrisés au niveau de l'EA Cithefor : -un modèle in vitro utilisant la lignée cellulaire Caco-2 se différentiant en formant une mono-couche de cellules épithéliales; -un modèle ex vivo consistant à isoler une partie de l'intestin de rat (ileum) dans une chambre de Ussing. Les résultats obtenus sur le modèle cellulaire en plus des résultats purement analytiques présentés dans la publication soumis à Rapid Communications in Mass Spectrometry, ont fait l'objet d'un article intitulé « Intestinal absorption of S-nitrosothiols: permeability and transport mechanisms » et soumis à Biochemical Pharmacology (155 (2018) 21-31). Les mesures de perméabilité apparente (P app ) du GSNO ainsi que d'autres RSNOs ont permis de les positionner dans la classification de la perméabilité des médicaments. Des indications sur le type de mécanisme assurant le passage des RSNOs et de ses métabolites à travers cette barrière physiologique sont également données. La place du GSNO dans la classification de la perméabilité des médicaments a été confirmée en utilisant le modèle ex vivo et la méthode de marquage à l'azote 15 associée à la technique LC-MS/MS. De plus, les principaux mécanismes, enzymatiques ou non, dénitrosant le GSNO ont été étudiés sur intestin isolé. Les résultats correspondants font l'objet d'un article intitulé «Degradation of S-nitrosoglutathione in rat intestine: roles of denitrosating enzymatic and chemical factors on absorption and permeation» en préparation pour soumission à Drug Metabolism and Disposition. La soumission de cet article ne pourra se faire qu'après des travaux complémentaires réalisés au sein de l'EA 3452 Cithefor. Les conclusions-perspectives résument les points les plus remarquables obtenus au cours de mes travaux de thèse sur le plan bioanalytique et perméabilité intestinale du GSNO ainsi que les perspectives attendues dans le cadre du projet de l'EA 3452 Cithefor et au delà.IntroductionNitric oxide (NO) is a radical molecule with a very short half-life playing important roles in physiology, pathophysiology and pharmacology. The detailed information can be easily found in numerous books and reviews such as:[START_REF] Moncada | Nitric oxide: physiology, pathophysiology, and pharmacology[END_REF]. S-nitrosothiols (RSNOs), such as S-nitrosoglutathione (GSNO), which can release NO without the drawbacks of NO donors present on the market (drug tolerance phenomenon and induction of oxidative stress), appear as promising candidate drugs. Related informations have been well detailed in the following reviews:[START_REF] Richardson | Potential therapeutic uses for S-nitrosothiols[END_REF](Gaucher et al. 2013) and PhD thesis maunscripts(Wu 2015)[START_REF] Belcastro | Inflammation et stress oxydant dans l'athérosclérose: rôle dans les réponses vasculaires des S-nitrosothiols[END_REF][START_REF] Ming | Développement de formulations polymériques de S-nitrosoglutathion comme traitement per os pour prévenir les maladies inflammatoires chroniques de l'intestin[END_REF]).

  ( 15 N) to label exogenous GSNO and taking advantage of MS to analyze 15 N labeled NO species. Related methods that have analyzed 15 N labeled NO species in the literature are summarized in the following review. et ses nombreux dérivés hydrogénés et oxygénés sont d'une importance primordiale dans notre environnement et dans les cellules vivantes tant sur le plan qualitatif que quantitatif. Leur surveillance est nécessaire pour évaluer toutes les perturbations se produisant dans le cycle de l'azote et dans les événements physiopathologiques liés aux variations de la biodisponibilité de l'oxyde nitrique (NO). De nombreuses méthodes d'analyse sont consacrées à la mesure des espèces azotées, en particulier celles liées au NO, dans les domaines environnemental, biologique et pharmacologique. Elles ont déjà été compilées et discutées dans de nombreuses revues. Le marquage des espèces azotées avec l'isotope 15 ( 15 N) stable et à faible abondance naturelle associé à la spectrométrie de masse (MS) donne lieu à plus d'informations mécanistiques et à des performances analytiques supérieures aux méthodes conventionnelles. La présente revue est consacrée au marquage 15 N des espèces azotées apparentées pour suivre leur interconversion et leur métabolisme, aux différentes sondes chimiques utilisées pour leur dérivation et aux méthodes séparatives correspondantes couplées à la MS pour l'analyse des adduits obtenus. Le mode de fragmentation des différents adduits et la sélectivité et la sensibilité qui en résultent sont discutés. Lors de la quantification de composés d'intérêt biologique dans les milieux complexes, quelle que soit leur origine, il est communément admis que le couplage de méthodes séparatives à la spectrométrie de masse en tandem (MS/MS ou MS²) est la plus performante des méthodes pour atteindre l'état de trace. En effet, outre sa haute sensibilité, cette technique de détection permet même souvent de s'affranchir d'une préparation d'échantillons compliquée grâce à sa plus grande spécificité au regard de la détection UV-Vis, voire fluorescence. Par ailleurs, la MS permet aussi de distinguer des compositions isotopiques différentes pour une même molécule, que cette composition soit naturelle (exemple 13 C naturellement présent à hauteur de 1,1% sur Terre) ou issue de synthèses par enrichissement isotopique pour des études de mécanisme de fragmentation, pour son utilisation en tant qu'étalon interne en quantification ou encore pour le suivi de flux métaboliques dans les organismes vivants (fluxomique).Dans ce travail, l'utilisation de la MS/MS (ou MS²) fait la synthèse d'un grand nombre de ces avantages puisque nous allons utiliser sa spécificité de détection pour nous affranchir au maximum de l'effet de matrice complexe, sa grande sensibilité de détection pour atteindre une détection des métabolites d'intérêt à des concentrations de l'ordre du nanomolaire, et enfin le marquage isotopique qui nous permettra de distinguer les sources naturelles de NO possédant l'isotope 14 N de celle du médicament étudié, marqué spécifiquement par synthèse avec l'isotope 15 N. La plupart du temps, les quantifications de composés d'intérêt à l'aide des techniques couplées à la MS/MS utilisent des spectromètres triple quadripôles (QqQ, Figure1A), ces derniers étant les plus performants dans la quantification à l'état de trace. Dans ce cas, l'ion moléculaire (radicalaire, protonné ou déprotonné selon le mode d'ionisation et la source utilisée) est sélectionné après filtrage par un premier quadripôle (Q 1 ) qui ne laisse passer que l'ion de rapport m/z désiré. Cet ion (dit précurseur) pénètre dans un deuxième quadripôle (Q 2 ) qui sert de cellule de collision où il percute plus ou moins violemment (selon l'énergie cinétique communiquée par l'expérimentateur) un gaz inerte (N 2 ou Ar). Il en résulte un accroissement d'énergie interne de l'ion moléculaire qui se fragmente en ions plus petits (les fragments neutres ne seront pas observables). Les ions fragments qui sortent de la cellule de collisions sont alors analysés par un troisième quadripôle (Q 3 ).

Figure 1 .

 1 Figure 1. (A) : Schéma d'un spectromètre de type triple quadripôle QqQ. Le quadripôle Q 0 et les lentilles IQ i servent de système de focalisation des ions. OR = orifice, lentille par laquelle les ions provenant de la source Electrospray (à pression atmosphérique) pénètrent dans le vide du spectromètre de masse pour y être analysés selon leur rapport masse sur charge (m/z). Le quadripôle Q 2 sert de cellule de collision (souvent notée « q ») en mode de fragmentation soit pour un suivi des ions produits, précurseurs ou des pertes de neutres en fonction de l'utilisation des quadripôles Q 1 et Q 3 . En Mode MRM (dosage), les quadripôles Q 1 et Q 3 sont réglés sur les rapports m/z de l'ion moléculaire et d'un fragment respectivement pour une plus grande spécificité et sensibilité de détection. (B) : Schéma du spectromètre LTQ Velos Pro (Thermo Scientific). Les quadripôles Q 00 , Q 0 et Q 1 servent de système de focalisation des ions provenant de la source Electrospray (à pression atmosphérique) après leur entrée dans le vide du spectromètre de masse et du système d'acheminement vers l'analyseur de type trappe linéaire (QLT pour Quadrupole Linear Trap). En mode CID, la fragmentation de l'ion moléculaire a lieu dans la trappe alors qu'en mode HCD, l'ion moléculaire sélectionné dans la trappe (étape A) est renvoyé en arrière pour être fragmenté dans la région du Q 00 (étape B). Les fragments reviennent dans la trappe (étape C) pour être analysés selon leur rapport m/z (étape D). D'après (McAlister et al. 2011).

Figure 2 .

 2 Figure 2. Scheme of (A) Caco-2 cell monolayer model, (B) intact isolated intestine model in Ussing chamber (oriented with mucosa toward donor compartment and serosa toward acceptor compartment) and (C) small intestine.

Figure 1 .

 1 Figure 1. Scheme of S-nitrosoglutathione (GSNO) metabolism by (A) γ-glutamyltransferase (GGT); (B) thiols (RSH) through transnitrosation reaction; and

  purification. Reduced L-glutathione (GSH), glutathione disulfide (GSSG), -glutamyl-3-carboxy-4-nitroanilide (GCNA), glycylglycine (glygly), L-serine, sodium borate, N-ethylmaleimide (NEM), 2,3-diaminonaphthalene (DAN), mercuric chloride (HgCl 2 ) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Sodium 15 N-nitrite (Na 15 NO 2 ) was supplied by Cambridge Isotope Laboratories (Tewksbury, MA, USA). Nitrate/Nitrite fluorometric assay kit (ref. 780051) was obtained from Cayman Chemical (Michigan, USA)

MgCl 2 (

 2 glygly-Mg 2+ ) in presence or absence of 20 mM SBC (20 mM serine-20 mM sodium borate) in 5 mL of 100 mM Tris buffer (pH 7.4) at 37°C under rotation (35 rpm, CAT Roller RM5-40, Dordrecht, Netherlands). Every 10 min during 30 min, the absorbance was read at 405 nm (ε (3-carboxy-4-nitroanilide) = 9 500 M -1 ×cm -1 ).

  Fluorometric Assay Kit(Kayman, (ref. 780051). Twenty µL of each standard (concentration range: 0.75 -25 µM) or sample (in triplicate) were derivatized according to the manufacturer protocol and fluorescence intensity was measured.

  2 and GSNO standards (blank, 6.25, 12.5, 25, 37.5, 50 nM) for three different days were built according to the literature(Damacena-Angelis et al., 2017)(Shin and Fung, 2011). Resulting 2,3-naphthotriazole (NAT) was extracted with acetonitrile and analyzed by reversed phase liquid chromatography coupled with fluorescence detection (LC-FL). Briefly, the rat intestine was frozen with liquid N 2 , next smashed into powder in liquid N 2 with a pestle in a mortar; 20 ± 2 mg intestine powder were spiked with 20 µL of NaNO 2 or GSNO standard solutions. Next, 600 μL DAN or DAN/Hg 2+ in 0.6 M HCl were added, followed by vortex for 30 s. The mixture was then incubated at 37 °C for 10 min under rotation (80 rpm, MX-RD-E Classic Rotator, Labtech, Cernusco, Italy). 40 µL of 10 M NaOH were added, and mixed by vortex for 10 s. Finally, 600 µL acetonitrile were added, followed by centrifugation at 18,000 x g at 4 °C for 15 min. The supernatant was collected and stored at 4 °C for a maximum period of 24 h before analysis by LC-FL. A ZORBAX SB-Aq C18 column (4.6 X 150 mm, 3.5 μm, Agilent) eluted with CH 3 CN-0.01 M ammonium acetate pH 7.2 (28:72, v/v), and a spectrofluorometric detector (RF-10A XL model, Shimadzu, Kyoto, Japan) with excitation and emission wavelengths set at 375 and 415 nm, respectively, were used. Background levels of NaNO 2 and RSNOs in Tris buffer and intestine without GSNO treatment corresponding to blanks were measured and subtracted from all values.

Figure 2 .

 2 Figure 2. Remaining amounts of (A) S-nitrosothiols (RSNOs) (250 nmol of S-nitrosoglutathione (GSNO) as starting amount) after 60 min of incubation with different additives in presence or absence of rat intestine; (B) NO species (RSNOs, nitrite and nitrate ions) repartition when rat intestine is incubating with GSNO in the presence of glycylglycine (Glygly) and MgCl 2 . Data are shown as mean ± sem, n = 3. P value (One-way ANOVA, Bonferroni's post-test): *p < 0.05 (additives: glygly, magnesium chloride (MgCl 2 ), serine-borate complex (SBC) and N-ethylmaleimide (NEM)).

  B). [Histology discussion] This may due to the low reproducibility of experiments (is it linked to the loss of mucus during washing steps of tissue preparation? Histological studies are needed to visualize mucus, bacteria and epithelium.

Figure 3 .

 3 Figure 3. Amounts of nitrite ions and S-nitrosothiols (RSNOs) quantified inside the isolated rat intestine, after incubation with (A) 25, 50 and 100 μM (equal to 125, 250 and 500 nmol, respectively) of S-nitrosoglutathione (GSNO) in Tris buffer (0.1 M, pH 7.4) for 60 min; (B) 100 µM (equal to 500 nmol) GSNO in the presence of different additives. Data are shown as mean ± sem, n = 3. P value (One-way ANOVA, Bonferroni's post-test): *p < 0.05 (additives: glycylglycine (glygly), magnesium chloride (MgCl 2 ), serine-borate complex (SBC), N-ethylmaleimide (NEM)).

Figure 4 . 3 -.

 43 Figure 4. Remaining amounts of (A) nitrite ions (NO 2 -), (B) S-nitrosothiols (RSNOs)

Figure 5 .

 5 Figure 5. Permeated amounts of (A) nitrite ions (NO 2 -), (B) S-nitrosothiols (RSNOs)

  diaminonaphthalene (DAN) assay relies upon the reaction between the non-fluorescent reagent DAN and nitrite ion in acidic medium to yield 2,3-naphthotriazole (NAT) adduct, which exhibits in alkaline medium fluorescence at 415 nm after excitation at 375 nm. For S-nitrosothiols (RSNOs), mercuric ions are used to cleave the S-NO bond, therefore allowing them to be measured with the DAN assay. Nitrate ions are reduced to nitrite ions by an enzymatic reaction, next they are measured by DAN assay.

Figure 1 .

 1 Figure 1. Reaction between nitrite ion and 2,3-diaminonaphthalene (DAN) producing 2,3-naphthotriazole (NAT) and corresponding fluorescence spectra at an excitation wavelength of 375 nm (panel-A) and at an emission wavelength of 415 nm (panel-B) (Jobgen et al. 2007).

  of sodium nitrite (NaNO 2 ) (Mw = 69 g.mol -1 ; ref Merck 6549) in a 50 mL volumetric flask, dissolve and adjust the volume with PBS. Mix the solution gently for one time. Prepare daily. of sodium nitrate (NaNO 3 ) (Mw = 85 g.mol -1 ; ref Sigma S8170)

  Transfer the content of the Assay Buffer vial (from the Nitrate/Nitrite fluorometric assay kit Cayman ref. 780051) to a 100 mL volumetric flask, rinse the vial three times with 1 mL of ultrapure water and fill up to 100 mL with ultrapure water. Store at 4 °C for no more than 2 months. Nitrate Reductase (Item N0. 780010): Reconstitute the content of the vial (from the kit) with 1.2 mL Assay Buffer. Keep on ice during use. Divide into 70 µL aliquots and store at -20 °C for no more than 2 months. Freezing and thawing should be only operated once. Enzyme Cofactors (Item N0. 780012): Reconstitute the contents of the vial (from the kit) with 1.2 mL Assay Buffer. Keep on ice during use. Divide into 70 µL aliquots and store at -20 °C for no more than 2 months. Freezing and thawing should be only operated once.

NaNO 2 /For

 2 GSNO daughter solution n° 2 (2.10 -6 M): transfer 500 µL of the NaNO 2 /GSNO daughter solution n°1 in a 25 mL volumetric flask, adjust the volume with sample buffer. Mix gently for one time. Dilutions for the calibration curve are prepared in 1.5 mL Eppendorf tubes (mix gently each tube for one time), as follows: each standard (concentration range: 0.1 -2 µM) or sample (in triplicate) is transferred into a black 96-well microplate, and 20 µL of 0.105 mM DAN solution is added. The mixture is shaken for 1 min at 500 rpm at 20-25 °C (Heidolph, VIBRAMAX 110, Schwabach, Germany) before incubation at 37 °C (incubator) in the dark for 10 min. Next, 20 µL of 2.1 M NaOH is added. The mixture is shaken for 5 min at 500 rpm at 20-25 °C, followed by analysis with a spectrofluorimeter (FP-8300iRM, JASCO, Tokyo, Japan) coupled with FMP micro-well plate reader. solution n°1 (2.5.10 -5 M): transfer 50 µL of 10 -2 M NaNO 3 solution in a 20 mL volumetric flask. Adjust the volume with the corresponding physiological medium. Dilutions for the calibration curve are prepared in Eppendorf tubes, as follows: kit protocol: 20 µL of each standard (concentration range: 0.75 -25 µM), or sample (in triplicate) is transferred into a black 96-well microplate, 60 µL of assay buffer and 10 µL enzyme cofactor mixture together with 10 µL nitrate reductase mixture are added. The mixture is shaken for 1 min at 500 rpm (Heidolph, VIBRAMAX 110, Schwabach, Germany) and incubated for 60 min at 20-25 °C in the dark. Next, 10 µL of kit DAN reagent (Item N0. 780070) is added, and the mixture is shaken for 1 min at 500 rpm at 20-25 °C before incubation at 37 °C (incubator) in the dark for 10 min. Finally, 20 µL of 2.8 M kit NaOH solution (Item N0. 780068) is added, followed by shaking for 5 min at 500 rpm and analysis with the spectrofluorimeter coupled with FMP micro-well plate reader.

-

  Nitrite concentration in samples is calculated with nitrite standard curve equation.

  diaminonaphthalene (DAN) assay relies upon the reaction between DAN and nitrite ion in acidic medium to yield 2,3-naphthotriazole (NAT) adduct. For S-nitrosothiols (RSNOs), mercuric ions are used to cleave the S-NO bond, therefore allowing them to be measured with the DAN assay. Nitrate ions are reduced to nitrite ions by an enzymatic reaction, next they are measured by DAN assay.

Figure 1 .

 1 Figure 1. Reaction between nitrite ion and 2,3-diaminonaphthalene (DAN) producing 2,3-naphthotriazole (NAT).

0 M

 0 NaOH titrated solution (commercial solution; ref Sigma S2770) in a 100 mL volumetric flask, and adjust the volume with ultrapure water to obtain 10 -2 M NaOH solution. Dilute 100-fold of NaOH solution in ultrapure water by transferring 1 mL of 10 -2 M NaOH in a 100 mL volumetric flask and adjust the volume with ultrapure water. of sodium nitrite (Na 15 NO 2 ) (Mw = 70 g.mol -1 ; ref Cambridge Isotope Laboratories 68378-96-1, purity: 98 %) in a 50 mL volumetric flask, dissolve and adjust the volume with PBS. Mix the solution gently for one time. Prepare daily. Weigh 43 mg of sodium nitrate (Na 15 NO 3 ) (Mw = 86 g.mol -1 ; ref Sigma 364606, purity: 98 %) in a 50 mL volumetric flask, dissolve and adjust the volume with PBS.

  Transfer the content of the Assay Buffer vial (from the Nitrate/Nitrite fluorometric assay kit Cayman ref. 780051) to a 100 mL volumetric flask, rinse the vial three times with 1 mL of ultrapure water and fill up to 100 mL with ultrapure water. Store at 4 °C for no more than 2 months. Nitrate Reductase (Item N0. 780010): Reconstitute the content of the vial (from the kit) with 1.2 mL Assay Buffer. Keep on ice during use. Divide into 70 µL aliquots and store at -20 °C for no more than 2 months. Freezing and thawing should be only operated once. Enzyme Cofactors (Item N0. 780012): Reconstitute the contents of the vial (from the kit) with 1.2 mL Assay Buffer. Keep on ice during use. Divide into 70 µL aliquots and store at -20 °C for no more than 2 months. Freezing and thawing should be only operated once. 2.12. 10 -2 M CH 3 COOH Dilute 0.285 mL of glacial acetic acid solution (volumetric mass = 1.05 g/mL;

  of 2-naphthylamine (Mw = 143 g.mol -1 , ref Sigma 91598) in a 10 mL volumetric flask, dissolve and adjust the volume with rinsing mobile phase to obtain 5 mM 2-naphthylamine solution. Dilute 250-fold 2-naphthylamine solution by transferring 0.04 mL of 5 mM 2-naphthylamine solution in a 10 mL volumetric flask and adjust the volume with rinsing mobile phase. Prepare daily. 2.15. 1.24 mM pure NAT Weigh 21 mg NAT (Mw = 169.2 g.mol -1 ; ref Chemodex 269-12-5; purity: 98.2 %) in a 100 mL volumetric flask, dissolve in 10 mL of methanol, and adjust the volume with 0.1 M HCl solution. Divide into aliquots of 2 mL and store at -80°C for 1 year.

  300 µL of each standard (concentration range: 5 -200 nM), quality controls, or sample are transferred into a 1.5-mL Eppendorf tube, and 60 µL of 0.105 mM DAN solution are added. The mixture is shaken for 0.1 min by vortex at 20-25 °C before incubation at 37 °C in the dark for 10 min. Next, 36 µL of 1 M and 4 µL of 10 -4 M NaOH are added. The mixture is shaken for 0.1 min by vortex at 20-25 °C, followed by freezing at -80 °C. 10 µL of 0.02 mM IS are added before analysis with LC-MS/MS. For RS 15 NO or (RS 15 NO + 15 NO 2 -) measurement, DAN reagent has to be replaced by DAN-HgCl 2 reagent.

3. 3 .

 3 Calibration curve for 15 NO 3 -determination Na 15 NO 3 daughter solution n°1 (10 -4 M): transfer 100 µL of 10 -2 M Na 15 NO 3 solution in a 10 mL volumetric flask. Adjust the volume with PBS. 5000 nM Na 15 NO 3 is prepared by transferring 0.5 mL of 10 -4 M Na 15 NO 3 in a 10 mL volumetric flask. Adjust the volume with HBSS; 2500 nM Na 15 NO 3 is prepared by mixing 0.6 mL of 5000 nM Na 15 NO 3 and 015 NO 3 as the quality control is prepared by mixing 0.8 mL of 5000 nM Na 15 NO 3 and 0.2 mL of HBSS. 2500 nM Na 15 NO 3 as the quality control is prepared by mixing 0.5 mL of 5000 nM Na 15 NO 3 and 0.5 mL of HBSS. 300 nM Na 15 NO 3 as the quality control is prepared by mixing 0.6 mL of 500 nM Na 15 NO 3 and 0.4 mL of HBSS. According to the kit protocol: 40 µL of each standard (concentration range: 250 -5000 nM), quality controls, or sample are transferred into a 1.5-mL Eppendorf tube, 120 µL of assay buffer and 20 µL enzyme cofactor mixture together with 20 µL nitrate reductase mixture are added. The mixture is shaken for 0.1 min by vortex and incubated for 60 min at 20-25 °C in the dark. Next, 40 µL of 0.105 mM DAN solution is added. The mixture is shaken for 0.1 min by vortex at 20-25 °C before incubation at 37 °C in the dark for 10 min. Then, 24 µL of 1 M and 3 µL of 10 -4 M NaOH are added.

  Gradient mobile phase: start at 40% B for 10 min (equilibrating the column) + 2 min; change linearly to 80% B over 3.5 min; hold at 80% B for 2.5 min (mobile phase A: 10 -2 M acetic acid; mobile phase B: CH 3 CN) -Flow rate: 0.2 mL.min -1 ; column temperature: 25 °C; resulting back pressure: 105 bars for 40% B, 65 bars for 80 % B. -Loop size: 20 µL -Software: Chromeleon (running time: 7 min; peak range: 5.3 min -5.5 min) -Rinsing conditions: use CH 3 CN-H 2 O (50/50, V/V) at a flow rate of 0.2 mL.min -1 and at 25 °C for 10 min. The operating conditions for MS/MS (LTQ Velos Pro, Thermo Fisher Scientific, San José, CA, USA) are as follows: -Ion source: ESI (source voltage: 5.00 kV; source current: 3.5 µA; source temperature: 280 °C; sheath / aux / sweep gas (N 2 ) flow rate: 10.00 / 5.00 / 0.05 arb; capillary temperature: 250 °C) -Analyzer: dual-pressure linear ion trap (monitor transition: m/z 171 → 156 for 15 N-NAT, m/z 170 →156 for 14 N-NAT) -Software: Thermo Tune Plus, (MRM; Act type: HCD; collision energy: 50 %; Act Q: 0.25; Act time: 10 ms) -A valve between LC and MS is used to throw sample for the first 3 min into waste and transfer sample for the next 4.5 min into detector.

  Column: ZORBAX SB-Aq C18 4.6 X 150 mm, 3.5 μm (Agilent) + precolumn (NUCLEODUR® EC4/3 C-18 Htec, 5 μm) -Mobile phase: CH 3 CN-CH 3 COONH 4 (10 -2 M, pH = 7.2) (28:72, V/V) -Flow rate: 0.8 mL.min -1 ; column temperature: 40°C; resulting back pressure: 95-105 bars. -Auto-sampler with cooling device set at 4 °C (loop size: 20 µL) -Spectrofluorometric detection: λ exc = 375 nm and λ em = 415 nm; sensitivity: low (RF-10A XL, Shimadzu, Kyoto, Japan) + software: LC Solution, (running time: 15 min; peak range: 9 -11 min) -Rinsing conditions: use CH 3 CN-H 2 O (50:50, V/V) at a flow rate of 0.8 mL.min -1

Figure 1 .

 1 Figure 1. Chromatograms of NAT resulting from derivatization of 12.5 μM GSNO in the (A) absence and (B) presence of rat intestine by LC-spectrofluorimetry at λ exc = 375 nm and λ em = 415 nm.

Figure 1 .

 1 Figure 1. Scheme of 6 Ussing chambers for studying drug permeability, e.g. S-nitrosothiols (RSNOs)

0. 1 M

 1 Tris buffer (pH = 7.4): weigh 1.576 g of Trizma-HCl (Mw = 158 g.mol -1 , ref Sigma T040), dissolve with 100 mL of ultrapure water; and adjust the pH to 7.40 ± 0.05 with 10 M NaOH solution. 0.1 M Phosphate buffer (pH = 7.4): Weigh 1.36 g of KH 2 PO 4 (Mw = 136.09 g.mol -1 , ref Merck 646A148973) and 0.074 g of EDTANa 2 •2H 2 O (Mw = 372.24 g.mol -1 ), dissolve with 100 mL of ultrapure water, and adjust the pH to 7.40 ± 0.05 with 10 M NaOH.

2. 2 .

 2 Substrate solution Weigh 6.6 mg of L-γ-glutamyl-3-carboxy-4-nitroanilide (Mw = 328.3 g.mol -1 , ref Sigma AC98), 40.6 mg of MgCl 2 •6H 2 O (Mw = 203.31 g.mol -1 , ref Sigma M9272) and 52.8 mg of glycylglycine (Mw = 132.1 g.mol -1 , ref Sigma G1002), dissolve with 20 mL of Tris buffer. Protect solution from light. The same solution is prepared without adding the substrate for the blank.

2. 3 .

 3 γ-glutamyltransferase (GGT) Weigh 1 mg of GGT (26 UI*/mL, bovine kidney, EC 2.3.2.2, ref G8040), dissolve with 1 mL of phosphate buffer, divide the solution in aliquots of 100 µL (1.1 UI/mL) and store them at -20°C for maximum five months (determine the GGT concentration by the BCA Protein Assay kit (Pierce™ BCA Protein Assay Kit, ref Thermo Fisher 23225)).

  g of sodium borate (Mw = 201.372 g.mol -1 , ref Sigma S4500) and 0.2665 g of serine (Mw = 105.09, ref Sigma G100), dissolve with 25 mL of Tris buffer, adjust the pH to 7.40 ± 0.05 with 10 M NaOH.

  Incubate for 30 min at r.t. with gentle stirring (Heidolph Polymax 1040 stirrer, Schwabach, Germany). Wash the membrane in TBST for 4 times (5 min for each wash) (Do not load the antibodies of Anti-PDI and the anti-Actin at the same time): antibody (Goat anti-Rabbit HRP, Santa Cruz Biotechnology) for 5000-fold in the TBST-Milk 5 % buffer (final volume = 20 mL), and use it to cover the membrane. Incubate for 60 min at r.t. with gentle stirring (Heidolph Polymax 1040

(

   Clarity Western ECL Substrate,) (use 2 different tips and don't mix these two solutions in the original vials). Use this mixture to cover the membrane completely and avoid bubbles. Incubate for 5 min in dark and take out the membrane with a forceps and drain it, next, wrap the membrane with cellophane (attention to bubbles). Place the membrane in the revelation cassette: fix the cellophane in the cassette with scotch (wearing gloves).Development: Take out the film, cut it to the proper size and cut off the top right corner; place the film on the diaphragm (align the film in the upper left corner); Expose (30 s for anti-Actin, 30 s for anti-PDI); Remove the film in a dry flash. Develop the film for 30 s and fix for 1.5 min. Rinse the film in ultrapure water and dry it. Place the film on the membrane again in the cassette: mark molecular weights according to the marker and note the contours of the membrane on the film. Rinse the membrane in TBST for 5 min under rotation, and store the membrane wrapped with cellophane at 4 ° C. The whole procedure is in dark.Stripping: Rinse the membrane in TBST for 2 h at r.t. under rotation, incubate for 30 min at 50 °C in stripping buffer; rinse the membrane in TBST for 5 min under rotation for 4 times, block the membrane again and label anti-actin as procedure3.5.; analyze proteins as procedure 3.6.. A representative result is shown in Figure1.

Figure 1 .

 1 Figure 1. Western blot analysis of PDI (55 kDa) in isolated rat intestine homogenate with different concentrations, and ᵦ-actin was used as a reference protein (43 kDa).

Figure 1 .

 1 Figure 1. Scheme of the Griess reaction.

Figure 2 .-

 2 Figure 2. Chromatograms corresponding to azo adduct resulting from derivatization of 10 -2 M NO 2 -in red or blank (ultrapure water) in black by LC-UV/Vis at λ = 220 nm.

Figure 3 .

 3 Figure 3. Chromatograms corresponding to NAT adduct resulting from derivatization of 5 mM NO 2 -in red or blank (ultrapure water) in black by LC-UV/Vis at λ = 330 nm.

Table 1 .

 1 Standard curves validation parameters for S-nitrosothiols (RSNO), nitrite ions

	(NO 2 -) and nitrate ions (NO 3 -) in HBSS with Ca 2+ /Mg 2+ . Mean ± SD; n=3 ....... 120

Table 2 .

 2 Values of apparent permeability coefficient (P app ) for NOx species ( + NO 2

	-+

-) and the RSNO molecular form after 4 h of permeation from the apical to the basolateral compartment. nd: not determined , LOQ: Limit of quantification. . 121 Table 3. Mass balance for each treatment (initial amount: 50 nmol) after 4 h of permeation from the apical to the basolateral compartment. .......................... 122

Table 4 .

 4 Values of apparent permeability coefficient (P app ) for NOx species (RSNO +

	NO 2 -+ NO 3

-) and the RSNO form after 4 h of permeation from basolateral to apical compartment. nd: not determined , LOQ: Limit of quantification. .................... 123 Table 5. Mass balance for each tested treatment (initial amount 150 nmol) after 4 h of permeability from the basolateral to the apical compartment.......................... 124

Table 6 .

 6 Values of apparent permeability coefficient (P app ) for NOx species (RSNO +

	NO 2 -+ NO 3 -

Table 2 .

 2 N-ethylmaleimide)) ......................................................................................... 141 Standard curves validation parameters for S-nitrosoglutathione (GSNO) and nitrite ions (NO 2 -). Mean ± SD; n ≥ 3. .............................................................. 143

	3-carboxy-4-nitroanilide;	CNA:	3-carboxy-4-nitroanilide;	SBC:

Table 3 .

 3 Values of apparent permeability coefficient (P app ) for NOx species (RSNOs + N-ethylmaleimide).. ......................................... 147

	NO 2 -+ NO 3 -), NO 2 -, RSNOs and NO 3 -molecular forms after 2 h of permeation through
	rat intestine; and recovery of NO species in Ussing chamber. nd: not determined.
	Mean ± sem of three independent experiments done in duplicate (GSNO:
	S-nitrosoglutathione; RSNOs: S-nitrosothiols; Glygly: glycylglycine; SBC:
	serine-borate complex; NEM:

3. Supplementary study:

Table

1

. Permeability of intestinal markers and S-nitrosothiols (RSNOs) in intestinal barrier models (GSNO: S-nitrosoglutathione; NACNO: S-nitroso-N-acetyl-cysteine; SNAP: S-nitroso-N-acetyl-penicillamine; TEER: Transepithelial electrical resistance; P app : Apparent permeability coefficient) .......................................................... 152

  SNAP at the same time; p < 0.05. ....................................................................................................................... 122

	Fig. 5. Apical to basolateral permeability -Intracellular quantifications of (A) RSNO,
	(B) NO 2 -, (subtracted from the control cells) after 1 h and 4 h of exposure to 50 nmol of
	each treatment. Results are shown as mean ± SD of four independent experiments
	done in duplicate and are compared using two-way ANOVA (p treatment (GSNO,
	NACNO, SNAP; excluding NaNO 2 ), p time (1 h, 4 h) and p interaction ) *vs. GSNO; # vs.
	SNAP at the same time; p < 0.05 (Bonferroni's multiple comparisons test). ... 122
	Fig. 6. Basolateral to Apical permeability -Quantification in the apical compartment of
	permeated (A) RSNO, (B) NO 2 -and (C) NO 3 -after 1 h and 4 h from 50 nmol of each
	treatment. Results are shown as mean ± SD of four independent experiments done in

3 -after 1 h and 4 h of exposure to 50 nmol of each treatment. Results are shown as mean ± SD of four independent experiments done in duplicate and are compared using one-way ANOVA (excluding NaNO 2 ) Bonferroni post-test. *vs. GSNO; # vs. duplicate and are compared using two-way ANOVA (p treatment (GSNO, NACNO, SNAP; excluding NaNO 2 ), p time (1 h, 4 h) and p interaction ). *vs. GSNO; # vs. SNAP at the same time; p < 0.05 (Bonferroni's multiple comparisons test) .................................. 123

  ....................................................................................................................... 146

	EPR LC-FL	Electron paramagnetic resonance LC coupled with fluorescence detection
	ESI IEC	Electrospray ionization Ion-exchange chromatography
	Et 3 O + [BF 4 -] iNOS	Triethyloxonium tetrafluoroborate Inducible NO synthase
	ADC FAB IS	Arginine decarboxylase Fast atom bombardment Internal standard
	ADMA FAD ITMS	Asymmetric dimethylarginine Flavin adenine dinucleotide Ion Trap Mass Spectrometer
	BCA FBS LC-MS	Bicinchoninic acid Fetal bovine serum LC coupled with mass spectrometry
	BCS BSA FL FMOCCI LC-ESI-MS/MS	Biopharmaceutical Classification System Bovine serum albumin Fluorescence 9-Fluorenylmethyl chloroformate Liquid chromatography-electrospray ionization-tandem mass spectrometry
	CAD FTIR LC-ESI-ITMS/MS	Collision-activated dissociation Fourier Transform InfraRed spectroscopy Liquid Chromatography-Electrospray Ionization -Ion Trap tandem Mass Spectrometry
	cAMP FTIRM	Cyclic adenosine monophosphate FTIR Microscopy
	cCOx GC LC-ITMS/MS	Cytochrome C oxidase Gas chromatography Liquid chromatography-tandem ion trap mass spectrometry
	CID GGT LC-MS/MS	Collisionally induced dissociation γ-glutamyltransferase Liquid chromatography-tandem mass spectrometry
	CL GIT LLOQ	Chemiluminescence Gastrointestinal tract Lower limit of quantification
	CNA Glygly LOD	3-Carboxy-4-nitroanilide Glycylglycine Limit of detection
	CVDs GCNA log P	Cardiovascular diseases L-γ-glutamyl-3-carboxy-4-nitroanilide Logarithmic value of partition coefficient
	Cys/Cu GSH LOQ	Cysteine and CuSO 4 Reduced glutathione Limit of quantification
	CysNO GSNO MeOH	S-nitrosocysteine S-nitrosoglutathione Methanol
	DAN GS 15 NO MES	2,3-Diaminonaphthalene 15 N labeled S-nitrosoglutathione 2-(N-morpholino)ethanesulfonic acid
	DeoxyHb GSSG MRM	Deoxygenated hemoglobin Glutathione disulphide Multiple-reaction monitoring
	DTNB Hb MS	5,5′-Dithiobis(2-nitrobenzoic acid) Hemoglobin Mass spectrometry
	DTPA HbFeNO MS/MS	Diethylene triamino pentaacetic acid Iron nitrosylhemoglobin Tandem mass spectrometry
	ECNICI HbSNO	Electron-capture negative-ion chemical ionization S-nitrosohemoglobin
	EDRF HBSS	Endothelium-derived relaxing factor Hank's Balanced Salt Solution
	EDTA HCD	Ethylene diaminotetraacetic acid Higher-energy Collision Dissociation
	EMEM HILIC	Eagle's Minimum Essential Medium Hydrophilic interaction liquid chromatography
	eNOS LC	Endothelial nitric oxide synthase Liquid chromatography

General introduction Titre: Oral bioavailability studies of S-nitrosoglutathione using intestinal barrier models by liquid chromatography coupled with mass spectrometry after labeling with the isotope nitrogen 15 Etude de la biodisponibilité orale du S-nitrosoglutathion au moyen de modèles de la barrière intestinale par chromatographie en phase liquide couplée à la spectrométrie de masse après marquage par l'isotope 15 de l'azote Introduction générale

	PFBBr n'ont cessé de croître, permettant de mieux comprendre son rôle ubiquitaire au-delà Pentafluorobenzyl bromide ou perlinguale (comprimé à croquer) étant préférable pour obtenir des concentrations
	de son action sur la relaxation des cellules musculaires lisses des vaisseaux dans le compartiment sanguin efficaces en thérapeutique. Mais dans ce cas, la durée
	RNS Les maladies cardiovasculaires (MCV) représentent la première cause de Reactive nitrogen species sanguins : inhibition de l'agrégation plaquettaire, rôle dans les systèmes immunitaire de l'effet est brève (demi-vie de l'ordre de 15 min). Dans le cas de traitements
	RPLC mortalité dans le monde, en particulier dans les pays occidentaux du fait du Reverse phase liquid chromatography et nerveux, etc... De plus, le NO, espèce radicalaire à demi-vie courte (de l'ordre de la chroniques, la prise des dérivés nitrés par voie orale offre une biodisponibilité très
	r² vieillissement de la population, de suralimentation ou de mauvaise alimentation, ou Coefficient of determination seconde) et à l'état gazeux dans les conditions standards de température et de faible (< 10 %), du fait d'un premier passage hépatique très important. Des
	RSD encore d'un manque d'activité physique. En outre, le tabagisme et l'usage nocif de Relative standard deviation pression, appartient à la classe des gazotransmetteurs qui incluent le sulfure formulations à libération prolongée permettent cependant d'obtenir un effet
	l'alcool sont des facteurs de risque aggravants. Il en résulte un réel enjeu sociétal et d'hydrogène (H 2 S) et le monoxyde de carbone (CO). Ces gaz ont des propriétés thérapeutique pendant 8 h (dinitrate d'isosorbide). La voie transdermique est
	RSNOs économique : maintenir une population vieillissante dans de bonnes conditions de vie S-nitrosothiols physiologiques proches et les interactions entre eux (en particulier H 2 S et NO) également utilisée. Après application du dispositif « patch » sur la peau, la trinitrine
	RS 15 NOs et diminuer les dépenses de santé liées aux MCV. 15 N labeled S-nitrosothiols constituent un domaine d'investigation très récent et en plein essor. pénètre directement dans la circulation sous forme active (pas d'effet de premier
	r.t. passage dans le foie). Les dérivés nitrés présentent cependant des effets Room temperature L'athérosclérose intervient pour une part importante dans la physiopathologie Il est actuellement établi que la production endogène du NO diminue avec l'âge
	des MCV, en particulier pour les syndromes coronariens aigus. Près de la moitié des (Loo et al. 2000) et au cours des MCV du fait d'une détérioration des fonctions de secondaires notables : phénomène de tolérance, apparition d'un stress oxydant
	SBC décès mondiaux sont dus à un infarctus du myocarde ; un tiers résulte d'un accident Serine-borate complex l'endothélium (monocouche cellulaire recouvrant la paroi interne des vaisseaux (consommation du pool de thiols en particulier du glutathion dans l'organisme). Ils
	S/B vasculaire cérébral ; viennent ensuite l'hypertension artérielle ou d'autres MCV Signal-over-blank ratio sanguins). En effet, les cellules endothéliales produisent le NO via l'oxydation de s'administrent donc de façon discontinue (par exemple en retirant le patch durant la
	SD comme l'embolie pulmonaire et les causes d'insuffisance cardiaque. Standard deviation l'arginine en citrulline catalysée par la NO synthase endothéliale (eNOS) ; le NO nuit), de manière à éviter tout échappement thérapeutique.
	SDS La prise en charge des MCV (diagnostic, thérapeutique, chirurgie, hygiène de vie) diffuse vers les cellules musculaires lisses sous-jacentes et active la guanylate II apparait que les composés donneurs de NO actuellement disponibles ont des Sodium dodecyl sulfate s'améliore régulièrement mais elle nécessite de nouvelles avancées pour faire reculer cyclase soluble (sGC), ce qui va permettre la synthèse de monophosphate de indications thérapeutiques restreintes et de nombreux inconvénients. Il est donc d'un
	Sem la fréquence de celles-ci et leur corollaire en termes de morbidité. Les classes Standard error of the mean guanosine cyclique (cGMP) induisant une vasodilatation. L'altération de l'endothélium intérêt majeur de développer de nouveaux donneurs de NO libérant celui-ci de façon
	SIM thérapeutiques des MCV sont nombreuses : bêtabloquants, inhibiteurs calciques, Selected-ion monitoring résultant de l'apparition d'une plaque athéromateuse, d'une inflammation et d'un prolongée et sans effets délétères.
	S 15 NACET diurétiques, médicaments ciblant le système rénine angiotensine, dérivés nitrés, 15 N labeled S-nitroso-N-acetylcysteine ethyl ester stress oxydant diminue l'activité de l'eNOS au profit de la production d'espèces
	digitaliques, antiarythmiques, inhibiteurs de la phosphodiestérase, antagonistes des réactives de l'azote délétères. Celles-ci résultent de l'expression de la NO synthase
	SNALB récepteurs à l'endothéline,…). Chacune de celles-ci bénéficie régulièrement de S-nitrosoalbumin inductible (iNOS) qui mène à la production d'importantes quantités de NO réagissant
	3 -S 15 NALB nouveaux développements. En outre, des combinaisons entre médicaments 15 N labeled nitrate ion 15 N labeled S-nitrosoalbumin avec l'anion superoxyde (O 2 -) également produit en grande quantité dans les
	NOx SNAP appartenant à différentes classes apportent des améliorations notables dans le Nitrogen oxides S-nitroso-N-acetyl-D-penicillamine conditions de stress oxydant. Il se forme ainsi l'anion peroxynitrite (ONOO -) très
	OCT S 15 NAP traitement des MCV. réactif vis-à-vis des constituants cellulaires, en particulier par la nitration des résidus Organic Cation Transporters 15 N labeled S-nitroso-N-acetyl-D-penicillamine Parmi ces différentes classes thérapeutiques, les dérivés nitrés ou nitrates tyrosine des protéines.
	OCTN S 15 NC organiques constituent l'une des plus anciennes puisque Alfred Nobel (1833-1896), Zwitterion Transporters 15 N labeled S-nitroso--L-cysteine Les travaux menés sur le NO en physiopathologie démontrent l'utilité d'une
	O 15 NOO -S/N inventeur de la nitroglycérine, s'est administré celle-ci pour combattre l'angor dont il 15 N labeled peroxynitrite ion Signal-over-noise ratio supplémentation en NO aussi bien dans des événements cardiovaculaires aigus ou
	P app SRM souffrait. La découverte de l'entité pharmacologique active, le monoxyde d'azote ou Apparent permeability coefficient Selected-reaction monitoring chroniques. L'alimentation (eau de boisson, légumes,…) est une source importante
	oxyde nitrique (NO), n'est intervenue que beaucoup plus tard. En effet, la nature d'ions nitrates qui sont réduits en ions nitrites par les bactéries du tube digestif, puis
	PDI TEER chimique de l'EDRF (endothelium-derived relaxing factor ou facteur relaxant dérivé de Protein Disulfide Isomerase Transepithelial electrical resistance assimilés et réduits en NO dans l'organisme. Cette source ne permet cependant pas
	HePI-MS UPLC l'endothélium) a été identifiée comme étant le NO en 1986 par Louis Ignarro, Ferid Helium-plasma ionization mass spectrometry Ultra performance liquid chromatography de compenser le manque de production de NO in situ au niveau du système
	PK XO Murad et Salvador Moncada, récipiendaires du prix Nobel de physiologie et de Pharmacokinetic Xanthine oxidase cardiovasculaire.
	pKa médecine en 1998. La trinitrine, appellation pharmaceutique de la nitroglycérine, est toujours Negative logarithmic value of acid ionisation constant
	Depuis la découverte du NO en physiologie, les travaux qui lui sont consacrés adaptée au traitement d'une crise d'angor, la voie d'administration sublinguale (spray)

Comment se positionnent mes travaux de thèse dans le projet scientifique de l'unité de recherche EA 3452 Cithefor dans laquelle j'ai réalisé ceux-ci, ainsi que dans la collaboration établie avec le Professeur Patrick Chaimbault (EA 4632 LCP-A2MC) ?

	Le projet de l'EA 3452 Cithefor s'intitule «Molécules

et nanoformulations innovantes donneurs de NO à visée vasculaire». Il est principalement axé sur les thionitrites ou S-nitrosothiols (RSNOs) capables

	pénicillamine, a fait l'objet de travaux au sein de l'EA Cithefor (Dahboul et al.
	2014)(Luo et al. 2016)(Bouressam et al. 2017). Ces molécules demeurent cependant
	sensibles aux conditions environnementales et elles nécessitent le développement de
	formulations adaptées réalisées au sein de l'EA Cithefor afin de limiter leur
	biotransformation et de faciliter leur diffusion vers les tissus vasculaires (Wu et al.
	2016a).
	La preuve du concept d'une libération prolongée de NO à partir de RSNOs
	formulés sous forme d'implants polymériques microparticulaires biodégradables
	sous-cutanés formés in situ a été apportée sur l'animal entier (rat), en observant à la
	fois la diminution de la pression artérielle par télémétrie, et l'effet antiagrégant
	plaquettaire, ceci sur plusieurs jours ; l'effet bénéfique du GSNO formulé en implants
	microparticulaires par rapport au GSNO libre a été démontré dans la récupération
	après un AVC. Il s'agit de la première étude de formulation retard de RSNOs testées
	in vivo dans des conditions physiologiques et pathologiques (modèle murin de
	thromboembolisme cérébral sans reperfusion (Parent et al. 2015).
	En vue d'accroître le champ d'application des RSNOs en thérapeutique en
	particulier dans les traitements chroniques, il est nécessaire de privilégier la voie
	d'administration orale et par conséquent d'étudier le passage de la barrière intestinale
	par ceux-ci. Pour ce faire, les méthodologies analytiques couramment utilisées au
	sein de l'EA Cithefor reposent sur des techniques spectroscopiques conventionnelles
	décrites dans la littérature mais leur limite de quantification (LOQ) située entre 1 et
	0,1 µM s'avère insuffisante. De plus, lors d'étude pharmacocinétique sur animal entier,
	il sera nécessaire de distinguer entre les différentes sources de NO : production
	endogène, alimentation et médicament. C'est en quoi les travaux de ma thèse doivent
	compléter le panel des méthodes bioanalytiques disponibles au sein de l'EA 3452
	Cithefor. La collaboration avec le Professeur P Chaimbault est liée au choix de l'outil
	de libérer NO sur de plus longues spectrométrie de masse (SM) pour remplir les objectifs en matière de méthodes
	durées que les médicaments actuels sans présenter leurs inconvénients. Le principal sélectives présentant une limite de quantification (LOQ pour Limit Of Quantification) la
	mode d'obtention des RSNOs consiste à faire réagir un thiol avec les ions nitrites en plus faible possible.
	milieu acide pour former une liaison S-NO. Les RSNOs utilisés au sein de l'EA
	Cithefor présentent soit un coeur cystéine (le S-nitrosoglutathion (GSNO), principal
	RSNOs endogène et la S-nitroso-N-acétylcystéine (NACNO)), soit un coeur
	pénicillamine (la S-nitroso-N-acétylpénicillamine (SNAP)). Une molécule originale, la
	S,S'-dinitrisobucillamine (Buc(NO) 2 ), possédant à la fois un coeur cystéine et

Comment est organisé mon manuscrit de thèse ?

	marquage des ions nitrites et des RSNOs d'intérêt par celui-ci va représenter dans le
	cadre de nos études un excellent moyen de suivre le devenir de ceux-ci dans
	l'organisme, en particulier après administration orale. La méthodologie analytique de
	choix pour ce faire sera la spectrométrie de masse associée à une méthode
	séparative. Cette revue de la littérature a fait l'objet d'une publication intitulée
	"Labeling
	L'introduction porte sur les approches méthodologiques utilisées dans la
	littérature pour suivre spécifiquement un dérivé azoté marqué par l'isotope 15 de
	l'azote ( 15 N). Ce dernier étant de faible abondance naturelle (0,37%) et stable, le

nitrogen species with the stable isotope 15 N for their measurement by separative methods coupled with mass spectrometry: a review" en

	cours de
	publication dans Talanta (191 (2018) 491-503).
	Le premier chapitre est axé sur les développements méthodologiques
	analytiques. Le NO et ses métabolites inorganiques (ions nitrites et nitrates) ont des
	faibles valeurs de masse molaire et leurs ions m/z correspondants sont de détection
	difficile en SM. Le recours à des réactions de dérivation s'avère indispensable pour
	augmenter la masse molaire et faciliter leur rétention en chromatographie en phase
	liquide (CPL), leur détection au moyen d'une fragmentation appropriée (SM/SM). J'ai
	eu recours à des techniques analytiques existantes largement utilisées dans la
	littérature et complètement maîtrisées au sein de l'EA 3452 Cithefor. Il s'agit de :

Experimental paper: "Comparison between two derivatization methods of nitrite ion labeled with 15 N applied to liquid chromatography-tandem mass spectrometry" in Analytical Methods (10 (2018) 3830-3836)

  procède comme sur les autres appareils de type trappe d'ions. Après avoir été ionisés dans la source, les composés pénètrent dans le spectromètre pour y rejoindre l'analyseur sous l'effet d'un gradient de vide et de tension. Ces ions rejoignent la trappe qui sur l'appareil est de type linéaire, d'où son nom « LTQ » pour Linear Quadrupole Trap (on trouve aussi l'abréviation QTL selon les sources). La trappe linéaire a une capacité de piégeage des ions plus élevée que les trappes classiques (circulaires) qui leur confèrent de meilleures limites de détection. En spectrométrie de masse tandem, l'ion moléculaire est d'abord isolé dans la trappe (on élimine tout les autres) puis fragmenté par excitation dans la trappe (collision avec de l'hélium généralement toujours présent même en spectrométrie de masse simple) puis après un certain temps de génération, les fragments sont à leur tour analysés par la trappe. Ces molécules de solvants, plus grosses communiquent plus d'énergie aux ions moléculaires à fragmenter mais ce n'est pas tout... Elles n'ont pas du tout la même inertie chimique et peuvent éventuellement réagir avec l'ion moléculaire en phase gazeuse provoquant des mécanismes de fragmentation originaux comme nous en avons été témoins lors de ce travail.

	1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter
	2.
	Detailed procedures used for experiments in this article are present in protocols (see
	appendices) entitled "Purification of azo adduct and 2,3-naphthotriazole adduct by LC
	coupled with UV/Vis detection", "Measurement of nitrite, nitrate ions and
	S-nitrosothiols using the 2,3-diaminonaphthalene assay by LC coupled with tandem
	mass spectrometry" and "Permeability studies of S-nitrosothiols in isolated rat
	intestine using Ussing chamber".
	76

Le spectromètre LTQ Velos Pro (Thermo Scientific) est conçu de manière à proposer plusieurs techniques de fragmentation dont la technique CID. Dans ce cas, celui-ci En mode MRM, pour le dosage, comme pour un QqQ, on suivra une transition mais les ions auront été générés dans le même analyseur. Sur ce spectromètre de masse, nous disposons également d'un mode HCD qui est en règle générale utilisé dans l'analyse des protéines et peptides pour générer des informations structurales complémentaires au mode CID. Le mode de fonctionnement de l'appareil est alors complètement différent. L'étape d'isolement (A de la figure 1 B) de l'ion moléculaire reste cependant la même mais la génération des fragments ne se fait plus dans la trappe. L'ion moléculaire est éjecté de la trappe pour un retour en arrière dans la région de l'interface Q00 (étape B de la figure 1 B) pour y être fragmenté et les ions fragments sont réinjectés vers la trappe (étape C de la figure 1 B) pour y être analysés (étape D de la figure 1 B). En mode MRM, on suivra toujours une transition, mais alors, d'où provient l'intérêt d'utiliser ce mode dans notre travail ? Comme il sera possible de le découvrir dans la deuxième partie de ce chapitre intitulée "Higher-energy Collision Dissociation for the quantification by liquid chromatography/tandem ion trap mass spectrometry of nitric oxide metabolites coming from S-nitrosoglutathione in an in vitro model of intestinal barrier" produite dans Rapid Communications in Mass Spectrometry, la différence entre CID et HCD peut être de taille. En effet, au fur et à mesure qu'on pénètre à l'intérieur du spectromètre, l'atmosphère de la source fait rapidement place au vide et dans la trappe, la collision en CID se fait avec un gaz inerte (hélium), a priori sans autre réaction qu'une simple fragmentation. Il n'en va pas de même avec le mode HCD pour lequel, le retour en arrière de l'ion s'accompagne de collisions avec les vapeurs de solvants issus de la source et principalement les solvants composant la phase mobile chromatographique. 1.

Experimental paper: "Higher-energy Collision Dissociation for the quantification by liquid chromatography/tandem ion trap mass spectrometry of nitric oxide metabolites coming from S-nitrosoglutathione in an in vitro model of intestinal barrier" in Rapid Communications in Mass Spectrometry (under minor revision)

	1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter 1 st Chapter
	Detailed procedures used for experiments in this article are present in protocols (see
	appendices) entitled "Measurement of nitrite, nitrate ions and S-nitrosothiols using the
	2,3-diaminonaphthalene assay by LC coupled with tandem mass spectrometry" and
	"Intestinal permeability studies of S-nitrosothiols using a Caco-2 cell monolayer
	model".
	94 98 111

Table 1 .

 1 Comparison of the physiological parameters in different parts of the gut (Bilat

	et al. 2017)
	Region	Average length (m)	Absorbing surface area (m 2 )	pH	Residence time	Mucus thickness	a	Bacterial cells (cells/mL)
								10 1 -10 4
	Duodenum 0.25-0.30	0.09	5.7-6.8	≈ 40 min	FAL: 16 ± 3 LAL: 154 ± 39	Aerobes and facultative
								anaerobes
								10 4 -10 8
	Jejunum	3	60	6.6-7.0	2-3 h	FAL: 15 ± 2 LAL: 108 ± 5	Facultative anaerobes and
								aerobes
								10 4 -10 8
	Ileum	3	60	7.0-7.3	3-4 h	FAL: 29 ± 8 LAL: 447 ± 47	Facultative anaerobes and
								aerobes
								10 10 -10 12
								Facultative
	Colon	1.5	0.3	5.7(caecum) to 6.6	16.6-19.0 h	FAL: 116 ± 51 LAL: 714 ± 109	aerobes to strict anaerobic
								bacteria (mainly
								Clostridia)
	needs two to three weekes to form the cell monolayer. The second step is to use an
	ex vivo model, taking advantage of the isolated tissue. Usually, O 2 -CO 2 (95:5, V/V)
	mixture bubbling is needed for tissue viability. This model is more relevant to the final
	real conditions than the in vitro model. The last step is performing experiments in vivo,

Abbreviations: LAL, loosely adherent layer; FAL, firmly attached layer. a Study performed in rats.

Usually, there are three steps to study intestinal permeability of drugs. The first step is to use an in vitro model, such as a monolayer of epithelial cells. This model is simple but does not fully correspond to the real conditions in the body; it can be improved by co-culturing colon cell lines HT-29-H or HT-29-MTX producing mucus

(Bilat et al. 2017)

. Noteworthy, the commonly used Caco-2 cell monolayer model but they are still suffering of drawbacks because of variations between animals and humans, and in human genotype and phenotype

nitrosoglutathione in rat intestine: roles of denitrosating enzymatic and chemical factors on absorption

2.3.2.2)

and other reagents of GSNO degradation (e.g. thiols) were studied. All related data are reported in the article draft "Degradation of Sand permeation". All experimental parts currently missing during manuscript deposit are indicated.

1.

Experimental paper: "Intestinal absorption of S-nitrosothiols: permeability and transport mechanisms" in Biochemical Pharmacology (155 (2018) 21-31)

	2.
	Detailed procedures used for experiments in this article are present in protocols (see
	appendices) entitled "Measurement of nitrite, nitrate ions and S-nitrosothiols using the
	2,3-diaminonaphthalene assay by spectrofluorometry" and "Intestinal permeability
	studies of S-nitrosothiols using a Caco-2 cell monolayer model".

Experimental paper: "Degradation of S-nitrosoglutathione in rat intestine: roles of denitrosating enzymatic and chemical factors on absorption and permeation" to be submitted in Drug Metabolism and Disposition after completion of additional experiments

	Detailed procedures used for experiments in this article are present in protocols (see
	appendices) entitled "Measurement of nitrite, nitrate ions and S-nitrosothiols using the
	2,3-diaminonaphthalene assay by spectrofluorometry", "Measurement of nitrite,
	nitrate ions and S-nitrosothiols using the 2,3-diaminonaphthalene assay by LC
	coupled with tandem mass spectrometry", "Measurement of nitrite, nitrate ions and
	S-nitrosothiols using the 2,3-diaminonaphthalene assay by LC coupled with tandem

mass spectrometry", "Measurement of γ-glutamyltransferase activity by spectrophotocolorimetry" and "Permeability studies of S-nitrosothiols in isolated rat intestine using Ussing chamber".

Detailed procedures used for the experiment concerning identification of protein disulfide isomerase (PDI) in rat intestine are present in protocols (see appendices) entitled "Western blot for protein disulfide isomerase identification in tissues".

Degradation of S-

nitrosoglutathione in rat intestine: roles of denitrosating enzymatic and chemical factors on absorption and permeation

	Yu Haiyan 1,* , Schmitt Romain 1,* , Leroy Pierre 1 , Gaucher Caroline 1 , Fries Isabelle 1 ,
	Chaimbault Patrick 2 , Sapin Anne 1,**
	1 Université de Lorraine, CITHEFOR, F-54000 Nancy, France
	2 Université de Lorraine, LCP-A2MC, F-570 00 Metz, France
	*Equal contribution to the present work

**Correspondence to: Sapin Anne, Université de Lorraine, CITHEFOR EA 3452, Faculté de Pharmacie, 5, rue Albert Lebrun, F-54001, NANCY, France (To be submitted to Drug Metabolism and Disposition (IF: 3.64) when the experimental part will be completed by other researchers of the lab)

Table 2 .

 2 Standard curves validation parameters for S-nitrosoglutathione (GSNO) and

	nitrite ions (NO 2 -). Mean ± SD; n ≥ 3.
	NO species	Linearity (6.25 -50 µM) Slope (a); intercept (b); determination coefficient (r 2 )	Concentration (µM)	Accuracy (%)	Precision (%) Intra-day Inter-day
			6.25	113.3	0.7	10.2
		a: 15308 ± 1160	12.5	97.0	8.2	11.9
	GSNO	b: -51365 ± 56834 r 2 : 0.996 ± 0.005	25 37.5	98.6 104.0	6.4 4.6	5.7 4.3
			50	100.3	4.5	5.6
			6.25	99.9	2.9	11.3
		a: 16572 ± 218	12.5	100.2	0.4	2.0
	NO 2 -	b: -40458 ± 29394	25	112.9	3.8	7.9
		r 2 : 0.995 ± 0.005	37.5	101.8	2.1	7.9
			50	98.4	6.5	4.7

Table 3 .

 3 Values of apparent permeability coefficient (P app ) for NOx species (RSNOs +

	NO 2 -+ NO 3 -), NO 2 -, RSNOs and NO 3 -molecular forms after 2 h of permeation through
	rat intestine; and recovery of NO species in Ussing chamber. nd: not determined.
	Mean ± sem of three independent experiments done in duplicate (GSNO:
	S-nitrosoglutathione; RSNOs: S-nitrosothiols; Glygly: glycylglycine; SBC:
	serine-borate complex; NEM: N-ethylmaleimide).
	Treatments		NOx	NO 2 -	P app (x 10 -6 cm.s -1 ) RSNOs	NO 3 -	Recovery (%)
	GSNO		5.3 ± 0.8	1.4 ± 0.2	0.3 ± 0.1	3.5 ± 0.8	96 ± 26
	GSNO + Glygly + MgCl	2	6.4 ± 1.7	4.0 ± 0.8	nd	2.4 ± 1.0	97 ± 18
	GSNO + Glygly + MgCl	2	3.4 ± 0.9	0.9 ± 0.3	2.4 ± 1.1	nd	97 ± 18
	+ SBC + NEM

Noteworthy, method using LC-ESI-ITMS/MS is needed to quantify permeated NO species, due to poor LOQ of conventional spectrofluorometry. Since LOQ of both methods are limited by blank, labeling drug candidate of GSNO with 15 N not only contributes to distinguish NO species from exo-and endo-genous RSNOs, but also results in studying GSNO with low concentration (e.g. 100 µM).

  .cm² for intestine). Caco-2 cell monolayer model has been well validated by both a high permeable molecule, i.e. propranolol, and a low permeable molecule, i.e.

	The influence of GSNO on the intestinal integrity was checked by measuring
	transepithelial electrical resistance (TEER) and calculating permeability values of
	related marker (sodium fluorescein) after GSNO treatment. As a matter of fact,
	Caco-2 cell monolayer model can be used for more than 4 h, but isolated rat intestine
	in Ussing chamber should not be used after 2 h due to a loss of tissue viability (TEER
	less than 30 Ω.cm²; work has been done by PhD students in EA 3452: Schmitt R. and
	Ming H.). Thus, the intestinal cells were firstly incubated with GSNO for 4 h, then
	cultured with sodium fluorescein for another 2 h; isolated rat intestine was incubated
	with GSNO and sodium fluorescein at the same time for 2 h.

2 cell monolayer model (experimental paper submitted to Rapid Communications in Mass Spectrometry in Chapter 1) (Bonetti et al. 2018), and for rat isolated intestinal model (experimental paper submitted to Rapid Communications in Mass Spectrometry in Chapter 1). Resulting data are shown in Table 1. The standard TEER values as a criterion to define integrity for both models are quite different (500 Ω.cm² for intestinal cells, while 30 Ω

Table 1 .

 1 Permeability of intestinal markers and S-nitrosothiols (RSNOs) in intestinal barrier models (GSNO: S-nitrosoglutathione; NACNO: S-nitroso-N-acetyl-cysteine;

	SNAP: S-nitroso-N-acetyl-penicillamine; TEER: Transepithelial electrical resistance;
	P app : Apparent permeability coefficient)
			Caco-2 cell monolayer model	Rat isolated intestinal model in Ussing chamber
		Samples	Initial concentration (µM)	Incubation time (h)	Papp (10 -6 cm.s -1 )	Initial concentration (µM)	Incubation time (h)	Papp -6 (10 cm.s -1 )
		Sodium fluorescein	-	-	-	1000	2	17.2 ± 1.3
	Model control	Furosemide Propranolol	100 50	1 1	0.08 -0.11 3.3 -41.9	-1000	-2	-*
		TEER (Ω.cm²)		730 ± 53			≥ 30
		GSNO	100	4	2.6 ± 0.3	100	2	-
	RSNOs permeability study	GS 15 NO NACNO	10 -100 100	1 4	2.3 ± 0.5 5.0 ± 0.7	100 -	2 -	7.1 ± 0.6 -
		SNAP	100	4	3.3 ± 0.5	-	-	-

*: under limit of quantification

Table 1 .

 1 15 N labeling is a powerful tracer methodology for a better understanding of nitrogen cycle process and metabolism of nitrogen oxides. Using MS to analyze 15 N labeled NO species can selectively monitor NO species released from GS 15 NO, and differentiate them from other sources (endogenous synthesis and diet). Summary of bioanalytical method performances for S-nitrosoglutathione (GS 15 NO)/ 15 N-nitrite ion measurement based on 2,3-diaminonaphthalene (DAN) derivatization (compositions of PBS/HBSS/Krebs solution are available in appendices)The bioanalytical work using 15 N labeling and LC-ESI-ITMS/MS is the preamble of future assays of NO species metabolized from labeled GS 15 NO or other NO donors in the cells or tissues. The transposition of our approach to other biological matrices (plasma, tissues, organs like aorta) using sample preparation protocols already described(Shin and Fung 2011)(Damacena-Angelis et al. 2016) constitutes a natural extension of the fields of application. Noteworwhy, sample pretreatment is a critical point. Consequently, SBC, NEM and EDTA are usually added to stabilize GSNO[START_REF] Tsikas | GC-ECNICI-MS analysis of S-nitrosothiols and nitroprusside after treatment with aqueous sulphide (S2-) and derivatization with pentafluorobenzyl bromide: Evidence of S-transnitrosylation and formation of nitrite and nitrate[END_REF] acetonitrile (Damacena-Angelis et al. 2016)

			Spectrofluorometry		LC-FL	LC-MS/MS
	Media	PBS, HBSS, Krebs solution
	Linearity range (nM)	100-2000		5-200
	Limit of quantification (nM) Based on S/B	100			5
	Throughput	ca. 3 min for 96 samples		15 min per sample	20 min per sample
	Storage	r.t. for 2 days	-80 °C for 3 months (Freezing/defreezing for two times)
		Initial
		GS 15 NO
		deposit	≥ 50		≥ 50	≥ 5 (5 -50)
	Application to Caco-2	amounts (nmol)
	cell monolayer	Incubation time (h)	1/4/24		1/4/24	≥ 1
		Quantification spots	Apical and basolateral compartments	Apical and basolateral compartments	Apical and basolateral compartments
		Initial
	to isolated Application	(nmol) GS 15 NO deposit range	> 250		> ca. 125	> ca. 50
	intestine in Ussing	Incubation time (h)	2		2	2
	chamber	Quantification spots	Donor and acceptor compartments	Donor compartment and intestine*	Donor and acceptor compartments
	*: to measure S-nitrosothiols (RSNOs) and NO 2	-in intestine, see protocol entitled
	"Measurement of S-nitrosothiols in the rat intestine with the 2,3-diaminonaphthalene assay by LC
	coupled with fluorescence detection".

Performances of bioanalytical methods for GS 15 NO/ 15 N-nitrite ion measurement based on DAN assay is summarized in Table

1

. Compositions of PBS, HBSS and Krebs solution are available in the protocol entitled "Measurement of nitrite, nitrate ions and S-nitrosothiols using the 2,3-diaminonaphthalene assay by spectrofluorometry".

Table 1 .

 1 Resulting calibration curves obtained in the different media without subtracting the blank signal (r 2 ≥ 0.99, n ≥ 3)

	Medium	Fluorescence intensity (AU) of key points (Mean ± SD)	Equation of calibration curve
		Nitrite	GSNO	(Mean ± SD)
		Blank: 433 ± 69	Blank: 378 ± 53	Nitrite: (1810 ± 138)x + 465 ± 73
	PBS	0.1 µM: 635 ± 55	0.1 µM: 632 ± 89	Nitrate: -
		2 µM: 4083 ± 209	2 µM: 3591 ± 163	GSNO: (1549 ± 156)x + 498 ± 173
		Blank: 495 ± 76	Blank: 533 ± 18	Nitrite: (1846 ± 143)x + 585 ± 88
	Krebs	0.1 µM: 797 ± 52	0.1 µM: 603 ± 126	Nitrate: -
		2 µM: 4373 ± 307	2 µM: 4677 ± 300	GSNO: (2262 ± 34)x -204 ± 83
		Blank: 468 ± 66	Blank: 380 ± 24	Nitrite: (1527 ± 96)x + 435 ± 89
	HBSS	0.1 µM: 572 ± 67	0.1 µM: 592 ± 83	Nitrate: (264 ± 22)x + 1719 ± 116
		2 µM: 3518 ± 230	2 µM: 3436 ± 253	GSNO: (1451 ± 236)x + 399 ± 98
		Blank: 364 ± 46	Blank: 368 ± 41	Nitrite: (891± 55)x + 510 ± 87
	Lysis buffer	0.25 µM: 668 ± 99	0.25 µM: 581 ± 40	Nitrate: -
		2 µM :2279 ± 103	2 µM: 1954 ± 96	GSNO: (787 ± 49)x + 337 ± 58
	Nitrate in HBSS: blank, 1652 ± 188; 0.75 µM, 1825 ± 152; 25 µM, 8322 ± 512

105 mM DAN with 1.05 mM HgCl 2

  The resulting NAT adduct is measured by a reversed phase LC method coupled with tandem mass spectrometry to differentiate the NAT adducts labeled Mix 1 volume of 0.316 mM DAN solution, 1 volume of 3.17 mM mercuric chloride solution and 1 volume of 0.6 M HCl. Prepare daily.

	2.3. 0.316 mM DAN
	Weigh 5 mg of DAN (Mw = 158 g.mol -1 ; ref Sigma D-2757) in a 100 mL
	volumetric flask, dissolve and adjust the volume with 0.6 M HCl. Store at 4°C
	protected from light for 1 month. Aliquot in 1 mL fractions.
	2.4. 0.105 mM DAN
	Dilute 3-fold of 0.316 mM DAN solution with 0.6 M HCl (500 µL of 0.316 mM
	DAN + 1000 µL of 0.6 M HCl). Prepare daily.
	2.5. 3.17 mM HgCl 2
	Weigh 86.0 mg of HgCl 2 (Mw = 271.5 g.mol -1 ; ref VWR 25384) in a 100 mL
	volumetric flask, dissolve and adjust the volume with 0.6 M HCl. Store at 4°C
	protected from light for 1 month. Aliquot in 1 mL fractions.
	2.6. 0.
	either	with	14 N	or	15 N,	as	already	reported	in	Table	1.

. Calibration curves and quality controls for 15 NO 2 -and RS 15 NO determination

  Na 15 NO 2 /GS 15 NO daughter solution n° 2 (2.10 -6 M): transfer 500 µL of the Na 15 NO 2 /GS 15 NO daughter solution n°1 in a 25 mL volumetric flask, adjust the volume with sample buffer. Mix gently for one time.200 nM Na 15 NO 2 /GS 15 NO is prepared by mixing 0.1 mL of 2.10 -6 M Na 15 NO 2 /GS 15 NO and 0.9 mL of HBSS.

	100 nM Na 15 NO 2 /GS 15 NO is prepared by mixing 0.6 mL of 200 nM
	Na 15 NO 2 /GS 15 NO and 0.6 mL of HBSS.
	Lower Na 15 NO 2 /GS 15 NO concentrations are prepared as follows:
	Tube n°		1	2	3	4	5	6
	Na 15 NO 2 /GS	15 NO solution
				0	50	100	200	500	320
	(100 nM) (μL)
					2.10 -6 M): defreeze 1.24 mM NAT solution,
	sonicate for 1 min, transfer 2 mL to a 200 mL volumetric flask, add 2.96 mL of HBSS (μL) 1000 950 900 800 500 0
	CH 3 CN-H 2 O (25/75, V/V), and adjust the volume with ultrapure water to obtain 0.012 [ 15 NO 2 -]/[GS 15 NO] (nM) -5 10 20 50 100
	mM NAT. Next, transfer 1.65 mL of 0.012 mM NAT to a 10 mL volumetric flask and
	adjust the volume with ultrapure water. 180 nM Na 15 NO 2 /GS 15 NO as the quality control is prepared by mixing 0.9 mL of
	The dilutions for the calibration curve are prepared in 1.5 mL Eppendorf tubes, 200 nM Na 15 NO 2 /GS 15 NO and 0.1 mL of HBSS.
	as follows: 50 nM Na 15 NO 2 /GS 15 NO as the quality control is prepared by mixing 0.5 mL of
	Tube n° 100 nM Na 15 NO 2 /GS 15 NO and 0.5 mL of HBSS. 1 2 3		4	5
	Pure NAT daughter solution (2.10 15 nM Na 15 NO 2 /GS 15 NO as the quality control is prepared by mixing 0.15 mL of 50 500 μL of n° 1 200 μL of n° 2 500 μL of n° 3 500 μL of n° 4 -6 M) [μL] 100 nM Na 15 NO 2 /GS 15 NO and 0.85 mL of HBSS.
	Ultrapure water [μL]	950	500	800		500	500
	[NAT] (nM)	100	50	10		5	2.5
	3.2Na 15 NO 2 /GS 15 NO daughter solution n°1 (10 -4 M): transfer 100 µL of 10 -2 M
	Na 15 NO 2 /GS 15 NO solution in a 10 mL volumetric flask, adjust the volume with PBS.
	Mix gently for one time.

Table 2 .

 2 Inter-day and intra-day variability of quality control samples for 15 N-nitrite

	S-nitrosoglutathione (GS 15 NO) and 15 N-nitrate analysis with IS. Accuracy was
	determined as the deviation (%) of calculated concentrations from the nominal
	concentrations. Precision was determined as the relative standard deviation (RSD) of
	the 6 measurements.
			Intra-day	Inter-day
	NO species Concentration (nM)
			Accuracy (%)	Precision (%)	Accuracy (%)	Precision (%)
		15	105.2 ± 6.4	6.1	104.7 ± 7.0	6.7
	15 N-nitrite	50	99.0 ± 6.8	6.9	94.7 ± 2.8	3.0
		180	98.6 ± 5.3	5.4	102.7 ± 7.3	7.1
		15	98.1 ± 13.1	13.4	93.3 ± 11.8	12.6
	GS 15 NO	50	99.1 ± 10.9	11.0	96.0 ± 7.4	7.7
		180	100.0 ± 12.7	12.7	101.5 ± 11.4	11.3
		300	105.2 ± 6.4	6.1	104.7 ± 7.0	6.8
	15 N-nitrate	2500	99.0 ± 6.8	6.9	94.7 ± 2.8	8.7
		4000	98.6 ± 5.3	5.4	102.7 ± 7.3	8.1

. 0.105 mM DAN with 1.05 mM HgCl 2

  IU.kg -1 heparin Choay, penis vein and sacrificed by exsanguination)).Rinse quickly with 1 mL cold PBS for 2 times, use tissue paper to dry the intestine each time; freeze the rat intestine with liquid N 2 . Smash the intestine in liquid N 2 with a

	Mix 1 volume of 0.316 mM DAN solution, 1 volume of 3.17 mM mercuric chloride solution and 1 volume of 0.6 M HCl. Prepare daily. 2.7. 10 M NaOH Weigh 10 g of NaOH (Mw = 40 g.mol -1 , ref Prolabo-28252.293) and dissolve in 25 mL of ultrapure water, use ice to cool the mixture. 2.8. 10 -2 M sodium nitrite Solutions are prepared, titrated and stored according to protocol entitled "Batch synthesis of S-nitrosoglutathione". 2.10. 1.24 mM pure NAT Weigh 21 mg NAT (Mw = 169.2 g.mol -1 ; ref Chemodex 269-12-5; purity: 98.2 %) in a 100 mL volumetric flask, dissolve in 10 mL of methanol and adjust the volume with 0.1 M HCl. Divide into aliquots of 2 mL and store at -80°C for 1 year. 2.11. Mobile phase CH 3 CN-CH 3 COONH 4 (10 -2 M, pH = 7.2) (28/72, V/V) Dilute 0.57 mL of glacial acetic acid solution (volumetric mass = 1.05 g/mL; Mw = 60.05 g.mol -1 ; ref Sigma 64-19-7) with 1 L of ultrapure water, and adjust the pH g/mL; Mw = 17.03 g.mol -1 ; ref Sigma 1336-21-6); mix 280 mL acetonitrile and 720 mL ammonium acetate solution and filter with 0.45-µm HV membrane (Durapore®). 3. Methods 3.1. Calibration curves for NO 2 -and GSNO determination 50 µM NaNO 2 or GSNO solutions are prepared by transferring 50 µL of 10 -2 M NaNO 2 or GSNO in a 10 mL volumetric flask. Adjust the volume with Tris buffer. Further dilutions for the calibration curve are prepared in 1.5 mL Eppendorf tubes (mix gently each tube for one time), as follows: Tube n° 1 2 3 4 5 6 NaNO 2 /GSNO solution (50 µM) (μL) 0 125 250 500 750 1000 Tris buffer (μL) 1000 875 750 500 250 0 [NO 2 -]/[GSNO] (µM) -6.25 12.5 25 37.5 50 Remove Wistar rat intestine (length: around 2 cm, weight: 100 ± 5 mg) after injection, Sanofi Santé Nutrition Animale, Libourne, France); administrated with heparin (1000 pestle in a mortar, weigh 20 ± 2 mg intestine powder in a 2-mL Eppendorf tube. Spike the powder with 20 µL of NaNO 2 or GSNO standard solutions. Add 600 μL DAN or DAN/Hg 2+ in 0.6 M HCl, followed by vortex for 30 s. Incubate the mixture in the dark in an incubator (37 °C, Jouan IG150) for 10 min under rotation (80 rpm, MX-RD-E Classic Rotator, Labtech, Cernusco, Italy), add 40 µL of 10 M NaOH, mix by vortex for 10 s, add 600 µL acetonitrile, mix by vortex for 30 s, and centrifuge at 18,000 x g for 15 min at 4 °C (VWR Hitachi, CT15RE); collect the supernatant and store at 4 °C for a Weigh 34.5 mg of sodium nitrite (NaNO 2.9. S-nitrosoglutathione (GSNO) to 7.2 with 10 times diluted ammonia solution (ca. 25% NH 3 ; volumetric mass = 0.91 maximum period of 24 h before measurement by LC-FL.
	sacrifice (anesthetized with sodium pentobarbitone (60 mg.kg -1 , intraperitoneal

2 ) (Mw = 69 g.mol -1 ; ref Merck 6549) in a 50 mL volumetric flask, dissolve and adjust the volume with PBS. Mix the solution gently for one time. Prepare daily.

Table 1 .

 1 Validation data for GSNO quantification in the rat intestine

	NO	Linearity (6.25 -50 µM, n ≥ 3)	Concentration		Precision (%) (n ≥ 3)
	species	Slope (a) ; intercept (b) ;		(µM)	Accuracy (%)
		determination coefficient (r	2 )			Intra-day	Inter-day
					6.25	113.3	0.7	10.2
		a : 15308 ± 1160		12.5	97.0	8.2	11.9
	GSNO	b : -51365 ± 56834
					25	98.6	6.4	5.7
		r	2 : 0.996 ± 0.005
					37.5	104.0	4.6	4.3
					50	100.3	4.5	5.6
					6.25	99.9	2.9	11.3
			a : 16572 ± 218		12.5	100.2	0.4	2.0
	Nitrite	b : -40458 ± 29394		25	112.9	3.8	7.9
		r	2 : 0.995 ± 0.005		37.5	101.8	2.1	7.9
					50	98.4	6.5	4.7

Table 1 .

 1 Measurement of purified GGT activity at different times (n = 3)

	Incubation time (min)	0	10	20	30
	Group 1 (blank)			Substrate without GGT
	Group 2			Substrate with GGT

Table 2 .

 2 Measurement of intestine GGT activity at different time intervals

	Incubation time (min)	0	10	20	30
	Group 1 (blank)		Substrate without intestine
	Group 2			Substrate with intestine
	Group 3		Without substrate but with intestine

On appelle « transition », le couple des rapports m/z suivis en Q 1  Q 3 . L'utilisation typique de ce mode de fonctionnement est le dosage à l'état de trace. La technique de fragmentation induite par collision décrite s'appelle CID pour « Collision ou Collisionally Induced Dissociation ». C'est la technique classiquement utilisée sur des spectromètres QqQ. Le mode MRM issu d'une CID peut aussi être utilisé sur des spectromètres de type trappe d'ions (ITMS pour IonTrap Mass Spectrometer). Ces spectromètres, plus intéressants pour des approches structurales sont un peu moins performants en termes de limite de quantification que leurs homologue QqQ. Dans le cas d'un ITMS, l'analyseur IT permet à la fois la sélection de l'ion moléculaire, sa fragmentation et l'analyse des fragments générés. Si cette description un peu longue vient d'être faite, c'est que pour bien comprendre le travail qui va suivre, il faut expliquer maintenant l'originalité du spectromètre de masse dont nous disposons. Il s'agît d'un appareil ITMS que le constructeur a doté de plusieurs techniques de fragmentation dont le CID bien entendu mais aussi de la technique dite HCD pour « Higher-energy Collision Dissociation ». Cette dernière fait l'originalité de la technique de dosage par chromatographie en phase liquide (LC) que nous avons couplée à notre spectromètre de masse à trappe d'ion en mode tandem (ITMS/MS). L'ionisation des composés à la sortie de la colonne chromatographique aura lieu par électronébulisation (ESI) comme les travaux déjà publiés dans le domaine (McAlister et al. 2011). Il convient donc maintenant de décrire le fonctionnement des ces deux modes sur notre appareil dont la conception est présentée à la figure 1 B.

Acknowledgements Acknowledgments

The authors acknowledge the program of China Scholarship Council. The authors also thank Dr Philippe Giummelly (CITHEFOR, Université de Lorraine) for the synthesis of GS 15 NO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the present manuscript.

Abbreviations

Abbreviations

Full names

Measurement of S-nitrosothiols in the rat intestine with the 2,3-diaminonaphthalene assay by LC coupled with fluorescence detection

Principle

The present analytical method is aimed at being applied to experiments on rat intestine isolated. S-nitrosothiols (RSNOs) were measured by the 2,3-diaminonaphthalene/mercuric ions (DAN/Hg 2+ ) assay; the derivatization step was directly operated on smashed tissue as previously described (Shin and Fung 2011) (Damacena-Angelis et al. 2016), and resulting naphthotriazole (NAT) adduct extracted with acetonitrile was analyzed by reversed phase LC coupled with spectrofluorimetric detection (LC/FL). The method should be further applied to other tissues and organs by using LC coupled with tandem mass spectrometry (LC-MS/MS) after GSNO labeling with 15 N.

resistance (TEER) value with electrodes (Electrode kit for Ussing chamber, WPI).

Discard tissue samples that have a TEER value less than 30 Ω.cm 2 directly after having placed the intestine.

Replace the Krebs solution in acceptor compartment with 2.5 mL of fresh one and the solution in donor compartment with 2.5 mL of RSNOs solution (e.g. 100 µM)

or Krebs solution. Next, incubate intestine at 37 °C with bubbling for maximum 2 h, harvest 0.25 mL of the medium from acceptor compartment at regular time intervals and replace it with pre-heated buffer. Measure also TEER values at these same times.

Quantification of nitrite, nitrate ions and RSNOs in both compartments

See protocol entitled "Measurement of nitrite, nitrate ions and S-nitrosothiols using the 2,3-diaminonaphthalene assay by spectrofluorometry" and "Measurement of nitrite, nitrate ions and S-nitrosothiols using the 2,3-diaminonaphthalene assay by LC coupled with tandem mass spectrometry".

Quantification of nitrite ion and RSNOs in the rat intestine

See protocol entitled "Measurement of S-nitrosothiols in the rat intestine with the 2,3-diaminonaphthalene assay by LC coupled with fluorescence detection".

Quantification of proteins in the rat intestine

Quantify intestinal proteins with BCA assay, see procedure 3.2 from protocol entitled "Western blot for protein disulfide isomerase identification in tissues".

Calculations

Calculate the cumulative amounts of RSNOs permeated through the intestine by the concentrations measured in the acceptor compartment. Calculate the apparent permeability coefficient (P app ) values using the following equation:

dQ/dt (mol•s -1 ) refers to the quantity of permeated NO related species (dQ) in the acceptor compartment at the time of quantification (dt), A refers to membrane diffusion 2.3. 0.5 M Tris buffer (pH = 6.8) Dilute 3-fold 1.5 M Tris buffer with ultrapure water (3 mL of 1.5 M Tris buffer + 6 mL of ultrapure water); and adjust the pH to 6.80 ± 0.05.

Laemmli Lysis-buffer

Use commercial Laemmli Lysis-buffer (ref Sigma 38733) made of 0.004 % bromophenol blue, 400 mM dithiothreitol glycerol (DTT), 20 % glycerol, 4 % SDS, and 0.125 M TRIS.

Lysis buffer

Dissolve 1 Medium Complete Mini (Roche) tablet in 7.8 mL of ultrapure water, add reagents as indicated in the following table and vortex for 0.5 min. Store at 4 °C for maximum 2 weeks.

Quantification of purified azo adduct by spectrophotometry

The purified azo adduct is 20-fold diluted with ultrapure water (50 µL sample in 1-mL ultrapure water), followed by spectrophotometry (UV-1800, Shimadzu, Lognes, France) at wavelength of 540 nm. Nitrite concentration is calculated by the Beer-Lambert law (A = ԑ x l x c, ԑ = 37700 ± 5000 M -1 .cm -1 ).

Operating conditions for purified NAT quantification

Purified NAT is diluted for 50-fold in mobile phase, 2 µM nitrite (1.5 µM NAT) in En outre, des études sur les mécanismes de dénitrosation du GSNO ont été menées sur intestin isolé, démontrant en particulier le rôle d'enzymes telles que la γ-glutamyltransférase et la protein disulfide isomerase.

Mots clés : donneurs de NO; S-nitrosothiols; azote 15; dérivation; chromatographie en phase liquide; spectrométrie de masse tandem.

Oral bioavailability studies of S-nitrosoglutathione using intestinal barrier models by liquid chromatography coupled with mass spectrometry after labeling with the nitrogen isotope 15

Abstract: The development of innovative nitric oxide (NO) donors for the chronic treatment of cardiovascular diseases implies their bioavailability studies after oral administration. S-nitrosothiols (RSNOs) look interesting drug candidates for this purpose and evaluating their intestinal permeability appears the first step to be realized. Thus, an analytical method offering high sensitivity is needed; moreover this method should be selective by differentiating between the endogenous production of NO, the intake of nitrite and nitrate ions via the diet, and the drug itself. Our work consisted in using S-nitrosoglutathione (GSNO) labeled with the stable nitrogen isotope 15 ( 15 N) as a model. Released 15 NO species were derivatized by two conventional methods: Griess method leading to the formation of an azo adduct; reaction with 2,3-diaminonaphthalene (DAN) producing 2,3-naphtotriazole (NAT); fragmentation studies of the two adducts by tandem mass spectrometry (MS/MS) allow the selection of DAN method because it provides the highest sensitivity. An original transition resulting from the NAT fragmentation in Higher-energy Collisional Dissociation (HCD) mode instead of the conventional Collisionally Induced Dissociation (CID) mode was pointed out and permitted to reach a limit of quantification of 5 nM (20 fold less than when using fluorescence). The LC-MS/MS method was validated and applied to the GS 15 NO intestinal permeability studies with two models: in vitro (a monolayer of Caco-2 epithelial cells), and ex vivo (isolated intestine of rat (ileum) in an Ussing chamber). The apparent permeability values calculated with concentrations of GS 15 NO metabolites (nitrite, nitrate ions and RSNOs) classify it as a middle permeable drug. Studies on GSNO denitrosating processes using isolated rat intestine demonstrate that the enzymes γ-glutamyltransferase and protein disulfide isomerase play a pivotal role.

Keywords:

NO donors; S-nitrosothiols; nitrogen 15; derivatization; liquid chromatography; tandem mass spectrometry.