Determination of Intracellular and Extracellular Nitrite and Nitrate by Anion Chromatography

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Abstract
A highly sensitive method for the simultaneous direct detection as well as the ratio of intracellular and extracellular nitrite (NO2 −) and nitrate (NO3 −) in human macrophage (Mφ), plasma and urine has been developed. Samples were deproteinized with the organic solvent acetonirile, lyophilized and reconstituted in buffer, determination and quantification of nitric oxide (NO) stable end products NO2 − and NO3 − was performed utilizing an isocratic high performance liquid chromatography (HPLC) with an anion exchange column. A mobile phase of 20 mM NaCl with 1mM mono sodium phosphate-NaH2PO4 at pH = 7.0 was used. Analyte anions were detected by direct UV-wavelength at 210 nm. Sensitivity in intracellular and extracellular fluids were 0.01 μmol/L for both anions with recovery rates of 99.6–99.4% for NO2 − and NO3 −. This method has successfully been applied to the determination of nitrite and nitrate in plasma and urine of normal human volunteers as well as in cell line U-937 macrophage cytosol/supernatant. The mean concentration of NO3 − in normal plasma (n=22) was 3.1 ± 0.4 μmol/L and NO3 − 10.3 ± 0.3 μmol/L with ratio of NO2 −:NO3 − = 0.3; in normal urine (n=22) NO2 − was 380 ± 61 μmol/L and NO3 − 1345 ± 270 μmol/L with ratio of NO2 −:NO3 − = 0.3; in intracellular human macrophage fluid (n=21) NO2 − was 0.66 ± 0.08 mol/L/106 Mφ, and NO3 − 0.68 ± 0.09 mol/L/106 Mφ with ratio NO2 −:NO3 = 1; and in extracellular human macrophage fluid (n=21) NO2 − was 0.03 ± 0.01 mol/L/106 Mφ and NO3 − 3.03 ± 0.33 mol/L/106 Mφ with ratio NO2 −:NO3 − = 0.01.