Hydroxyproline assay

Hydroxyproline content was measured using a hydroxyproline assay kit (Biovision) according to the manufacturer's instructions with slight modification. The lung tissues were weighed, homogenized in sterile water (10 mg of tissue per 100 μl H₂O) and hydrolyzed in 12 M HCl in a pressure-tight, Teflon capped vial at 120°C for 3 h followed by filtration through a 45 μm Spin-X Centrifuge Tube filter (Corning). Ten µl of the hydrolyzed samples was dried in a Speed-Vac for 2 h, followed by incubation with 100 μl of Chloramine T reagent for 5 min at room temperature and 100 μl of 4-(Dimethylamino)benzaldehyde (DMAB) for 90 min at 60°C. The absorbance of oxidized hydroxyproline was determined at 560 nm. Hydroxyproline concentrations were calculated from a standard curve generated using known concentrations of trans-4-hydroxyl-L-proline. The total amount of protein isolated from the weighed tissues was determined by using a protein assay kit (Bio-Rad, absorbance at 595 nm). The amount of collagen was expressed in μg/mg total protein.