N-Acetyl-β-D-Glucosaminidase Activity in Bronchoalveolar Lavage Fluid: Characterization and Response to Ozone Exposure

Abstract

N-Acetyl-β-±-glucosaminidase (NAGA) activity in cell-free bronchoalveolar fluid of Fischer 344 rats was characterized in terms of assay reliability, pH response, stability, and activity relative to that in serum and the cellular fraction of the fluid. A linear relationship was observed between the volume of lavage fluid sample in the assay and the measured activity at 8 mM substrate (4-nitrophenyl-N-acetyl-β-±-glucosaminide) concentration, with no evidence of interference by lavage components. The stability of the enzyme activity in the cell-free lavage fluid over a 24-h incubation at 37±C and neutral pH was intermediate between that seen for serum (lowest stability) and lavage cells (highest stability). However, all three fractions showed an initial rapid inactivation of NAGA activity, with about 50% loss in activity after 5 h of incubation. Exposure of rats to 0.8 ppm ozone for 6 h, on a single day or 4 consecutive days, resulted in a twofold increase in total protein and NAGA activity in lavage fluids obtained two hours post-exposure. No qualitative differences were noted in the protein banding patterns on sodium dodecyl sulfate (SOS) polyacrylamide gels between air-control and ozone-exposed animals, and the electrophoretic profiles were consistent with an intraalveolar transudation of plasma protein. The higher activity of NAGA in lavage fluids of ozone-treated rats, when compared to serum, argues against a plasma origin of the excess activity.