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

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 (NO2-) and nitrate (NO3-) 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 NO2- and NO3- 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 mu 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 nM and 1 mM. Recoveries of NO2- and NO3- from spiked samples (1-100 muM) and from the extracted standards (1-100 muM) 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 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 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.

Figure 1

HPLC analysis of nitrite and nitrate. Chromatograms of an aqueous standard containing 25 μM nitrite and nitrate (A) and washed and spiked Anopheles culicifacies midguts obtained under control conditions (B) and Anopheles culicifacies haemolymph.

Figure 2

Haemolymph nitrite/nitrate of blood fed uninfected and blood fed Plasmodium vivax-infected Anopheles culicifacies species B at 0, 7, 9–10 and 14–15 days pBM using a high performance anion liquid chromatography method. Means were analysed using a 2-way ANOVA (p < 0.05).

Figure 3

Midgut nitrite/nitrate of blood fed Anopheles culicifacies species A and species B at 0, 7, 9–10 and 14–15 days pBM using a high performance anion liquid chromatography method. Means were analysed using a 2-way ANOVA (p < 0.05).