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

Effect of cocoa flavanols and exercise on cardiometabolic risk factors in overweight and obese subjects

Copyright © 2008 Nature Publishing GroupObjective: Impaired endothelial function in obesity may reduce blood flow to sites of metabolism, contributing to impaired fat oxidation and insulin resistance. This study investigated the effects of cocoa flavanols and regular exercise, interventions known to improve endothelial function, on cardiometabolic function and body composition in obese individuals. Design: Overweight and obese adults were randomly assigned to high-flavanol cocoa (HF, 902 mg flavanols), HF and exercise, low-flavanol cocoa (LF, 36 mg flavanols), or LF and exercise for 12 weeks (exercise duration was 3 times 45 min per week at 75% of age-predicted maximum heart rate). Body composition was assessed by dual-energy X-ray absorptiometry at 0 and 12 weeks. Brachial artery flow-mediated dilatation (FMD), supine blood pressure (BP) and fasting plasma insulin, and glucose levels were assessed at 0, 6 and 12 weeks, respectively. Insulin sensitivity/resistance was determined using the modified homeostasis model assessment of insulin resistance (HOMA2). Results: A total of 49 subjects (M=18; F=31) completed the intervention. Baseline averages were as follows: body mass index=33.5 kg/m2; BP=123/76 mm Hg; HOMA2=2.4; FMD=4.3%; rate of fat oxidation during exercise=0.34 g min-1; abdominal fat=45.7% of total abdominal mass. Compared to LF, HF increased FMD acutely (2 h post-dose) by 2.4% (P<0.01) and chronically (over 12 weeks; P<0.01) by 1.6% and reduced insulin resistance by 0.31% (P<0.05), diastolic BP by 1.6 mm Hg and mean arterial BP by 1.2 mm Hg (P<0.05), independent of exercise. Regular exercise increased fat oxidation during exercise by 0.10 g min-1 (P<0.01) and reduced abdominal fat by 0.92% (P<0.05). Conclusion: Although HF consumption was shown to improve endothelial function, it did not enhance the effects of exercise on body fat and fat metabolism in obese subjects. However, it may be useful for reducing cardiometabolic risk factors in this population.K. Davison, A. M. Coates, J. D. Buckley and P. R. C. How