Measurement of nitric oxide in murine Hepatoma Hepa1c1c7 cells by reversed phase HPLC with fluorescence detection.

PURPOSE. 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. METHODS. 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-mum 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. RESULTS. 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. CONCLUSION. This fluorometric HPLC assay offers a sensitive and reliable measurement of NO production in cell culture medium.