Collected by rinsing the bronchoalveolar surface with PBS pH 7.4, and bringing the volume of

Collected by rinsing the bronchoalveolar surface with PBS pH 7.4, and bringing the volume of the pooled washings to 10 ml with additional PBS pH 7.4. Immediately thereafter, the lungs were surgically removed, washed without delay with ice-cold physiologic saline, patted dry with filter paper,Bhavsar et al. Journal of Biomedical Science 2010, 17(Suppl 1):S19 http://www.jbiomedsci.com/content/17/S1/SPage 3 offrozen in Chaetocin cost liquid nitrogen, and kept at -20 until used in an assay.Preparation of lung homogenatesFollowing their removal, the lung samples were rinsed immediately with physiological saline, patted dry with filter paper, weighed, and perfused with ice-cold physiologic saline. A portion of lung sample was mixed with PBS pH 7.4 in a 1:30 (w/v) ratio and made into a fine homogenate with a hand held tissue homogenizer (Tissue-Tearor? BioSpec Products, Inc., Bartlesville, OK) while keeping the mixture cold with the help of an ice bath. After a short sonication, the suspension was centrifuged at 14,000 rpm for 30 min, and the supernatant used for the assays of MDA, GSH, CAT, SOD and GPx.Assay of MDApH 7.4 and of 30 mM H 2 O 2 in a PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28549975 spectrophotometric quartz cuvette, and the absorbance of the reaction mixture read without delay at 240 nm twice, immediately after mixing and 1 min later. The activity of CAT, in U/ min/mg of protein, was calculated from the equation [OD c f/0.071 s ], where OD is the difference between the first and second absorbance readings; Vc is the volume of the spectrophotometric cuvette in ml; Vs is the volume of sample taken in ml; df is the dilution factor; and 0.071 is the molar extinction coefficient of H2O2.Assay of GPx activityThe concentration of MDA in the lung was measured as TBARS using the method of Buege and Aust [32]. An aliquot of lung homogenate was mixed with a reagent containing 15 TCA (w/v)-0.375 TBA (w/v)-0.25 N HCl, and the mixture heated at 90 o C for 1 hr. After allowing the mixture to cool to room temperature, and a brief centrifugation step to remove insolubles, the absorbance of the clear supernatant was read on a spectrophotometer at 535 nm. The concentration of MDA was derived from a standard curve prepared from serial dilutions of a 3.2 stock solution of TEP that were treated in identical manner as the lung homogenate samples. The concentration of MDA was expressed as nmol/mg of protein.Assay of GSHThe activity of GPx was measured indirectly by a coupled reaction with glutathione reductase using the spectrophotometric method of Gnzler and Floh?[35]. The reaction mixture contained an aliquot of lung homogenate, glutathione reductase solution (54 U/ml), 10 mM GSH, and 15 mM b-NADPH in PBS pH 8.0. After standing at room temperature, the reaction mixture was mixed with 3 mM H2 O 2 , and the change in absorbance of the reaction mixture at 340 nm was measured for 1 min. The results are expressed in U/min/mg of lung.Assay of SOD activityThe concentration of GSH in the lung homogenate was measured following its reaction with DTNB according to Ellman [33]. An aliquot of lung homogenate was mixed with 5 metaphosphoric acid, the mixture centrifuged at 2000 x g for 5 min, and an aliquot of the clear supernatant was mixed with 1.9 ml of 0.1 M phosphate buffer pH 8.0 and 20 of 0.02 M DTNB in 0.1 M phosphate buffer pH 8.0, and the absorbance of the resulting product read at 412 nm on a spectrophotometer. The concentration of GSH in the sample was derived by reference to a calibration curve of GSH p.