DETERMINATION OF INTRACELLULAR AND EXTRACELLULAR NITRITE AND NITRATE BY ANION CHROMATOGRAPHY

A highly sensitive method for the simultaneous direct detection as wel l as the ratio of intracellular and extracellular nitrite (NO2-) and n itrate (NO3-) in human macrophage (M phi), plasma and urine has' been developed. Samples were deproteinized with the organic solvent acetoni rile, lyophilized and reconstituted in buffer, determination and quant ification of nitric oxide (NO) stable end products NO2- and No-3(-) wa s performed utilizing an isocratic high performance liquid chromatogra phy (HPLC) with an anion exchange column. A mobile phase of 20 mM NaCl with 1 mM mono sodium phosphate-NaH2PO4 at pH = 7.0 was used. Analyte anions were detected by direct UV-wavelength at 210 nm. Sensitivity i n intracellular and extracellular fluids were 0.01 mu mol/L for both a nions with recovery rates of 99.6-99.4% for NO2- and NO3-. This method has successfully been applied to the determination of nitrite and nit rate in plasma and urine of normal human volunteers as well as in cell line U-937 macrophage cytosol/supernatant. The mean concentration of NO2- in normal plasma (n=22) was 3.1 +/- 0.4 mu mol/L and NO3- 10.3 +/ - 0.3 mu mol/L with ratio of NO2-:NO3- = 0.3; in normal urine (n=22) N O2- was 380 +/- 61 mu mol/L and NO3- 1345 +/- 270 mu mol/L with ratio of NO2-:NO3- = 0.3; in intracellular human macrophage fluid (n=21) NO2 - was 0.66 +/- 0.08 mol/L/10(6) M phi, and NO3- 0.68 +/- 0.09 mol/L/10 (6) M phi with ratio NO2-:NO3 = 1; and in extracellular human macropha ge fluid (n=21) NO2- was 0.03 +/- 0.01 mol/L/10(6) M phi and NO3- 3.03 +/- 0.33 mol/L/10(6) M phi with. ratio NO2-:NO3- = 0.01.