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

• Published in: Malaria Journal
• Main Authors: Adak Tridibesh, Raghavendra Kamaraju, Sharma Arun, Dash Aditya P
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2008
• Subjects: Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1186/1475-2875-7-71; https://doaj.org/article/651852c9fd8f48ddb50b742cce878bfe

Abstract Background 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 (NO 2 - ) and nitrate (NO 3 - ) are produced by the action of an inducible Anopheles culicifacies NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes. Method While exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of An. culicifacies , 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 NO 2 - and NO 3 - from mosquito mid-guts and haemolymph. Results 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 NO 2 - and NO 3 - in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 n M and 1 m M . Recoveries of NO 2 - and NO 3 - from spiked samples (1–100 μ M ) and from the extracted standards (1–100 μ M ) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO 2 - and NO 3 - in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO 2 - and NO 3 - in midguts and haemolymph of An. culicifacies sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology. Conclusion HPLC is a sensitive and accurate technique for ...