Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness

<p>Abstract</p> <p>Background</p> <p>The diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO₂^-) and nitrate (NO₃^-) are produced by the action of an inducible <it>Anopheles culicifacies </it>NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes.</p> <p>Method</p> <p>While exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of <it>An. culicifacies</it>, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO₂^-and NO₃^-from mosquito mid-guts and haemolymph.</p> <p>Results</p> <p>This method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 μ SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO₂^-and NO₃^-in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 n<it>M </it>and 1 m<it>M</it>. Recoveries of NO₂^-and NO₃^-from spiked samples (1–100 μ<it>M</it>) and from the extracted standards (1–100 μ<it>M</it>) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO₂^-and NO₃^-in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO₂^-and NO₃^-in midguts and haemolymph of <it>An. culicifacies </it>sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology.</p> <p>Conclusion</p> <p>HPLC is a sensitive and accurate technique for identification and quantifying pmole levels of NO metabolites in mosquito midguts and haemolymph samples that can be useful for clinical investigations of NO biochemistry, physiology and pharmacology in various biological samples.</p

Post-transplantation plasma malondialdehyde is associated with cardiovascular mortality in renal transplant recipients:a prospective cohort study

Background. In renal transplant recipients (RTRs), cardiovascular mortality is the most common cause of long-term renal graft loss. Oxidative stress (OS) has been associated with cardiovascular disease and is known to be enhanced in RTRs. We aimed to prospectively investigate whether the concentration of the OS biomarker malondialdehyde (MDA) is associated with long-term risk of cardiovascular mortality in a large cohort of RTRs. Methods. The plasma MDA concentration was measured using the thiobarbituric acid reaction assay in 604 extensively phenotyped RTRs with a functioning allograft for >= 1 year. The association between MDA and cardiovascular mortality was assessed using Cox proportional hazard regression analyses in the overall cohort and within subgroups according to significant effect modifiers. Results. Median circulating MDA concentration at baseline was 5.38 [interquartile range (IQR) 4.31-6.45] mu mol/L. During a follow-up period of 6.4 (IQR 5.6-6.8) years, 110 (18%) RTRs died, with 40% of deaths due to cardiovascular causes. MDA concentration was significantly associated with the risk for cardiovascular mortality {hazard ratio [HR] 1.31 [95% confidence interval (CI) 1.03-1.67] per 1-SD increment}, independent of adjustment for potential confounders, including renal function, immunosuppressive therapy, smoking status and blood pressure. The association between MDA concentration and the risk for cardiovascular mortality was stronger in RTRs with relatively lower plasma ascorbic acid concentrations [<= 42.5 mu mol/L; HR 1.79 (95% CI 1.30-2.48) per 1-SD increment] or relatively lower estimated glomerular filtration rates [<= 45 mL/min/1.73 m(2); HR 2.09 (95% CI 1.45-3.00) per 1-SD increment]. Conclusions. Circulating MDA concentration is independently associated with long-term risk for cardiovascular mortality, particularly in RTRs with relatively lower ascorbic acid concentrations or renal function. Further studies are warranted to elucidate whether OS-targeted interventions could decrease cardiovascular mortality in RTRs.Dutch Kidney Foundation C00.1877 Comision Nacional de Investigacion Cientifica y Tecnologica F 7219011

The effects of lung protective ventilation or hypercapnic acidosis on gas exchange and lung injury in surfactant deficient rabbits

Background: Permissive hypercapnia has been shown to reduce lung injury in subjects with surfactant deficiency. Experimental studies suggest that hypercapnic acidosis by itself rather than decreased tidal volume may be a key protective factor. Objectives: To study the differential effects of a lung protective ventilatory strategy or hypercapnic acidosis on gas exchange, hemodynamics and lung injury in an animal model of surfactant deficiency. Methods: 30 anesthetized, surfactant-depleted rabbits were mechanically ventilated (FiO2 = 0.8, PEEP = 7cmH2O) and randomized into three groups: Normoventilation-Normocapnia (NN)- group: tidal volume (Vt) = 7.5 ml/kg, target PaCO2 = 40 mmHg; Normoventilation-Hypercapnia (NH)-group: Vt = 7.5 ml/kg, target PaCO2 = 80 mmHg by increasing FiCO2; and a Hypoventilation- Hypercapnia (HH)-group: Vt = 4.5 ml/kg, target PaCO2 = 80 mmHg. Plasma lactate and interleukin (IL)-8 were measured every 2 h. Animals were sacrificed after 6 h to perform bronchoalveolar lavage (BAL), to measure lung wet-to-dry weight, lung tissue IL-8, and to obtain lung histology. Results: PaO2 was significantly higher in the HH-group compared to the NN-group (p<0.05), with values of the NH-group between the HH- and NN-groups. Other markers of lung injury (wetdry- weight, BAL-Protein, histology-score, plasma-IL-8 and lung tissue IL-8) resulted in significantly lower values for the HH-group compared to the NN-group and trends for the NHgroup towards lower values compared to the NN-group. Lactate was significantly lower in both hypercapnia groups compared to the NN-group. Conclusion: Whereas hypercapnic acidosis may have some beneficial effects, a significant effect on lung injury and systemic inflammatory response is dependent upon a lower tidal volume rather than resultant arterial CO2 tensions and pH alone

On the dynamics of nitrite, nitrate and other biomarkers of nitric oxide production in inflammatory bowel disease

Nitrite and nitrate are frequently used surrogate markers of nitric oxide (NO) production. Using rat models of acute and chronic DSS-induced colitis we examined the applicability of these and other NO-related metabolites, in tissues and blood, for the characterization of inflammatory bowel disease. Global NO dynamics were assessed by simultaneous quantification of nitrite, nitrate, nitroso and nitrosyl species over time in multiple compartments. NO metabolite levels were compared to a composite disease activity index (DAI) and contrasted with measurements of platelet aggregability, ascorbate redox status and the effects of 5-aminosalicylic acid (5-ASA). Nitroso products in the colon and in other organs responded in a manner consistent with the DAI. In contrast, nitrite and nitrate, in both intra- and extravascular compartments, exhibited variations that were not always in step with the DAI. Extravascular nitrite, in particular, demonstrated significant temporal instabilities, ranging from systemic drops to marked increases. The latter was particularly evident after cessation of the inflammatory stimulus and accompanied by profound ascorbate oxidation. Treatment with 5-ASA effectively reversed these fluctuations and the associated oxidative and nitrosative stress. Platelet activation was enhanced in both the acute and chronic model. Our results offer a first glimpse into the systemic nature of DSS-induced inflammation and reveal a greater complexity of NO metabolism than previously envisioned, with a clear dissociation of nitrite from other markers of NO production. The remarkable effectiveness of 5-ASA to abrogate the observed pattern of nitrite instability suggests a hitherto unrecognized role of this molecule in either development or resolution of inflammation. Its possible link to tissue oxygen consumption and the hypoxia that tends to accompany the inflammatory process warrants further investigation

Nitric oxide production and monoamine oxidase activity in cancer patients during interferon-α therapy

Both increased and decreased nitric oxide (NO) synthesis have been reported in patients treated with interferon-α (IFN-α). Animal studies showed that IFN-α administration results in increased levels of biogenic amines, subsequent activation of monoamine oxidases (MAOs), and finally in a change in NO production due to the H2O2 generated by MAOs. We examined the potential relationship between NO production in plasma and MAO-B activity in platelets of 43 cancer patients during 8 weeks of treatment with IFN-α. NO synthesis was quantitated by measuring both the ratio of citrulline and arginine (CIT/ARG-ratio) and total nitrite/nitrate (NOx) levels. Compared to baseline, MAO activity and NOx increased, while the CIT/ARG-ratio decreased. No associations were found between NOx, MAO and CIT/ARG-ratio. Only few associations were observed between changes in the biochemical parameters and changes in psychopathology induced by IFN-α, of which the association between changes in CIT and lassitude was the most consistent. The results suggest that peripheral NO production and MAO activity are unrelated to each other, and that peripheral changes in these biochemical parameters induced by IFN-α are unlikely to contribute to definite psychiatric disturbance

Separation and Determination of Fatty Acids from Lipid Fractions by High-Performance Liquid Chromatography: Cholesterol Esters of Umbilical Cord Arteries

Preeclampsia is accompanied by an extensive remodeling of the extracellular matrix of umbilical cord. It is associated with an increase in collagen content in the umbilical cord artery. Furthermore, preeclampsia distinctly reduces proteolytic and gelatinolytic activity, especially after activation with various agents

Pharmaceutical electrochemistry: the electrochemical detection of aspirin utilising screen printed graphene electrodes as sensors platforms.

A sensitive electrochemical sensor was designed for acetyl salicylic acid detection using graphene modified Screen Printed Electrodes. The electrochemical response of the sensor with graphene was improved compared to Screen Printed Electrodes without graphene and displayed an excellent analytical performance for the detection of acetyl salicylic acid. The high acetyl salicylic acid loading capacity on the electrode surface and the outstanding electric conductivity of graphene were also discussed in this manuscript. When a range of different concentrations of acetyl salicylic acid from 0.1 to 100 μM into a pH 4 buffer solution (N defined as the sample size N = 9) were plotted against the oxidation peak a linear response was observed. The detection limit was found to be 0.09 μM based on (3-σ/slope). Screen Printed Graphene electrodes sensors methodology is shown to be useful for quantifying low levels of acetyl salicylic acid in a buffer solution as well as in biological matrixes such as human oral fluid. A linear response was obtained over a range of concentrations from 10 to 150 μM into a human oral fluid solution (N = 10) giving a detection limit of 8.7 μM

Results, meta-analysis and a first evaluation of UNOxR, the urinary nitrate-to-nitrite molar ratio, as a measure of nitrite reabsorption in experimental and clinical settings

Diabetes mellitus: pathophysiological changes and therap