Hydroxyproline determination
Hydroxyproline content was determined as previously described (Woessner, 1961). Briefly, the lung was removed and homogenised in 1.0 ml of 5% trichloroacetic acid. After centrifugation, pellets were hydrolysed in 0.5 ml of 10 N HCl for 16 h at 110°C. Each sample was incubated for 20 min at room temperature with 0.5 ml of 1.4% w/v chloramine T solution, and then incubated at 65°C for 10 min with 0.5 ml of Ehrlich’s reagent (1 M DMBA, 70% v/v isopropanol and 30% v/v perchloric acid). The absorbance of each sample was then measured at 550 nm to determine the amount of hydroxyproline present.