DNA quantification and fragmentation in sputum after inhalation of recombinant human deoxyribonuclease.
BACKGROUND
Inhaled rhDNase may improve sputum viscosity and mucociliary clearance by cleavage of extracellular DNA derived for instance from dead leukocytes in purulent, highly viscous patient sputum.


METHODS
Here we established a method to quantify rhDNase-mediated DNA fragmentation in sputum using gel electrophoresis. Sputum of Pseudomonas aeruginosa colonized cystic fibrosis (CF) patients with (CF+) or without (CF-) rhDNase treatment or mechanically ventilated non-CF patients receiving rhDNase (non-CF+) or not (non-CF-) was analyzed. DNA measurements from T-lymphocytes served as controls. Absolute DNA content and the relative quantity within eight molecular mass ranges (12000 to 200 bp) was determined by gel electrophoresis and densitometric analysis.


RESULTS
Geometric mean sputum DNA concentrations were 0.41 mg/dl for CF- (n=54), 0.78 mg/dl for CF+ (n=60), 0.053 mg/dl for non-CF- (n=41) and 0.049 mg/dl for non-CF+ (n=28). Treatment with rhDNase resulted in fragmentation of DNA that was quantified by separation and densitometric analysis of the DNA on agarose gels. The new analysis method permits analysis of DNA cleavage with high accuracy.


CONCLUSIONS
This new monitoring method facilitates DNA quantification and in vitro monitoring of rhDNase in sputum.
Riethmueller
Joachim
J
Department of Pediatrics, University Hospital, Tübingen, Germany. joachim.riethmueller@med.uni-tuebingen.de
Vonthein
Reinhard
R
Borth-Bruhns
Thomas
T
Grassmé
Heike
H
Eyrich
Matthias
M
Schilbach
Karin
K
Stern
Martin
M
Gulbins
Erich
E