Capillary electrophoresis analysis of nitrite and nitrate in sub-microliter quantities of airway surface liquid

We developed a simple capillary electrophoresis (CE) method to measure nitrite and nitrate concentrations in submicroliter samples of rat airway surface liquid (ASL), a thin (10-30 microm) layer of liquid covering the epithelial cells lining the airways of the lung. The composition of ASL has been poorly defined, in large part because of the small sample volume (approximately 1-3 microl per cm2 of epithelium) and difficulty of harvesting ASL. We have used capillary tubes for ASL sample collection, with microanalysis by CE using a 50 mM phosphate buffer (pH 3), with 0.5 mM spermine as a dynamic flow modifier, and direct UV detection at 214 nm. The limit of detections (LODs), under conditions used, for ASL analysis were 10 microM for nitrate and 30 microM for nitrite (SIN= 3). Nitrate and nitrite were also measured in rat plasma. The concentration of nitrate was 102+/-12 microM in rat ASL and 70+/-1.0 microM in rat plasma, whereas nitrite was 83+/-28 microM in rat ASL and below the LOD in rat plasma. After instilling lipopolysaccharide intratracheally to induce increased NO production, the nitrate concentration in ASL increased to 387+/-16 microM, and to 377+/-88 microM in plasma. The concentration of nitrite increased to 103+/-7.0 microM for ASL and 138+/-17 microM for plasma.