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通过阴离子交换高效液相色谱法测定库氏按蚊中肠和血淋巴中的一氧化氮代谢产物、硝酸盐和亚硝酸盐:难治性的可能机制

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.

作者信息

Sharma Arun, Raghavendra Kamaraju, Adak Tridibesh, Dash Aditya P

机构信息

Protein Biochemistry Laboratory, National Institute of Malaria Research (ICMR), 22 Sham Nath Marg, Delhi- 110 054, India.

出版信息

Malar J. 2008 Apr 28;7:71. doi: 10.1186/1475-2875-7-71.

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.

摘要

背景

一氧化氮在针对多种细胞内和细胞外病原体的固有免疫防御中具有多种生理和病理作用,这促使人们开发了各种测定一氧化氮(NO)合成的方法。NO代谢产物亚硝酸盐(NO2-)和硝酸盐(NO3-)是由按蚊中肠中诱导型库蚊按蚊一氧化氮合酶(AcNOS)的作用产生的,可能是这些蚊子抗寄生虫武器库的核心。

方法

在探索基于按蚊近缘种中一氧化氮合酶生理学的一种合理的抗性机制时,开发了一种灵敏、特异且经济高效的高效液相色谱(HPLC)方法,该方法不受生物胺存在的影响,用于测定蚊子中肠和血淋巴中的NO2-和NO3-。

结果

该方法基于提取效率、测定重现性和污染物最小化。它需要通过Ultracel 3 K过滤器进行离心超滤去蛋白,并通过配备214 nm紫外检测的高效阴离子交换液相色谱(Sphereclone,5μm SAX柱)进行分析。该测定方法在所有中肠和血淋巴样品中的检测下限为50皮摩尔。标准品和中肠样品中NO2-和NO3-的保留时间分别为3.42分钟和4.53分钟。标准品的测定线性范围为50 nM至1 mM。加标样品(1 - 100μM)和提取标准品(1 - 100μM)中NO2-和NO3-的回收率计算为100%。加标和未加标中肠样品中NO2-和NO3-的批内和批间变异及相对标准偏差(RSD)均为5.7%或更低。与A种相比,按蚊近缘种B的中肠和血淋巴中NO2-和NO3-水平升高,这反映了基于AcNOS生理学的一种抗性机制。

结论

HPLC是一种灵敏且准确的技术,可用于鉴定和定量蚊子中肠和血淋巴样品中皮摩尔水平的NO代谢产物,这对于各种生物样品中NO生物化学、生理学和药理学的临床研究可能有用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/78ea/2390569/bc78fd91331a/1475-2875-7-71-1.jpg

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