Gharavi Negar, El-Kadi Ayman O S
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada.
J Pharm Pharm Sci. 2003 May-Aug;6(2):302-7.
Nitric oxide (NO) is produced by various cell types in picomolar to nanomolar range and has important roles in a variety of biological functions. The aim of the present study was to investigate a sensitive and reproducible fluorometric HPLC method for measuring nitrite, one of the stable oxidation products of nitric oxide, in murine hepatoma Hepa 1c1c7 cells.
Hepa 1c1c7 cells were incubated with vehicle or recombinant murine tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml) in Hanks' balanced salt solution (HBSS) for 10 hours. Thereafter, the HBSS medium was collected for nitrite analysis. NO production was examined by measuring the conversion of 2, 3-diaminonaphthalene (DAN) to its fluorescent product, 2, 3-naphthotriazole (NAT). NAT was analyzed after elution with 60% of 15 mM sodium phosphate buffer (pH = 7.5) and 40% methanol through a 10-microm reversed-phase C18 column (250 x 4.00 mm, I.D.) at a flow rate of 1 ml /min. Fluorescence was monitored with excitation at 375 nm and emission at 415 nm.
NAT appeared in approximately 16 min with no interference peaks. The assay yielded linear response within the examined range of 10 - 200 pM (r(2) >0.99) with an intra and inter-day variability of <10 % and accuracy of > 90%. The detection limit for nitrite was 10 pM. NO production by TNF- alpha treated Hepa 1c1c7 cells is estimated to be approximately 2 folds more than untreated cells.
This fluorometric HPLC assay offers a sensitive and reliable measurement of NO production in cell culture medium.
一氧化氮（NO）由多种细胞类型产生，浓度范围在皮摩尔至纳摩尔之间，在多种生物学功能中发挥重要作用。本研究的目的是探究一种灵敏且可重复的荧光高效液相色谱法，用于测定小鼠肝癌Hepa 1c1c7细胞中一氧化氮的稳定氧化产物之一亚硝酸盐。
将Hepa 1c1c7细胞在汉克斯平衡盐溶液（HBSS）中与载体或重组小鼠肿瘤坏死因子-α（TNF-α，10 ng/ml）孵育10小时。之后，收集HBSS培养基用于亚硝酸盐分析。通过测量2,3-二氨基萘（DAN）向其荧光产物2,3-萘三唑（NAT）的转化来检测NO的产生。用60%的15 mM磷酸钠缓冲液（pH = 7.5）和40%的甲醇通过10微米反相C18柱（250×4.00 mm，内径）以1 ml/min的流速洗脱后分析NAT。在激发波长为375 nm和发射波长为415 nm下监测荧光。
NAT在约16分钟出现，无干扰峰。该测定法在10 - 200 pM的检测范围内产生线性响应（r(2)>0.99），日内和日间变异性<10%，准确度>90%。亚硝酸盐的检测限为10 pM。经TNF-α处理的Hepa 1c1c7细胞产生的NO估计比未处理的细胞多约2倍。
这种荧光高效液相色谱测定法为细胞培养基中NO的产生提供了灵敏且可靠的测量方法。
