Quantification of nitric oxide by high-performance liquid chromatography-fluorometric method in subgenomic hepatitis C virus-replicon expressing Huh7 cells upon treatment with acetylsalicylic acid

摘要

As nitric oxide (NO) expression levels are lower in hepatocytes compared with other cell types, it is difficult to quantify this compound via Griess assay. The aim of the present study was to quantify NO concentration in the cell culture medium from a subgenomic hepatitis C virus (HCV)-replicon expressing Huh-7 cell system using a high-performance liquid chromatography (HPLC)-fluorescence detector in the presence or absence of acetylsalicylic acid (ASA) treatment. HCV-replicon cells were incubated with ASA (4 mM) for 24, 48 and 72 h. Thereafter, the medium was collected to measure nitrites (NO2-) as an indirect indicator of NO levels using diaminonaphtalene as a derivate agent. NO levels were significantly higher (1.7-fold) in Huh-7 replicon cells treated with ASA (72 h post-treatment) than untreated cells (P0.05); NO inhibitor reduced 30% the level of NO in Huh-7 replicon cells treated with ASA (48 h post-treatment; P0.05). The findings suggested that the HPLC-fluorescence method provided an accurate and efficient measurement of NO production in Huh-7-HCV-replicon cells culture medium.