Kristofer F Nilsson
email: kristofer.nilsson@ki.se

Per Michael Lundgren
email: michael.lundgren@akzonobel.com

L Christofer Agvald
email: per.agvald@sll.se

Dag Adding

Lars E Linnarsson

Gustafsson

L Christofer Adding

Dag Linnarsson
email: dag.linnarsson@ki.se

Lars E Gustafsson
email: lars.gustafsson@ki.se

Formation of new bioactive organic nitrites and their identification with gas chromatography-mass spectrometry and liquid chromatography coupled to nitrite reduction

Keywords: nitric oxide donors, nitrites, pulmonary circulation, hypertension, pulmonary, chromatography, high pressure liquid

Formation of new bioactive organic nitrites and their

identification with gas chromatography-mass spectrometry and liquid chromatography coupled to nitrite reduction.

Introduction

Nitric oxide donors are used to supply a patient with exogenous nitric oxide (NO) and can be used in conditions and diseases where increased NO is thought to be beneficial. There is a wide range of NO donors with different chemistry and biochemistry available for experimental use [START_REF] Wang | Nitric oxide donors: chemical activities and biological applications[END_REF]. The list of conditions and diseases is extensive for where experimental studies have suggested beneficial effects, but only a few compounds are used in clinical practice [START_REF] Miller | Recent developments in nitric oxide donor drugs[END_REF]. Nitroglycerin and isosorbide mononitrate are used in acute and chronic anginal pain respectively and nitroglycerin may be used in the treatment for anal fissure [START_REF] Miller | Recent developments in nitric oxide donor drugs[END_REF].

Isosorbide dinitrate together with hydralazine is used for heart failure in African-Americans [START_REF] Miller | Recent developments in nitric oxide donor drugs[END_REF]. Inhaled amyl nitrite has been used in anginal pain and as an antidote in life-threatening cyanide intoxication [START_REF] Lavon | Does amyl nitrite have a role in the management of pre-hospital mass casualty cyanide poisoning?[END_REF]. Organic nitrates and organic nitrites are converted to NO in vivo which can be seen as increased exhaled NO and decreased systemic blood pressure [START_REF] Persson | Detection of nitric oxide in exhaled air during administration of nitroglycerin in vivo[END_REF][START_REF] Cederqvist | Direct demonstration of NO formation in vivo from organic nitrites and nitrates, and correlation to effects on blood pressure and to in vitro effects[END_REF]. A significant clinical problem with organic nitrates is the rapid tolerance development which can be demonstrated experimentally with decreased generation of NO to exhaled air [START_REF] Agvald | Nitric oxide generation, tachyphylaxis and cross-tachyphylaxis from nitrovasodilators in vivo[END_REF][START_REF] Husain | Exhaled nitric oxide as a marker for organic nitrate tolerance[END_REF].

Organic nitrites exhibit little or no tolerance [START_REF] Bauer | Vascular and hemodynamic differences between organic nitrates and nitrites[END_REF], and recently ethyl nitrite has been suggested as an inhaled therapeutic [START_REF] Moya | Inhaled ethyl nitrite gas for persistent pulmonary hypertension of the newborn[END_REF]. However, the use of ethyl nitrite and other organic nitrites have been questioned due to significant methaemoglobin formation and other serious adverse effects [START_REF] Abman | Inhaled ethyl nitrite gas for persistent pulmonary hypertension in infants[END_REF][START_REF] Lavon | Does amyl nitrite have a role in the management of pre-hospital mass casualty cyanide poisoning?[END_REF]. Sodium nitroprusside is used in hypertensive crises but is associated with tolerance development and risk of cyanide intoxication and its use is sometimes therefore discouraged [START_REF] Varon | Treatment of acute severe hypertension: current and newer agents[END_REF]. Inhaled NO is used in persistent pulmonary hypertension of the newborn [START_REF] Clark | Low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. Clinical Inhaled Nitric Oxide Research Group[END_REF] whereas use of inhaled NO in acute lung injury and acute respiratory distress syndrome is not recommended [START_REF] Afshari | Inhaled nitric oxide for acute respiratory distress syndrome (ARDS) and acute lung injury in children and adults[END_REF]. Other conditions where treatment with exogenous NO has been suggested from experimental studies include myocardial ischemia reperfusion [START_REF] Schulz | Nitric oxide in myocardial ischemia/reperfusion injury[END_REF][START_REF] Bolli | Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research[END_REF], splanchnic ischemia reperfusion and acute intestinal inflammation [START_REF] Lefer | Nitric oxide. II. Nitric oxide protects in intestinal inflammation[END_REF], stroke [START_REF] Willmot | The potential of nitric oxide therapeutics in stroke[END_REF],

A c c e p t e d M a n u s c r i p t Page 5 of 50 pulmonary hypertension [START_REF] Tilton | Therapeutic potential of endothelin receptor antagonists and nitric oxide donors in pulmonary hypertension[END_REF] and sepsis [START_REF] Lamontagne | Nitric oxide donors in sepsis: a systematic review of clinical and in vivo preclinical data[END_REF]. The reasons for the lack of development of new NO donors that actually reach clinical practice are complex and may include side-effects in the form of systemic hypotension, headache, lack of specificity, tolerance development and toxicity [START_REF] Miller | Recent developments in nitric oxide donor drugs[END_REF]. Altogether there is a quest for new NO donors [START_REF] Miller | Recent developments in nitric oxide donor drugs[END_REF]. We believe that there is a specific need for intravenous NO donors with selectivity for the pulmonary circulation and that can be used, for example, in acute pulmonary embolism, where recent data indicate a protective role of endogenous NO [START_REF] Nilsson | Increase in exhaled nitric oxide and protective role of the nitric oxide system in experimental pulmonary embolism[END_REF]. We hypothesised that new lung-selective NO donors can be identified by utilizing exhaled NO as measure for pulmonary NO donation and systemic arterial pressure to monitor hypotension and tolerance development. We find that NO donors are formed, in a solution suitable for intravenous delivery, when deoxygenated aqueous solutions of alcohols and carbohydrates are reacted with NO gas and that these NO donors have different selectivity towards the pulmonary and systemic circulations.

Material and Methods

Preparation of reaction products between compounds and nitric oxide gas.

Aqueous solutions of alcohols and sugars and related compounds (please refer to Table 1 and supplementary Table A.1) were prepared and placed in gas tight cylinders (volume 20-150 ml) with taps in both ends and a sideway port with a septum plug (F-145, Alltech Biotechnology, Ireland) in the middle portion. The cylinders were filled to 75% with solution, allowing a gaseous head-space. The solutions were deoxygenated by means of helium bubbling for 10 min and purged with pure nitric oxide gas for 3 min. Samples and infusion solutions were drawn via the septum plug.

The HPLC system

The following reverse phase HPLC systems were used in all the analyses: a C 18 column (ReproSil-Pur 120 C 18 -AQ, 4x150 mm, 5 µm, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany or Nova-Pak C18, Radial-Pak 8x100 mm, 4 µm, Waters Inc., Milford, MA, USA) or a CN column (Nova-Pak CN, 8x100 mm Radial-Pak, 4 µm, Waters Inc.), two pumps (ConstaMetric III; LDC/Milton Roy, Riviera Beach, FL, USA), an injector (9125; Rheodyne, Cotati, CA, USA) with an injector loop of 2.2 ml, a variable UV-absorbance detector measuring at 227 nm or 254 nm (SpectroMonitor III; LDC/Milton Roy), and a scanning UV absorbance detector (2140 Rapid Spectral Detector; LKB, Bromma, Sweden).

The absorbance from the SpectroMonitor III detector was recorded and the pumps were controlled by commercial hardware and software (CSW32; DataApex, Prague, The Czech Republic) and the signal from the scanning detector was recorded by Wavescan software (LKB) run in DosBox (open source; www.dosbox.com). Samples and standards were injected at volumes from 2 µl to 2 ml. The flow rate of the mobile phase was 0.5-1 ml min -1 depending on the size of the column and consisted of A) 5-10 mM ammonium formate in water followed by increasing proportions of B) methanol or acetonitrile. Isocratic elution or a linear gradient starting with 100% A and with an increase of 3% min -1 of B was used. The effluent from the HPLC system could then either be sampled or continuously be passed into a nitrite reduction system for online measurement of NO/NO 2 -/organic nitrites (see section 2.3). Aliquots or effluent from the HPLC were intermittently respectively continuously injected in a temperature-controlled reaction chamber (total volume 300 ml) containing 75 ml of 1% (w/v) sodium iodide in hot (89° C) acetic acid. Inorganic and organic nitrites are reduced to NO in this environment but not inorganic nitrate and organic nitrates [START_REF] Walters | A rapid method for the determination of nitrate and nitrite by chemiluminescence[END_REF]. The reaction solution was purged with N 2 -gas (200 ml min -1 ) to deoxygenate the solution and to carry NO gas to a chemiluminescence NO analyzer system. In the NO analyzer ozone was produced in an oxygen flow of 100 ml min -1 and made to react in a vacuum chamber with the NO in the incoming gas from the reducing chamber (sample flow rate 150-200 ml min -1 ). In the NO+ozone reaction, excited NO 2 is generated which emits one photon per molecule. The photons are counted by a photomultiplier tube and are linearly proportional to the NO content in the incoming gas. The signal from the photomultiplier tube was collected by a computerized data acquisition system (MP100 system with AcqKnowledge software, BIOPAC Inc., Goleta, CA, USA). The system was intermittently calibrated with aliquots (10-500 µl) of known amounts of sodium nitrite either by direct injection or via the HPLC system (detection limits: 5 pmol and 10 pmol respectively). The sensitivity of the photomultiplier tube was adjusted in order to measure in the linear range. Quantification of NO content was made by comparing peak area measurements with the peak area of a known amount of standard. In experiments where the HPLC effluent was introduced into the reaction chamber, the volume in the reaction chamber increased slowly and intermittent withdrawal of liquid was necessary. In order to minimise the interference of different volumes in the chamber, calibration was always done before and after each analysis. These calibrations were very similar and the mean between the two sets was used in the calculation of quantity. The HPLC together with the online continuous nitrite reduction is henceforth referred to as LC-NO. ). The gas chromatograph was an Agilent 6890 Series GC system with a 7673 autosampler (Agilent Technologies, Inc.). Data were collected and analyzed with an Enhanced Chemstation G1701

Online measurement of NO/NO

Gas chromatography coupled to mass spectrometry

BA computer program with a Wiley 7n library of mass spectra (Agilent Technologies, Inc.).

Alternatively, 1 µl of reaction mixture consisting of 25-100 % propanol and NO gas was injected directly in the gas chromatography column thus bypassing the extraction procedure and the autosampling system. Commercial propyl nitrite was diluted in toluene and injected in the same way for comparison. In addition, 0.5 µl samples of 25-100% 1,2-propanediol treated with NO gas were injected in the gas chromatography-mass spectrometry (GC-MS) system, now equipped with an Agilent DB-1701 column (0.25 mm i.d., 30 m length, 0.25 µm phase thickness, Agilent Technologies, Inc.), in a total He flow of 30 ml min -1 , split fraction 1:5.

Temperature gradient was as above. Mass spectrometry was either performed in electron impact mode, or in chemical ionisation mode using a chemical ionisation interface supplied with a methane flow. The experiments were approved by the local animal ethics committee. Male white New Zealand rabbits (body weight: 2.0-3.5 kg, n=34) were anaesthetized with sodium pentobarbital via an ear vein (6 mg ml -1 in saline, 50-70 mg kg -1 ). Another catheter was inserted in the right jugular vein for administration of drugs. In experiments where the NO donating capabilities of the NO solutions were tested the preparation was complete at this point and the animals were allowed a 30-60 min intervention free period. In experiments with pharmacologically induced pulmonary hypertension a mid thoracotomy was performed and a flow probe around the ascending aorta (T201 system, Transonic Systems Inc., Ithaca, NY, USA) measured cardiac output. A heparinised catheter was introduced via the left jugular vein and advanced to the pulmonary artery for measurement of pulmonary artery pressure. Another catheter was inserted through the left atrial wall for left atrial pressure measurements, and central venous pressure was measured via the catheter in the right jugular vein. Pulmonary vascular resistance (PVR), systemic vascular resistance (SVR) and PVR/SVR index were calculated with the common formulas.

Anaesthesia and surgical preparation

After the surgery, the animals were allowed a 30-60 min intervention-free period to reach stable circulatory conditions and exhaled NO values. A continuous infusion containing glucose (26 g l -1 ), dextran 70 (Macrodex, 28 g l -1 ) and NaHCO 3 (6.6 g l -1 ) was administered.

In experiments where the NO donating capabilities of the NO solutions were tested sodium ) was added to the infusate which was given at a rate of 5 ml kg -1 h -1 . In experiments with induced pulmonary hypertension sodium pentobarbital was 2.1 g l -1 in the infusate and the infusion rate was 10 ml kg -1 h -1 .

NO measurements in exhaled breath

The fraction of NO in mixed exhaled gas (FENO) was continuously measured by means of a chemiluminescence based system (NIOX, Aerocrine AB, Solna, Sweden) sampling at 100-140 ml min -1 through a Naphion sampling catheter at the end of a mixing chamber (size: 5 tidal volumes) connected to the ventilator exhaust. Calibration was performed using certified NO standard gas in nitrogen (AGA Specialgas, Lidingö, Sweden).

Experimental protocol for in vivo experiments on NO donors

Rabbits received the candidate NO donor solutions (described in section 2.1) intravenously (syringe pump CMA/100, Carnegie Medicin AB, Stockholm, Sweden) at pre-determined flow rates into a standardized carrier flow (864 Syringe Pump, Univentor LTD, Zejtun, Malta) via the right jugular vein catheter. Each rabbit received more than one of the NO donor solutions and was allowed ample intervention-free periods (at least 30 min) between infusions to reach stable baseline conditions and to exclude cross-tolerance. Initial experiments included also trials with NO-saturated isotonic saline and a lipid emulsion purged with NO as means of administering NO parenterally. Arterial blood samples were intermittently drawn from the arterial catheter and analyzed for blood gases, acid-base status, methaemoglobin and total haemoglobin (ABL 520, Radiometer A/S, Copenhagen, Denmark). In some experiments the In experiments with pulmonary circulatory measurements (see section 2.5) the thromboxane mimetic U46619 was administered intravenously to the animals in a continuous infusion (10-30 µg ml -1 in saline, 150-750 ng kg -1 min -1 ) in order to induce a stable pulmonary hypertension (target: increase in PVR by approximately 150%). Thereafter one of the NO donor solutions or nitroglycerin (1 mg ml -1 ) were administered in cumulatively increasing doses (200-1600 nmol kg -1 min -1 , calculated for NO donating groups, 10 min for each dose)

while monitoring haemodynamics and respiratory gases, including FENO as well as intermittent checks of blood gases. Several NO donors were investigated in each animal in random order with at least 30 min in between.

Computation of physicochemical descriptors

The calculator plugin modules of MarvinSketch (version 5.2.6, ChemAxon Kft, Budapest, Hungary), accessed through ChemIDplus at the US National Library of Medicine, were used to obtain calculated molecular descriptors of the NO donor molecules. These descriptors included octanol/water partition coefficient (Log P), molecular size descriptors (molecular weight, MW; molecular surface area, MSA; molar refractivity, maximal and minimal projection radii and projection area, polarizability), molecular shape descriptors (the ratios between maximal and minimal projection radii and projection area respectively), polar and non-polar surface area (PSA and NSA respectively) from which a relative polar surface area of each donor molecule (PSA(%)) was calculated by dividing PSA with MSA. These descriptors were then used as independent variables in multiple linear regression models using the log (-MAP/FENO) as the dependent variable. Pure NO and calibration gases were from AGA gas AB (Lidingö, Sweden).

Statistics

Descriptive data are presented as mean ± standard error of the mean. Comparisons between groups were made by one way ANOVA and repeated comparisons within one group were made by repeated measures ANOVA or a paired t-test. Multiple linear regressions were made San Jose, CA, USA). P<0.05 was considered as significant.

Results

NO treated lipid emulsion and glycerol infused intravenously in vivo

Initial experiments showed that bolus infusions intravenously (0.5-2.5 ml) of a lipid emulsion (Intralipid) saturated with pure NO gas transiently increased FENO paralleled with a transient drop in MAP (data not shown). These responses could not be reproduced by only the native lipid emulsion (data not shown). In another experiment a rabbit with inhibited endogenous NO production (L-NAME 30 mg kg -1 ) first for 30 min intravenously received deoxygenated saline equilibrated with NO gas (0.5 ml kg -1 min -1 ) and after a recovery period of 3 hours a 30 min intravenous infusion of deoxygenated lipid emulsion (0.5 ml kg -1 min -1 ) equilibrated with NO. Saline with NO gas yielded a small increase in FENO and a very high methaemoglobin concentration whereas the lipid emulsion treated with NO gas gave a large increase in FENO whereas methaemoglobin was essentially not changed (Figure 1). MAP decreased with both infusions (data not shown). However the lipid emulsion turned yellow indicating oxidation.

When studying the table of content of the lipid emulsion it was noted that it contained glycerol, and therefore deoxygenated NO-equilibrated glycerol dissolved in water (glycerol-NO) was infused intravenously in a rabbit with inhibited NO production (L-NAME 30 mg kg - ). This resulted in increased FENO and decreased MAP. Thereafter the glycerol-NO solution was infused intravenously at increasing doses (3-100 µl kg -1 min -1 ) in a group of rabbits (n=4-5). FENO increased and MAP decreased dose-dependently (Figure 2a) during glycerol-NO infusions whereas neither saline treated with NO nor unreacted glycerol in the same doses had these effects (data not shown). Heart rate increased with 36±2 beats min -1 from 283±28 beats min -1 during infusion (100 µl kg -1 min -1 ) of glycerol-NO. When studying the pattern of FENO and MAP during the infusion, both these parameters reached a stable plateau, thus indicating absence of tolerance development (Figure 3). It was also found that there was a curvilinear relationship between the increments in FENO and the change in MAP (Figure 2b), a relationship that was linear when FENO increased by less than 20 ppb (this analysis was continued in section 3.4). Methaemoglobin levels remained low (data not shown) and other parameters did not change. These experiments suggested that an NO donor solution was created when deoxygenated solutions of lipid emulsion or glycerol were treated with NO gas.

Identification of other starting materials exhibiting NO donation when reacted with NO gas

More than 20 different alcohols and carbohydrates ( 3 and data not shown). A common feature for these compounds and groups are that they contain at least one hydroxyl group. Some compounds that were tested did not exhibit NO donating capacity in vivo despite NO equilibration: 2-Amino-1,3-propanediol, 3-Amino-1,2propanediol, lactic acid, alanine, glycine, and glucosamine (Table 1). This indicates that amine or carboxyl groups close to the hydroxyl-group in the substrate molecule prevent the formation of the NO donor compounds.

Chemical characterization and identification of NO donors.

Identification of reaction products with liquid chromatography coupled to on-line nitrite reduction

In order to further characterize and identify the NO donating compounds HPLC coupled to on-line nitrite reduction (LC-NO) was developed. Analyses of water saturated with NO gas and water with inorganic nitrite showed that the NO gas/inorganic nitrite appeared with the recording was synchronized by adjusting for the time delay between the two detection methods. This peak was denoted peak I. The same synchronized front peak was seen in the LC-NO of all the analysed NO solutions and was thus regarded as emanating from the NO gas/inorganic nitrite of these solutions. In addition, in all the solutions investigated with HPLC or LC-NO (Table 1) there appeared UV absorbing peaks with UV absorbance maxima near 225 nm, and with faint but for organic nitrites characteristic vibrational spectral peaks in the range 330-370 nm. No other such novel peaks were seen when deareated but non-NO treated organic starting materials were chromatographed. In the LC-NO it was found that all these chromatographic peaks were capable to generate NO in refluxing conditions (Figure 456, Table 1 and data not shown). None of the investigated parent substrate compounds yielded such NO chemiluminescence signals in the nitrite-reducing LC-NO analysis. Furthermore, the number of eluted organic nitrite-like peaks correlated with the number of nitrosylation sites in the form of hydroxyls in the parent compounds. Thus, 1,2-propanediol treated with NO gas (PDNO) exhibited 2 peaks (II and III in Figure 4) and glycerol-NO gave two peaks (II and III in Figure 5) the second of which was several times larger than the other, compatible with glycerol having two equivalent primary hydroxyls and one secondary. NO-treated glucose (glucose-NO) gave 4 organic nitrite-like peaks discernible in the UV recording and which despite considerable coelution, due to peak broadening in the transfer capillary, were still discernible as three peaks when reaching the NO/nitrite analysis (Figure 6). HPLC analysis of sucrose (60% in water, w/v) reacted with NO gas yielded 8 peaks including the front peak (data not shown). LC-NO analyses of these solutions were also made in reversed phase with gradients of methanol and acetonitrile, however no additional peaks were seen (data not shown). the propanol-NO must be less than 2.5% of the content of propyl nitrite. Further comparisons with some commercial organic nitrites in HPLC and LC-NO were completely compatible with formation of the corresponding nitrites in the NO gas treated hydroxyl precursor solutions (Table 1). In the experiments with authentic organic nitrites it was also found that the stability of the authentic organic nitrites was poor in water and was markedly increased by dissolving them in deoxygenated water or more preferable in deoxygenated NO gas-saturated solution (data not shown).

In the LC-NO experiments the content of NO donor compounds in the NO donor solutions could be estimated, by comparison with aliquots of inorganic nitrite. The content was determined to be 1-5 mM when the concentration of the carrier solutions were 3.3 M (1,2propanediol, glycerol and glucose). If the formed compounds were considered reacted with one NO molecule the relation between reacted carrier molecules to unreacted carrier molecules would be approximately 1:1000. Detection of the organic nitrites by e.g. NMR against this background was thus considered nearly futile, and attempts at identification with LC-MS yielded no conclusive data (unpublished results). The 3 solutions which appeared having the highest content of organic nitrites were therefore chosen for GC-MS analysis. For mass spectrometry the C 18 HPLC fractions corresponding to the 27.4 min peak from gradient elution of propanol-NO were injected on the GC-MS system and several peaks eluted.

Identification of reaction products with gas chromatography-mass spectrometry

At the front (1.5 min) the expected air gas peaks (nitrogen, oxygen and CO 2 ) were observed, together with a NO peak. At 3 min a peak exhibiting mass spectral characteristics for propyl nitrite was identified by the mass detector. Propanol was eluted and identified at 3.5 min in the GC-MS system, followed by small amounts of benzene (breakdown product of toluene) at 5.5 min, and then a large toluene peak at 8 min followed (data not shown). Direct on-column injection of commercial propyl nitrite confirmed the elution time (3 min) and mass spectral characteristics of propyl nitrite. Direct on-column injection of propanol-NO, obtained from the gas-tight reaction vessel, gave similar results but with a very large peak of propanol followed by toluene (data not shown). A solution of PDNO was similarly analyzed, using manual injection on an Agilent DB-1701 column (Figures 789). Two peaks were identified in the gas chromatogram (retention times 5.29 min and 6.15 min respectively) in the PDNO sample that were not found when injecting only 1,2-propanediol (Figure 7). The mass spectrum of the first of these peaks showed fragments of 30, 45 and 60 which are suggested to arise from the cleavage of the mononitrite of 1,2-propandiol when the hydroxyl at position 1 is nitrosylated (2-hydroxy propyl nitrite, Figure 8). The mass spectrum of the second peak showed fragments of 30, 31, 59 and 74 which are suggested to arise from cleavage of the mononitrite of 1,2-propandiol when the hydroxyl at position 2 is nitrosylated (2-hydroxy-1methylethyl nitrite, Figure 9). A major abundance of mass fragment 30, a common fragment for organic nitrites, was found in the 2 chromatographic peaks for compound 1 and 2 (data not shown). Mass spectrometry using chemical ionization demonstrated the M-1 and M+1 molecular ions in the mass spectra for both compound 1 and 2 (data not shown), as should be expected from the structures shown with the mass spectra (Figures 89). Attempts at Infusions of organic nitrites of PDNO, glycerol-NO and glucose-NO were studied in several rabbits and at different doses. By using increases of FENO less than 20 ppb, significant linear correlations were found between the increases in FENO (delta ppb from baseline) and the changes in MAP (% of baseline) (data not shown). Linear regressions (forced to start at 0)

showed that the slopes of the lines were different: PDNO -2.4±0.5, glycerol-NO -2.9±0.5 and glucose-NO -8.6±0.9. The slope was denoted MAP/FENO-index and was used as a measure of selectivity of NO donation in the pulmonary circulation versus systemic circulation. By dividing the change in MAP (% of baseline) with the increase in FENO from baseline (delta ppb), this index was generated for all NO donor solutions (supplementary Table A A.4). The second best fit showed that the more non-polar surface area and the higher Log P value of the NO donor molecules gave more effects on FENO compared with MAP and vice versa (supplementary Table A.4).

The third best fit indicated that the less polar surface area and the more non-polar surface area of the NO donor molecule predicted more effects on FENO versus MAP (supplementary Table A .4). Taken together the results from multiple linear regressions revealed that the polarity of the NO donor molecules was important for the selectivity towards the pulmonary versus the systemic circulation. Another important notion was that the very non-polar organic nitrites (e.g. propyl nitrites) and therefore theoretically the most lung selective compounds produced significant methaemoglobinemia thus limiting their use as therapeutics.

Reversal of pharmacologically induced pulmonary hypertension

The effects of PDNO, glycerol-NO, glucose-NO and nitroglycerin on pharmacologically

Discussion

For more than a century, organic nitrites have been used as volatile NO donors for relieving anginal pain. Further use has been limited due to problems with administration, dosing and adverse effects.

The present study introduces a method for producing potent NO donor solutions, evidenced by increases in exhaled NO and decreases of systemic blood pressure and by the ability to deliver NO gas in a nitrite reduction system after HPLC. The NO donor molecules were identified to be organic nitrites where two of them for the first time are described and identified with GC-MS. The selectivity of the NO donation towards the pulmonary and systemic circulation was dependent on the relative polarities of the NO donor molecules. The NO donor solutions were also potent in reversing pharmacologically induced pulmonary hypertension.

The present study provides further evidence that the rabbit in vivo model [START_REF] Persson | Detection of nitric oxide in exhaled air during administration of nitroglycerin in vivo[END_REF][START_REF] Cederqvist | Direct demonstration of NO formation in vivo from organic nitrites and nitrates, and correlation to effects on blood pressure and to in vitro effects[END_REF][START_REF] Adding | Direct gas measurements indicate that the novel cyclooxygenase inhibitor AZD3582 is an effective nitric oxide donor in vivo[END_REF], with measurements of exhaled NO and systemic blood pressure, is an excellent way to explore the action of NO donors. In the present work we used it as a bioassay system in order to find new NO donors. Previously the model has been used to study the acute tolerance development to organic nitrates and other NO donors [START_REF] Agvald | Nitric oxide generation, tachyphylaxis and cross-tachyphylaxis from nitrovasodilators in vivo[END_REF]. In the present study, the response on exhaled NO and systemic blood pressure to the organic nitrites showed a plateau pattern over time, indicating no acute tolerance development. This is in contrary to the response to nitroglycerin where a peak and plateau pattern was observed and taken as an indication of acute tachyphylaxis [START_REF] Agvald | Nitric oxide generation, tachyphylaxis and cross-tachyphylaxis from nitrovasodilators in vivo[END_REF]. The lack of acute tolerance of other organic nitrites has previously been shown both in vivo and in vitro [START_REF] Bauer | Vascular and hemodynamic differences between organic nitrates and nitrites[END_REF], constitutes a very important pharmacological difference Furthermore, our present experimental data on exhaled NO and systemic blood pressure indicated that different organic nitrites had differential effects on exhaled NO and systemic blood pressure. Therefore we used a MAP/FENO index previously used [START_REF] Agvald | Nitric oxide generation, tachyphylaxis and cross-tachyphylaxis from nitrovasodilators in vivo[END_REF] to describe these differential effects and the data were compared with calculated molecular descriptors of the NO donor molecules. By screening multiple linear regression models it was found that the best fits included measures of polarity of the NO donor molecules, i.e. that more polarity of the NO donor molecules affected systemic blood pressure more and that less polarity of the donor molecules affected exhaled NO more. This novel structure activity comparison approach for organic nitrites might be useful in the future in designing organic nitrites with wanted effects and selectivity as has been suggested for rational design of other classes of compounds [START_REF] Van De Waterbeemd | Estimation of blood-brain barrier crossing of drugs using molecular size and shape, and H-bonding descriptors[END_REF] and as used to understand important substructures for organic nitrates [START_REF] Koenig | NO donors. Part 16: investigations on structure-activity relationships of organic mononitrates reveal 2nitrooxyethylammoniumnitrate as a high potent vasodilator[END_REF].

The experiments with pharmacologically induced pulmonary hypertension indicate that certain organic nitrites will be more selective for the pulmonary circulation (e.g. organic nitrites of 1,2-propanediol and glycerol) compared to the systemic circulation (e.g. organic nitrites of glucose). However this observation needs to be confirmed and extended in a future study.

A study of the exact mechanisms behind the relationship between the selectivity for the pulmonary circulation and the polarity of the organic nitrite molecules have to be confined to a future study, but some considerations can be made. Relative polarity can be regarded as a measure of lipophilicity. Since several measures of compound lipophilicity correlate well with cell membrane passage [START_REF] Kramer | Absorption prediction from physicochemical parameters[END_REF], one potential mechanism could be that non-polar organic nitrites have increased permeability through cell membranes and therefore are cleared by the lungs and reach the systemic circulation in smaller amounts. This idea would also explain why the A c c e p t e d M a n u s c r i p t

Page 24 of 50 most non-polar organic nitrites produced methemoglobin, since a fast passage into the red blood cells should be expected. Other properties which have been shown to differ between organic nitrites, and may affect their half-lives and relative lung selectivity, are differences in efficacy in methaemoglobin formation [START_REF] Doyle | Structural effects in alkyl nitrite oxidation of human hemoglobin[END_REF], rate of hydrolysis [START_REF] Doyle | Hydrolysis, Nitrosyl Exchange, and Synthesis of Alkyl Nitrites[END_REF], rate of transnitrosylation [START_REF] Doyle | Hydrolysis, Nitrosyl Exchange, and Synthesis of Alkyl Nitrites[END_REF] and rate of enzymatic bioactivation the lung [START_REF] Meyer | Human glutathione transferase catalysis of the formation of S-nitrosoglutathione from organic nitrites plus glutathione[END_REF]. A short half-life of organic nitrites seems to be a general property of this group of compounds, although the extent of it may differ between the different individual compounds. In a study of intravenous infusions of isobutyl nitrite in rats the half-life, systematic clearance and distribution volume of this organic nitrite were determined to 1.3 min with a monoexponential decline, clearance being 10 times larger than cardiac output and with a distribution volume of 5.8 l kg -1 [START_REF] Kielbasa | Pharmacokinetics of a model organic nitrite inhalant and its alcohol metabolite in rats[END_REF]. In the present study, the effect half-lives of the studied organic nitrites are very short (approximately 1.5-3.0 min), as can be seen by the off response for glycerol-NO and PDNO in Figure 3. The short half-lives of organic nitrites simplify the use of them in an intensive care setting, since intravenous infusion of them will be highly controllable and therefore safe and easy to manage.

The present work introduces a method for analyzing inorganic and organic nitrites in HPLC coupled to a nitrite reduction system. The nitrite reduction system alone has previously been used in our laboratory in for example studying NO release from guinea-pig colon [START_REF] Iversen | Nitric oxide-like activity in guinea pig colon as determined by effector responses, bioassay and chemiluminescence analysis[END_REF] and from rabbit corpus cavernosum [START_REF] Hallen | Nerve-induced release of nitric oxide from the rabbit corpus cavernosum is modulated by cyclic guanosine 3',5'-monophosphate[END_REF]. Sen et al. [START_REF] Sen | Rapid and sensitive determination of nitrite in foods and biological materials by flow injection or high-performance liquid chromatography with chemiluminescence detection[END_REF] used a partly similar system (HPLC coupled to nitrite reduction) to analyze inorganic nitrite in food, saliva and urine. One development in the present study compared to Sen et al. [START_REF] Sen | Rapid and sensitive determination of nitrite in foods and biological materials by flow injection or high-performance liquid chromatography with chemiluminescence detection[END_REF] is the use of gradient elution in the HPLC. The method offers both high sensitivity (at least 10 pmoles, by the chemiluminescence) and specificity (different retention times by HPLC). We used the method for identifying and quantifying formed organic nitrites when NO gas was reacted with alcohols and carbohydrates, especially the method was of great use for the organic nitrites that could not be identified with GC-MS or LC-MS. When using this method some caution must be taken in that some other compounds will give a signal in the ozone reaction (e.g. ethers)

and that the nitrite reduction system can reduce both inorganic nitrite and O-nitrosylated compounds, as well as N-nitrosylated and S-nitrosylated compounds [START_REF] Jobgen | Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography[END_REF].

By using GC-MS and LC-NO, the reaction product between propanol and NO gas was identified to be propyl nitrite, a previously known organic nitrite. By using the same methods the reaction products between NO gas and 1,2-propanediol were identified to be the mononitrites of 1,2-propanediol (2-hydroxy propyl nitrite and 2-hydroxy-1-methylethyl nitrite). Although we were not able to identify the reaction products with GC-MS or LC-MS of the other NO donor solutions it is most likely that these NO donor compounds also are the organic mononitrites of the carrier molecules. The evidences are: 1) They were produced with the same method that produced organic nitrites unequivocally identified by GC-MS; 2) They showed similar NO donor capabilities in vivo; 3) The number of reaction products was associated with the number of hydroxyl groups in the carrier molecule. 4) The UV absorbance maxima were around 219-234 nm for all the reaction products which is around the same UV absorbance as authentic organic nitrites have as shown in this study and previously [START_REF] Ungnade | Ultraviolet Absorption Spectra of Nitroparaffins, Alkyl Nitrates, and Alkyl Nitrites[END_REF], and they exhibited characteristic vibrational spectra; 5) They released NO in the nitrite reduction system similar to authentic organic nitrites. 6) The starting material for the reaction was known.

Most previously known organic nitrites and that are commercially available include mononitrites of monohydric alcohols. Furthermore it has been shown that peroxynitrite is capable of nitration (adding NO 2 ) and nitrosation (adding NO) of glycerol thus forming glycerol mononitrate and glycerol mononitrite [START_REF] White | Formation of the NO donors glyceryl mononitrate and glyceryl mononitrite from the reaction of peroxynitrite with glycerol[END_REF]. Similar reactions between peroxynitrite and glucose, fructose and glyceraldehyde have been suggested. Although the reaction products were not identified they displayed biological activities similar to organic nitrites or organic nitrates [START_REF] White | Formation of the NO donors glyceryl mononitrate and glyceryl mononitrite from the reaction of peroxynitrite with glycerol[END_REF][START_REF] Moro | The formation of nitric oxide donors from peroxynitrite[END_REF]. It has also been suggested that glucose scavenges NO, particularly in hyperglycemia, and that treatment of glucose with acidified sodium nitrite led to monoaddition of NO to the glucose molecule [START_REF] Brodsky | Glucose scavenging of nitric oxide[END_REF].

The present study does not aim to elucidate the mechanism by which the organic nitrites were formed in the reaction with NO gas. There are several classical ways to generate organic nitrites which include reaction of alcohols with nitrous acid or other nitrosating agents such as nitrosyl chloride and nitrosonium salts [START_REF] Williams | Nitrosation reactions and the chemistry of nitric oxide[END_REF][START_REF] Noyes | n-butyl nitrite. Organic Syntheses[END_REF] or nitrosyl exchange between an alcohol and an alkyl nitrite [START_REF] Doyle | Hydrolysis, Nitrosyl Exchange, and Synthesis of Alkyl Nitrites[END_REF]. The latter principle is interesting for the present study as that means that in a compound with several hydroxyl-groups there will be an equilibrium and thus a mixture between the mononitrites of that compound and therefore it will not be possible to produce one specific organic mononitrite from for example 1,2-propanediol [START_REF] Doyle | Hydrolysis, Nitrosyl Exchange, and Synthesis of Alkyl Nitrites[END_REF]. Grossi and Strazzari [START_REF] Grossi | A New Synthesis of Alkyl Nitrites: The Reaction of Alkyl Alcohols with Nitric Oxide in Organic Solvents[END_REF] produced cycloalkyl nitrites from the reaction of NO gas with parent cycloalkyl alcohols in the presence of oxygen. They suggested that the reaction in this situation is not through the NO molecule itself but rather from nitrous anhydride (N 2 O 3 ) which is formed in the reaction between NO and molecular oxygen (4 NO + O 2  2 N 2 O 3 ). From the present work we can not determine the exact mechanism for the formation of organic nitrites in our system, and it is difficult to exclude trace amounts of oxygen in the reaction chamber. Therefore the reaction may involve a direct action of NO on the hydroxyl groups of the carrier molecules or it may involve a nitrosating agent such as N 2 O 3 (formed by the reaction of NO gas and trace amounts of oxygen) or it may be another, for us unknown, mechanism.

We conclude that new organic nitrites suitable for intravenous administration are produced by reacting NO gas and certain hydroxyl-containing compounds in aqueous solutions. In this work we have also developed a new method where liquid chromatography is coupled to nitrite reduction, which can be of great use, due to high sensitivity, in identifying possible endogenous organic nitrites in body fluids. Selectivity of different organic nitrites to the A c c e p t e d M a n u s c r i p t Note that for infusions that increased FENO with less than 20 ppb, the relationship is linear between increase in FENO and decrease in MAP.

A c c e p t e d M a n u s c r i p t Page 8

 8 of 50 Eluted peaks from the HPLC with the reaction mixture of propanol and NO gas were collected in gas tight syringes and extracted by an equal volume of toluene in a small sealed sample vial, shaken briefly, and quickly separated by centrifugation, if necessary. Fractions were injected at 1 µl onto an Agilent DB-5MS gas chromatography column (0.25 mm i.d., 30 m length, 1 µm phase thickness, Agilent Technologies, Inc., Santa Clara, CA, US) at an initial temperature of 40° C, thereafter the column temperature was raised by 10° C per min until a temperature of 200 or 250° C was reached. Detection of eluted compounds was made by an Agilent 5973 Mass Selective Detector (quadrupole MS, Agilent Technologies, Inc.

A c c e p t e d M a n u s c r i p t

  Page 11 of 50 endogenous NO production was inhibited with N G -nitro-L-arginine methyl ester (L-NAME, 30 mg kg -1 ).

A c c e p t e d M a n u s c r i p t

  Page 13 of 50 by using the best subsets function. Models including variables with non-significant variables (P>0.05) or with severe multicollinearity (Variance Inflation Factor>4) were excluded. Linear correlations were made by using Pearson Product Moment Correlation. All statistical analyses were made by means of SigmaStat (v. 3.10.0) and SigmaPlot (v. 9.01, Systat Software Inc.,

  A c c e p t e d M a n u s c r i p t Page 14 of 50 1

  A c c e p t e d M a n u s c r i p t Page 15 of 50 NO-treated monohydric alcohols (1-propanol, 2-propanol), dihydric alcohols (1,2-propanediol, 1,3-propanediol), trihydric alcohols (glycerol) and polymers of alcohol (polyethylene glycol) donated NO to exhaled air and decreased MAP (

A c c e p t e d M a n u s c r i p t

  Page 16 of 50 front of the chromatogram and at the corresponding elution time when the NO/nitrite

A c c e p t e d M a n u s c r i p t

  Page 17 of 50Further experiments were performed to verify the formation of organic nitrites. When analyzing commercially available propyl nitrite in methanol gradient elution there was a perfect match between the elution time(27.4 min) and UV absorbance between peak II in propanol-NO and authentic propyl nitrite. Propyl nitrate analyzed in the same system had a retention time of 23.7 min (approximately 55% of methanol, detection limit: 0.5 nmoles) and a UV maximum of 205 nm with no other specific UV absorbances. A corresponding peak was not found in the analysis of propanol-NO and therefore the content of propyl nitrate, if any, in

A c c e p t e d M a n u s c r i p t 3 . 4 .

 34 Page 19 of 50 analyzing glycerol-NO by the same method failed due to chromatographic interference from the large amounts of unreacted glycerol. Determination of important molecular properties for selectivity to the pulmonary and systemic circulation respectively.

A c c e p t e d M a n u s c r i p t

  Page 20 of 50 regression, relative polarity of the NO donor molecule, i.e. polar surface area divided with molecular surface area, showed to be the best predictor of the MAP/FENO index, i.e. the more relative polarity of the NO donor molecule, the more effects on MAP compared to FENO and vice versa (Figure10and supplementary Table

  induced pulmonary hypertension (obtained by means of i.v. infusion of the throboxane A 2mimetic U46619 in 8 rabbits) in vivo were investigated. Hemodynamic parameters and blood gases were normal and essentially similar in each group at baseline and during U46619 infusion. U46619 increased mean pulmonary arterial pressure (mPAP; 15±1 to 23±1 mmHg), PVR (26±2 to 62±4 mmHg min l -1 ), SVR (203±14 to 244±16 mmHg min l -1 ) and PVR/SVR (0.13±0.1 to 0.26±0.1) and slightly decreased FENO (10.7±0.5 to 10.4±0.4 ppb), cardiac output (330±20 to 280±10 ml min -1 ) and arterial partial pressure of oxygen (PaO 2 , 12.4±0.4 to A c c e p t e d M a n u s c r i p t Page 21 of 50 11.8±0.4 kPa). Other parameters did not change due to U46619 (data not shown), including MAP (70±4 versus 74±3 mmHg). PDNO (n=4, 200-1200 nmol kg -1 min -1 , Figure 11), glycerol-NO (n=4, 200-1200 nmol kg -1 min -1 ), glucose-NO (n=4, 200-1200 nmol kg -1 min -1 )and nitroglycerin (n=3, 200-1600 nmol kg -1 min -1 calculated on NO donating groups) dosedependently reversed the effects of U46619 on mPAP, PVR, cardiac output and PaO 2 (data not shown) to normal or almost normal at the highest doses. The order of efficacy differed and was PDNO>glycerol-NO>nitroglycerin>glucose-NO. All four NO donors also decreased MAP and SVR dose-dependently but on these parameters the efficacy order was the reverse i.e. glucose-NO>nitroglycerin>glycerol-NO>PDNO. Notably, in two out of four experiments the highest infusion rate of glucose-NO (1200 nmol kg -1 min -1 ) had to be terminated due to extreme systemic hypotension and signs of left heart failure, probably due to inadequate perfusion of the myocardium. All four NO donors also increased FENO dose-dependently (Figure12a), but their efficacy differed, i.e. PDNO>glycerol-NO>nitroglycerin>glucose-NO.Most importantly the four NO donors differently changed the PVR/SVR index, i.e. an index of selectivity towards the pulmonary and systemic circulations. PDNO decreased the PVR/SVR index (Figure12b), thus indicating increased selectivity for the pulmonary circulation. Glucose-NO was the most potent in increasing the PVR/SVR index (Figure12b), thus indicating increased selectivity for the systemic circulation. Glycerol-NO (Figure12b) and nitroglycerin (data not shown) affected the PVR/SVR index similarly by increasing it slightly. PDNO very slightly increased the methemoglobin level (from 0.4±0.2 to 0.85±0.1%) at the end of the cumulative infusions, whereas the three other NO donors left the methemoglobin concentration unchanged. Taken together these experiments clearly showed that the four investigated NO donors differed in selectivity for the pulmonary and systemic circulations respectively, and that FENO is an adequate marker for this difference.

A c c e p t e d M a n u s c r i p t

  Page 23 of 50 between organic nitrites and organic nitrates and may therefore encourage the use of organic nitrites instead of organic nitrates in clinical settings.

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  Page 27 of 50 pulmonary and systemic circulation, respectively, is dependent on the relative polarity of the specific organic nitrite. The nature of organic nitrite will also determine the tendency for methaemoglobin formation. Thus, by careful choice of NO donor molecule methaemoglobin formation may be avoided in therapeutically active doses. The present study shows that PDNO is relatively more selective to the lung compared with one clinically used NO donor, nitroglycerin, which should encourage future investigations on organic nitrites in disease models of pulmonary hypertension. We thus believe that the present study will stimulate further research in the field of organic nitrites as NO donors.

Figure 1 .

 1 Figure 1. Ventilated and anesthetized rabbit. Exhaled NO (FENO, panel A) and methaemoglobinemia (Met Hb, panel B) during infusion (0.5 ml kg -1 min -1 ) of first NO gas

A c c e p t e d M a n u s c r i p t Page 35 of 50 Figure 2 .

 502 Figure 2. Ventilated anaesthetized rabbits (n=4-5). Dose-response curves (panel A) of

A c c e p t e d M a n u s c r i p t Page 36 of 50 Figure 3 .

 503 Figure 3. Ventilated and anesthetized rabbits (n=3-5). Exhaled NO (FENO, panels A and B),

A c c e p t e d M a n u s c r i p t Page 37 of 50 Figure 4 .

 504 Figure 4. LC-NO, i.e. HPLC (panel A) and reflux NO/nitrites analysis (panel B) for

A c c e p t e d M a n u s c r i p t Page 38 of 50 Figure 5 .

 505 Figure 5. LC-NO, i.e. HPLC (panel A) and reflux NO/nitrites analysis (panel B) for

A c c e p t e d M a n u s c r i p t Page 39 of 50 Figure 6 .

 506 Figure 6. LC-NO, i.e. HPLC (panel A) and reflux NO/nitrites analysis (panel B) for

Figure 8 .Figure 7 ,

 87 Figure 8. Electron impact ionisation spectrum of peak 1 from a similar chromatography as in

A c c e p t e d M a n u s c r i p t Page 42 of 50 Figure 9 .Figure 7 ,

 5097 Figure 9. Electron impact ionisation spectrum of peak 2 from a similar chromatography as in

A c c e p t e d M a n u s c r i p t Page 43 of 50 Figure 10 .

 5010 Figure 10. Anesthetized and ventilated rabbits subjected to intravenous infusions of different

A c c e p t e d M a n u s c r i p t Page 44 of 50 Figure 11 .

 5011 Figure 11. Ventilated and anesthetized rabbit. The effects of intermittent infusions of NOtreated 1,2-propanediol (PDNO, doses of 130 and 450 nmoles kg -1 min -1 respectively) on

A c c e p t e d M a n u s c r i p t

Figure 1 Page

 1 Figure 1

Figure 4 Page

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Figure 5 Page

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Figure 6 Page

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 9 Figure 9

  Table 1 and supplementary Table A.1) were dissolved in water, deoxygenated 10 min with helium, bubbled 3 min with pure NO gas, transferred to a syringe and administered intravenously (100 µl kg -1 min -1 ) to rabbits in order to test NO donating capabilities (see following text, Table 1 and supplementary Table A.1).

	The capacity of NO donation in vivo was determined positive if FENO increased and MAP
	decreased.

  Table 1 and supplementary Table A.1). NO-

	treated monosaccharides (glucose, galactose, fructose, and ribose), monosaccharide alcohols
	(sorbitol and mannitol) and certain modified monosaccharides (fucose, 2-deoxy-ribose, 1-O-
	methyl-ribose) all had NO donor capabilities. Also NO-treated disaccharides (sucrose,
	lactobionic acid) and carbohydrate polymers (inulin, dextran, fucoidan) increased FENO and
	lowered MAP. Similar with glycerol-NO, all these compounds exhibited a rapid onset and

offset in effects without acute tolerance as illustrated by NO-treated 1,2-propanediol (Figure

Table A .

 A [START_REF] Wang | Nitric oxide donors: chemical activities and biological applications[END_REF]. Each point represents one NO donor (Table1 and supplementaryTable A.1). Shown is also the best linear regression found (expression 1 in supplementary Table A.4) by exploring the dependent variable Log (-MAP/FENO index) against the independent variable relative polarity (PSA/MSA in %, calculated as polar surface area divided with molecular surface area) for the different NO donors (Refer to supplementary Table A.2 for the list of compounds included) showing that relative polarity is a strong predictor for MAP versus FENO effects.

Acknowledgements and role of funding sources

This study was supported by the European Space Agency, the Swedish National Space Board, Fraenckel's Foundation for Medical Research, the Lars Hierta Foundation, the Swedish Science Council project 07919, the Swedish Heart-Lung Foundation and the Karolinska Institutet (MD/OD/PhD program grant). The funding sources solely provided financial support and were not involved in any part of the conduct of the research.

Statement of conflicts of interest

The authors (except ML) have filed patent applications on organic nitrites. LEG is a minority shareholder and member of the board of directors of Aerocrine AB (publ.) and is an inventor of patents concerning the use of exhaled nitric oxide.

The capacity of NO donation in vivo was determined positive if both exhaled NO increased and systemic blood pressure decreased (+).denotes no NO donation capacity in vivo. UV absorbance in HPLC was denoted (+) if treatment with NO gas of parent compounds resulted in new chromatographic peaks exhibiting characteristic absorbance around 225 nm and vibrational absorbance in the range 330-370 nm [START_REF] Ungnade | Ultraviolet Absorption Spectra of Nitroparaffins, Alkyl Nitrates, and Alkyl Nitrites[END_REF]. *denotes that the new chromatographic peaks in HPLC had the same retention time and UV characteristics in HPLC as the corresponding authentic and commercially available organic nitrite. NO release in HPLC coupled to online nitrite reduction (LC-NO) was indicated (+) if new chromatographic peaks also were confirmed to release NO in the nitrite reduction system. # indicates that authentic organic nitrite of the parent compound also released NO at identical retention time in the LC-NO. Some of the formed compounds (+) were identified with gas chromatography-mass spectrometry (GC-MS). Non-filled spaces in the table indicate that the corresponding analysis was not performed for that compound in that system. Absence of markers # and * means that commercial reference compounds were not available. A c c e p t e d M a n u s c r i p t

Revised Figure 12