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A GC-MS method is reported for the quantitative analysis of S-nitrosothiols (RSNO) derived from endogenous low- and high-molecular mass thiols (RSH) including hemoglobin, cysteine, glutathione, N-acetylcysteine, and the exogenous N-acetylcysteine ethyl ester. The method is based on the conversion of RSNO to nitrite by aqueous Na 2 S (S 2- ). 15 N-Labelled analogs (RS 15 NO) or 15 N-labelled nitrite and nitrate were used as internal standards. The nitrite ( 14 NO 2 - and 15 NO 2 - ) and nitrate (O 14 NO 2 - and O 15 NO 2 - anions were derivatised by pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone and their PFB derivatives were separated by gas chromatography. After electron-capture negative-ion chemical ionization, the anions were separated by mass spectrometry and detected by selected-ion monitoring of m/z 46 for 14 NO 2 - , m/z 47 for 15 NO 2 - , m/z 62 for O 14 NO 2 - , and m/z 63 for O 15 NO 2 - . The expected thionitrites ( - S 14 NO and - S 15 NO) were not detected, suggesting that they are intermediates and rapidly exchange their S by O from water, presumably prior to PFB-Br derivatization. The reaction of S 2- with RSNO and sodium nitroprusside (SNP) resulted in the formation of nitrite and nitrate as the major and minor reaction products, respectively. The novel Na 2 S procedure was compared with established procedures based on the use of aqueous HgCl 2 or cysteine/Cu 2+ reagents to convert the S-nitroso group to nitrite. Our results provide evidence for an equilibrium S-transnitrosylation reaction between S 2- with RSNO in buffered solutions of neutral pH. Use of Na 2 S in molar excess over RSNO shifts this reaction to the right, thus allowing almost complete conversion of RSNO to nitrite and nitrate. The Na 2 S procedure should be useful for the quantitative determination of RSNO as nitrite and nitrate after PFB-Br derivatization and GC-MS analysis. The Na 2 S procedure may also contribute to explore the complex reactions of S 2- with RSNO, SNP and other NO-containing compounds., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sodium nitroprusside is a potent vasodilator employed intraoperatively and within critical care areas. The photolabile pharmaceutical agent has been used for decades and various stability studies have been executed. Due to potential shortages and the desire to batch compound sodium nitroprusside at a concentration of 1 mg/mL in polypropylene syringes, a new stability study was performed. Chromatographic analysis was conducted on a C18 column, with elution via an aqueous phase of 0.01 M sodium phosphate monobasic, adjusted to pH 6.5 with sodium hydroxide, and methanol (97.5:2.5) at a rate of 1 mL/min, and subsequent ultraviolet detection at 210 nm. Triplicate determinations of four samples, stored under refrigeration at 4°C, were obtained initially and on days 2, 5, and 9. Turbidity and pH measurements were performed in conjunction with visual observation on days of chromatographic analysis. Results demonstrate that sodium nitroprusside compounded in 5% dextrose at a concentration of 1 mg/mL, stored at 4°C protected from light in polypropylene syringes, is physically and chemically stable for at least 9 days., (Copyright© by International Journal of Pharmaceutical Compounding, Inc.)

Background: Hypertension is a public health problem and increases the incidence of cardiovascular diseases., Objective: To evaluate the effects of a resistance exercise session on the contractile and relaxing mechanisms of vascular smooth muscle in mesenteric arteries of NG-nitro L-arginine methyl ester (L-NAME)-induced hypertensive rats., Methods: Wistar rats were divided into three groups: control (C), hypertensive (H), and exercised hypertensive (EH). Hypertension was induced by administration of 20 mg/kg of L-NAME for 7 days prior to experimental protocols. The resistance exercise protocol consisted of 10 sets of 10 repetitions and intensity of 40% of one repetition maximum. The reactivity of vascular smooth muscle was evaluated by concentration‑response curves to phenylephrine (PHEN), potassium chloride (KCl) and sodium nitroprusside (SNP)., Results: Rats treated with L-NAME showed an increase (p < 0.001) in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) compared to the initial period of induction. No difference in PHEN sensitivity was observed between groups H and EH. Acute resistance exercise reduced (p < 0.001) the contractile response induced by KCl at concentrations of 40 and 60 mM in group EH. Greater (p < 0.01) smooth muscle sensitivity to NPS was observed in group EH as compared to group H., Conclusion: One resistance exercise session reduces the contractile response induced by KCl in addition to increasing the sensitivity of smooth muscle to NO in mesenteric arteries of hypertensive rats.

1. The aim of the study was to determine the effects of dietary supplementation with sodium nitroprusside (SNP), a nitric oxide (NO) exogenous donor, and N(G)-nitro-L-arginine methyl ester (l-NAME), a NO inhibitor, on growth performance, some biochemical parameters and ovarian primordial and primary follicles of quail. 2. A total of 480 Japanese quail (Coturnix coturnix japonica), one-day-old, including both males and females, were randomly allocated into one control group and 4 treatment groups each consisting of 96 birds. The control group was fed on the basal diet, whereas the experimental groups were fed on the basal diet supplemented with 50 mg SNP/kg, 200 mg SNP/kg, 50 mg L-NAME/kg or 200 mg L-NAME/kg. In the group receiving 200 mg SNP/kg, BW was lower on d 28 and d 42 compared to the control group and body weight gain (BWG) was lower between weeks 2 and 4 compared to the control group. In the same group, BWG and feed consumption were lower compared with the control group. 3. In the group receiving 200 mg L-NAME/kg, BW on d 42 and BWG were lower, whereas feed consumption and FCR was higher than in the control group. 4. In the groups supplemented with SNP at 50 and 200 mg/kg, serum total protein and albumin were higher than the control group; however, serum lipid profile, and liver and kidney enzymes were not affected by supplementation with SNP or l-NAME. 5. The numbers of ovarian primordial and primary follicles were greater in the group fed on the diet supplemented with 200 mg SNP/kg compared with the control group. Supplementation at 200 mg L-NAME/kg diet reduced the number of primary follicles compared to the controls, whereas the diameter of primordial and primary follicles increased. 6. In conclusion, supplementation with SNP and L-NAME depressed quail growth. Furthermore, the increase in NO following dietary supplementation with the NO-donor SNP delayed the growth process from primordial to primary and primary to secondary follicle transition in quail.

Background: The development of long-term vascular disease can be linked to the intrauterine environment, and maternal nutrition during gestation plays a critical role in the future vascular health of offspring. The purpose of this investigation was to test the hypothesis that a high-energy (HE) gestational diet, HE post-weaning diet, or their combination will lead to endothelial dysfunction in offspring., Methods: Duroc × Landrace gilts (n = 16) were assigned to either a HE (10,144 Kcal/day, n = 8) or normal energy (NE: 6721 Kcal/day, n = 8) diet throughout pregnancy. Piglets were placed on either a NE or HE diet during the growth phase. At 3 months of age femoral arteries were harvested from offspring (n = 47). Endothelial-dependent and -independent vasorelaxation was measured utilizing wire-myography and increasing concentrations of bradykinin (BK) and sodium nitroprusside (SNP), respectively., Results: BK and SNP induced vasorelaxation were significantly reduced in the femoral arteries of gestational HE offspring. However, no effect for the post-weaning diet on BK and SNP induced vasorelaxation was seen. This investigation demonstrates that a HE diet prenatally diminishes both BK and SNP induced vasorelaxation in swine., Conclusions: These findings suggest that a HE gestational diet can play a critical role in the development of offspring's vascular function, predisposing them to endothelial dysfunction. This dysfunction may lead to atherosclerotic disease development later in life.

Macrophages are key cells of the immune system. Immunologically activated macrophages are known to release a cocktail of reactive oxygen and nitrogen species. In this work, RAW 264.7 macrophages were activated by interferon-gamma and lipopolysaccharide, and the reactive mixture released by single cells was analyzed, in real time, by amperometry at platinized carbon microelectrodes. In comparison with untreated macrophages, significant increases in amperometric responses were observed for activated macrophages. Nitric oxide (NO*), nitrite (NO2*-), and peroxynitrite (ONOO-) were the main reactive species detected. The amounts of these reactive species were quantified, and their average fluxes released by a single, activated macrophage were evaluated. The detection of ONOO- is of particular interest, as its role and implications in various physiological conditions have been widely debated. Herein, direct evidence for the formation of ONOO- in stimulated macrophages is presented. Finally, the presence of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitor, led to an almost complete attenuation of the amperometric response of activated RAW 264.7 cells. The majority of the reactive species released by a macrophage are thus likely to be derived from NO* and superoxide (O2*-) co-produced by iNOS.

Rationale: Exposure to combustion-derived air pollution is associated with an early (1-2 h) and sustained (24 h) rise in cardiovascular morbidity and mortality. We have previously demonstrated that inhalation of diesel exhaust causes an immediate (within 2 h) impairment of vascular and endothelial function in humans., Objectives: To investigate the vascular and systemic effects of diesel exhaust in humans 24 hours after inhalation., Methods: Fifteen healthy men were exposed to diesel exhaust (particulate concentration, 300 microg/m(3)) or filtered air for 1 hour in a double-blind, randomized, crossover study. Twenty-four hours after exposure, bilateral forearm blood flow, and inflammatory and fibrinolytic markers were measured before and during unilateral intrabrachial bradykinin (100-1,000 pmol/min), acetylcholine (5-20 microg/min), sodium nitroprusside (2-8 microg/min), and verapamil (10-100 microg/min) infusions., Measurements and Main Results: Resting forearm blood flow, blood pressure, and basal fibrinolytic markers were similar 24 hours after either exposure. Diesel exhaust increased plasma cytokine concentrations (tumor necrosis factor-alpha and interleukin-6, p < 0.05 for both) but appeared to reduce acetylcholine (p = 0.01), and bradykinin (p = 0.08) induced forearm vasodilatation. In contrast, there were no differences in either endothelium-independent (sodium nitroprusside and verapamil) vasodilatation or bradykinin-induced acute plasma tissue plasminogen activator release., Conclusions: Twenty-four hours after diesel exposure, there is a selective and persistent impairment of endothelium-dependent vasodilatation that occurs in the presence of mild systemic inflammation. These findings suggest that combustion-derived air pollution may have important systemic and adverse vascular effects for at least 24 hours after exposure.

Objective: To test the hypothesis that microvascular vasodilation is impaired in patients with systemic sclerosis (SSc) compared with patients with primary Raynaud's phenomenon (PRP) and healthy controls, using the technique of laser Doppler imaging to quantify blood flow responses to iontophoresis of vasoactive agents., Methods: Microvascular blood flow was measured by laser Doppler imaging before, during and after 120 s iontophoresis (30 microA) of 1% acetylcholine chloride (ACh, endothelium-dependent) and 1% sodium nitroprusside (NaNP, endothelium-independent). Two adjacent fingers of the left hand were studied, and the procedure then repeated on the right. Ten patients with limited cutaneous SSc (LCSSc), 10 patients with PRP and 11 healthy control subjects were studied., Results: Vasodilation in response to both ACh and NaNP iontophoresis, as measured by 'area under the blood flow.time curve' (AUC), normalized for baseline flux, was similar in the control and PRP groups, but was diminished in the LCSSc group compared with both control and PRP groups (ACh results: control vs LCSSc P = 0.028, PRP vs LCSSc P = 0.005; NaNP results: control vs LCSSc P = 0.004, PRP vs LCSSc P = 0.005). There were no differences between groups in baseline flux values nor in voltages required to drive the 30 microA current., Conclusions: Both endothelium-dependent and endothelium-independent vasodilation are impaired in patients with LCSSc. Vasodilatory responses in patients with PRP are similar to those in controls. If reproducibility is confirmed to be satisfactory, then these techniques could be used to examine disease progression over time and responsiveness to vasoactive treatment, thus facilitating clinical trials.

The present study investigates the renal vascular responsiveness to vasoactive agents in diabetic rats which present an early stage of renal failure. Adult male Wistar rats were administered alloxan (150 mg kg(-1), s.c.). Seven days later the right kidneys were isolated and perfused. Renal perfusion pressure was measured continuously. Concentration-response curves were plotted for noradrenaline (NA), sodium nitroprusside (SNP) and carbachol. In basal conditions, kidneys from diabetic rats presented a decreased vascular resistance compared with those from control rats. The vasoconstrictor response to NA showed decreased EC50 values in preparations from diabetic rats compared with control ones (EC50 nmols, control: 2.03 +/- 0.44, n = 8; diabetic: 0.84+/-0.18, n = 6, P < 0.05). This enhanced sensitivity to NA could be in line with the decreased glomerular filtration rate and cortical renal plasma flow previously described in vivo in our laboratory (Garcia, Girardi, Ochoa, Torres & Elias, 1998). Vasoconstrictor responses to phenylephrine were not however, different between diabetic and control rat kidneys. This suggests that the increased sensitivity to NA was due to impaired neuronal uptake since phenylephrine is not a substrate for neuronal uptake. After precontraction with phenylephrine, both endothelium-dependent (carbachol) and endothelium independent (SNP) vasodilator agents caused similar response in the preparations taken from the two groups of animals. So, the enhanced sensitivity to NA is not associated with a deficient dilator responsiveness of the renal vasculature. The vasodilator response to carbachol was the same in absence or presence of L-arginine in the perfusate, suggesting no alteration in its availability at this stage of diabetes. Diabetic animals showed increased plasma level of fructosamine and glycosylated haemoglobin (Hb A1c), indicating the presence of early glycated products at this stage of diabetes, which could be involved in a possible structural alteration of the vessels.

In the present investigation we purpose a simple, rapid and accurate method for the evaluation of sodium nitroprusside. In alkaline medium and on the presence of thiourea, a new complex is formed. The acidification of medium induced a blue solution (lambda m = 600 nm) that could be determined by spectrophotometry.

A new method of quantitative analysis of sodium nitroprusside (SNP) was developed according to the principle of a photodegradation analytical method that has been reported by the authors previously. After irradiated of solution of SNP under fluorescent lamp, the increase in absorbance at 394 nm belonged to nonlinear kinetics, but the absorbance increment with concentration was linear in certain concentration range of SNP. When irradiated for 30 min or 60 min, SNP in solution can be quantitated accurately by delta A394. The standard curve of this method was linear from 50 to 1000 mg/L. The within-day and day-to-day precisions (RSD) were 1.9% and 2.6% respectively, with recoveries of 99.0-100.1%. No interference from small amount of serum protein, stearyl alcohol, propylene glycol, azone, m-nifedipine, nitrendipine and verapamil was observed. This method has been successfully applied to study percutaneous absorption.

A simple method has been developed for the determination of nitroprusside in human serum and pharmaceutical preparations. The new method is based on the strong inhibitory effect of nitroprusside on the photochemical reduction of phloxin by ethylenediaminetetra-acetic acid. The rate measurements are accomplished very simply by measuring the time needed for the absorbance to be reduced to 1/10th of its initial value. Optimal conditions for the determination of nitroprusside at concentrations of 15-200 ng ml-1 are described.

The apparent intravascular decomposition of nitroprusside (SNP) has been attributed to photolysis and artefactual generation of cyanide (HCN) during assay, leading some workers to believe that large doses of SNP may be infused safely if light is excluded. However, we have shown that HCN is not produced from SNP during analysis. Significant amounts of HCN were formed only when SNP was first incubated with blood. The yield of HCN was a function of the temperature, pH and time of incubation. The time for release of 50% of the HCN from SNP 5 mumol litre-1 at 37 degrees C in blood was 26.6 min with greater than 90% yield in 2 h, and in plasma the optimum pH was about 7.5. A u.v. method for measuring SNP suggests that, at clinically appropriate blood concentrations, SNP is confined to plasma.

Clinical experience suggests that nitroprusside (SNP) concentrations decay more rapidly in vivo than in vitro. Plasma concentrations of SNP were measured therefore in 20 patients at the end of an infusion, with mean arterial pressure (MAP) and cyanide (HCN) concentrations. Plasma SNP concentrations (20-243 micrograms litre-1; mean = 123.5 micrograms litre-1), were related to infusion rate (r = 0.66, P less than 0.001), and declined rapidly to a mean (SD) of 7.7 (4.5) micrograms litre-1 in 15 min. The decay of SNP correlated closely with the increase in arterial pressure (mean MAP vs log mean plasma SNP concentrations: r = -0.993, P less than 0.001), and was probably biphasic: mean (SD) T1/2 alpha = 0.89 (0.62) min, T1/2 beta = 14.3 (12) min. Mean plasma HCN and mean plasma SNP concentrations decreased together (r = 0.955, P less than 0.001), thus confirming that in vivo decomposition of the drug is the source of HCN.

Ion-pair chromatography has been used for the separation of nitroprusside ion and its photochemical hydrolytic and metabolic products. Organic modifier and pH were adjusted for maximum separation of the ions. Methanol was selected as the organic modifier in a pH range 5-8 and tetrabutylammonium perchlorate was used as the ion-pairing reagent. Ions were detected with a photoelectrochemical detector as described by Krull. A modification of this procedure was used to detect nitroprusside ion in spiked serum samples.

A sensitive high-performance liquid chromatographic assay for nitroprusside using an ion-exchange column and UV detection was developed to evaluate the stability of aqueous solutions of sodium nitroprusside in light-protected glass and plastic containers and during simulated infusions. The results showed that sodium nitroprusside is stable in 5% dextrose, normal saline, and lactated Ringer's solutions in light-protected glass or plastic containers. In addition, there was no decrease in the delivered potency of sodium nitroprusside solutions during simulated infusions lasting up to 24 h.

The photodecomposition of sodium nitroprusside (SNP) in aqueous and 5% glucose solutions results in the accumulation of cyanide and ferricyanide ions. Methods for the determination of these ions in such solutions have been devised. The continuous-flow method for cyanide involved the generation of cyanogen chloride, ClCN, by oxidation with chloramine T and subsequent reaction of the ClCN with barbituric acid and pyridine to produce a chromophore measurable by spectrophotometry at 580 nm. This cyanide method had a detection limit and sensitivity of 10-12 mumol/L and 1.40 X 10(-3) absorbance units (AU)/mumol/L, respectively, with a working concentration range of 0-800 mumol/L. This method employed mild reaction conditions, pH = 4.1, which rendered it suitable for use in time-course studies of cyanide production from SNP solutions exposed to 366 nm UV light. A simple method for the determination of ferricyanide in SNP solutions was developed which involved the addition of a 1% FeSO4 X 7 H2O in 1% H2SO4 reagent to the SNP solutions under test followed by spectrophotometry at 750 nm. The detection limit and sensitivity of the method were 2-3 mumol/L and 4.05 X 10(-3) AU/mumol/L, respectively, with a working concentration range of 0-100 mumol/L. This method was considered suitable for use as a quality control test of SNP solutions close to the site of their clinical use.

A rapid high-performance liquid chromatographic method for the determination of nitroprusside in commercial lyophilized products or in intravenous admixture solutions is described. The method is stability-indicating. Reversed-phase liquid chromatography was performed using a microparticulate (10 micrometer) phenyl column with a mobile phase acetonitrile--phosphate/tetrabutylammonium hydroxide buffer (pH 7.1) (30:70) and detection at 210 nm. A coefficient of variation of less than 3.1% was achieved over the concentration range studied (10--50 microgram/ml). Total analysis time was 9 min. This method was used to show that there is a small loss of nitroprusside due to photodegradation during intravenous infusion, even when the admixture container is wrapped in foil as recommended and used expeditiously.

The concentration of cyanide, a toxic metabolite of sodium nitroprusside, in solutions other than 5% dextrose in water, has not been reported. In this study, cyanide ion levels were measured by a cyanide ion-specific electrode in 250 ml of six different intravenous solutions (5% dextrose in water, 10% dextrose in water, distilled water, 0.9% sodium chloride, and lactated Ringer's solution with and without 5% dextrose) exposed to 300 foot candles of fluorescent light for 72 hours after sodium nitroprusside was dissolved in each solution. The rates of the increase in cyanide ion concentration in all six solutions were fairly constant between 4 and 24 hours. At 24 hours, there were no statistically significant differences in cyanide ion concentration among the six solutions. After 24 hours, the rate of the increase in cyanide ion concentration in the electrolyte solutions decreased more than that in the nonelectrolyte solutions. At 72 hours, the electrolyte-containing solutions had statistically significant lower mean cyanide ion concentrations than 5% dextrose, often the recommended diluent for sodium nitroprusside. There was no difference in mean cyanide ion concentration between lactated Ringer's solution with and without 5% dextrose. Solutions containing electrolytes are preferable to 5% dextrose for the dilution of sodium nitroprusside.

This study examined the stability of concentrated sodium nitroprusside infusions during administration by a motorized syringe-pump system. Sodium nitroprusside and the free-cyanide degradation product were determined in infusion samples collected over periods of up to 24 h. Measures which were taken to protect the delivery system from light, ensured satisfactory stability of the infusion for at least 12 h from the time of preparation.

Plasma cyanide concentrations were measured potentiometrically in 12 patients before and after infusion of sodium nitroprusside (SNP). A close correlation was found between the increase in plasma cyanide level and the total dose of SNP and between the increase and the mean rate of SNP infusion. After a total dose of 3.1 mumol SNP/kg (0.924 mg/kg) the maximum plasma cyanide concentration amounted to 2.2 mumol/1 (57 micrograms/l) which is lower than the recommended upper limit of 3 mumol/l plasma (78 micrograms/l); but indicates the importance of the recommended maximum dose of 5 mumol SNP/kg (1.5 mg/kg) and the control of plasma cyanide levels under SNP treatment. The direct potentiometric evaluation of the plasma cyanide level using the indicator method seems to be useful for such a control because it is more sensitive and faster than spectroscopic methods. The time needed for performing one measurement is about 35 minutes.

A specific spectrophotometric method of analysis for sodium nitroprusside was developed utilizing the molar absorptivity values at the maxima appearing in the electronic spectrum at 394 and 498 nm that may be attributed to metal d leading to pi* NO transitions. These measurements appear to be indicative of the unaltered nitroferricyanide complex. A reaction sequence is postulated for aqueous solutions of nitroprusside exposed to normal artificial light. Evidence suggests that one species in the sequence is nitroferricyanide in a higher energy state but with the nitrosyl moiety still present in the coordination sphere, which exhibits an increased molar absorptivity at 394 nm. The molar absorptivity at the 498-nm maximum remains unchanged. Corroborating polarographic data are presented.

The stability of esmolol hydrochloride and sodium nitroprusside in an admixture containing both drugs was studied. Solutions containing sodium nitroprusside in a final concentration of approximately 200 micrograms/mL and esmolol hydrochloride in a final concentration of 10 mg/mL in 5% dextrose injection were prepared in a 250-mL volumetric flask. The flask was wrapped with a light-protective cover, stored at ambient room temperature (15-30 degrees C), and protected from light. All experiments were conducted in triplicate with samples taken at 0, 2, 4, 8, and 24 hours. Testing included measurement of pH and absorbance at 400 and 600 nm. High-performance liquid chromatography was used to measure esmolol hydrochloride and sodium nitroprusside concentrations. No changes were observed in the physical appearance, pH, or absorbance of the admixtures. Neither the esmolol hydrochloride nor the sodium nitroprusside concentrations varied by more than 4% during the study. Under the conditions studied, esmolol hydrochloride is compatible with sodium nitroprusside in an admixture containing both drugs.

The colour formation of cysteine (I), acetophenone (II) and sulfite (III) with the sodium and tetrabutylammonium (TBA) salt of nitroprusside (NP2-) in aqueous solution was studied. The intensity of colour formation depends strongly on the nature and concentration of the cations and increases in the order TBA less than Li less than Na less than K less than Rb less than Cs (in case of cysteine TBA and Li are interchanged). This specific cation effect was known for the Boedeker reaction (III) and is now also demonstrated for the Legal reaction (I and II). The adduct formation between NP2- and I to III is based on an anion-anion interaction. The role of the cation is to reduce the Coulombic repulsion between the reactants by ion-pair formation. The efficiency of ion-pair formation corresponds with the order given before except for TBA, which behaves divergently.

A review of physical, chemical, analytical and pharmacological properties of nitroprusside is presented. In view of the pharmaceutical applications of nitroprusside special attention is given to the discussion of the (photo)degradation, the stability of the pharmaceutical formulations, the application as a reagent in pharmaceutical analysis and the redox behaviour.