Study On New Analytical Methods Of Nitrate/Nitrite In ViVo And Their Applications

Nitric oxide(NO)is a signal molecule with broad and unique properties in vivo,the study of NO has developed for a hot spot of research of cardiovascular,tumor pathology and other disciplines for the past few years.NO,a chemical substance involved in sending signals in the nervous system,is an important carrier of information exchange among the cells which play an important role in platelet activity,immune regulation,fight ulcer and regulating function etc.Low concentration of NO make the bronchial smooth muscle and pulmonary vessels relax and its effective release plays an important role in maintaining normal blood pressure to the animal.Since NO chemical nature very lively,it only has a half-life of 3-5 s in the body and extremely easy to oxidation which has more difficulty to directly measure.In recent 10 years,as relatively stable metabolites of NO,the content of nitrite and nitrate in body fluids have become the world hot spots in medical to reflect the NO content in vivo and as an important index to reflect a number of body functions or disease diagnosis.Therefore,it has important significance to establish accurate and simple detection method for biological nitrite and nitrate.This study established sensitive and efficient analysis methods for the concentration of NO in the complicated biological sample matrix.This will lay the foundation for the study of correlation between the biological NO level and disease.In this work,the established detection methods were applied to study the changes of NO in the plasma and urine of people who live in different altitude.The significance in the plateau hypoxia adaptation was discussed by combining with statistical analysis.The main research contents of this thesis were as follows:(1)Reversed phase ion pair high performance liquid chromatography method for simultaneous determination of nitrite and nitrate in blood and urineThe ion-pairs formed between reagent tetrabutyl ammonium hydroxide and nitrate in the weak acid mobile phase and detection wavelength was set as 230 nm Azo compound generated by diazotization coupling reaction for nitrite and detection wavelength was set as 510 nm.Due to the large difference of the polarity of the two products,they will not interfere with each other under the same chromatographic conditions.Under the optimized experimental conditions,the nitrite and nitrate azo product fully separated within 12 min by Agilent TC-C18 chromatographic column and the detection sensitivity of nitrite was greatly improved simultaneously.Under the optimum conditions,there were good linear relationships between peak area of two ions and the concentration in linear range of nitrite from 10 to 1000 ng/mL and nitrate from 5 to 200 μg/mL,respectively.The limits of detection were 4.5 ng/mL and 0.5μg/mL for nitrite and nitrate,respectively,with the relative standard deviations from 2.8 to 4.9%.This method can simultaneous determine nitrite and nitrate in biological samples with high detection sensitivity and precision(2)Cloud point extraction(CPE)combined microplate method for the determination of nitrite in human urine and bloodIn this study,decolorization treatment of urine and blood was applied to overcome the interference of matrix by active carbon and ZnSO4-NaOH.The method was based on the Griess reaction and the reaction product was extracted into nonionic surfactant Triton X-114 using CPE technique.Multi-sample can be simultaneously detected thanks to a 96-well microplate technique.4%(v/v)Triton X-114 was adopted as the extractant.The proper extraction condition was placed in the 45 ℃ water bath for 5 min.Then,the solution was detached by adding 0.2 g(NH4)2SO4 and centrifugation at 3000 rpm for 8 min.The enrichment factor was higher than 24.5.Under the optimum conditions,a linear calibration graph was obtained in the range of 10-1000 ng/mL of nitrite with limit of detection(LOD)of 2.5 ng/mL and recoveries from 90.5 to 95.9%.Compared with uv-vis spectrophotometry,the microplate method was easer to operate.The enriched surfactant can be determined directly without dilution.(3)Aqueous two-phase extraction and spectrofluorimetric determination of nitrite with o-phenylenediamine enhanced by hydroxypropyl-β-cyclodextrinDerivatization of nitrite with o-phenylenediamine(OPD)and fluorescence intensity of derivatives were studied by fluorescence spectroscopy.Hydroxypropyl-β-cyclodextrin(HP-β-CD)was used for inclusion of derivative.The spectral property and fluorescence intensity of inclusion complex were investigated.A new method based on inclusion of nitrite derivative by HP-β-CD coupled with fluorescence spectrometry for analysis of nitrite in urine sample was established.Under the optimized conditions of derivatization and inclusion,a good linear relationship existed between the fluorescence intensities of nitrite and the concentration in the range of 10-400 ng/mL,the limit of detection is 0.5 ng/mL.By adding standards of known concentration in urine sample,the recovery of nitrite was much more than 89.1%and the relative standard deviation was below 6.2%.(4)Surfactant enhanced emulsification microextraction(SEME)method combined with microplate reader for the determination of nitrite in blood samplesIn this study,1-octanol was used as extraction solvent with density lower than water and cationic surfactant(CTAB)used as the dispersant and emulsifier to extact nitrite derivative.Subsequently,the extractant determined by microplate reader.The effects of different operating parameters such as type of extractant and surfactant,concentration of surfactant and volume of extractant were studied.Under the optimum conditions,a linear calibration graph was obtained in the range of 10-800 ng/mL of nitrite with limit of detection of 2.1 ng/mL.The recovery was above 90.1%and relative standard deviation was below 4.7%.As surfactant has both hydrophobic group and hydrophilic group,it is used as emulsifier to enhance the dispersion of the water-immiscible phase into the aqueous phase by reducing the interfacial tension between the two phases.(5)Developing of testing kit for determination of nitriate and nitrite in urine and blood samplesIn this study,high sensitivity and accuracy testing kit was completed a preliminary build on the basis of the former four chapters to overcome the problems such as complicated biological samples matrix,matrix interference and the low level of nitrite in vivo.The successful design of the kit not only has high efficient economy,but also has sensitity,accuracy and precision.Combining with the microtiter plateinstrument analysis,the kit can maximize their advantages of simplicity and speediness.The aim of this study was meet the needs of clinical research and scientific research andapplied to related disease diagnosis by using 96 well plates for determination.(6)The health people living in the plain and entering mountain rapidly men were used as the researched object.By combining microplate reader with the developed testing kit in the fifth chapter study to analyze the concentration changes of nitriate and nitrite in serum and urine.The result showed that the level of NO in the entering mountain rapidly man significantly was lower than the plain residents and the level of NO decrease with the increase of altitude.The experimental results that obtained are consistent with previous results of related studies demonstrated the nitric oxide of entering mountain rapidly man decreased significantly for lack of oxygen.It is obvious that the contents of nitrite in plasma drastically reduced of people from the plains who entering mountain rapidly.Thus,it is significant to analysis of nitriate and nitrite for preventing and healing altitude diseases.