Bronchoalveolar Lavage in Liquid Paraffin Pneumonitis: Methods (2)

Bronchoalveolar Lavage in Liquid Paraffin Pneumonitis: Methods (2)Electron microscopic examination was performed after fixation of the cell pellet for one hour with 3 percent glutaraldehyde and washing in phosphate buffer pH 7.3. The cells were subsequently postfixed in 2 percent osmium tetraoxide in phosphate buffer pH 7.3. Ultrathin sections of representative regions chosen from semithin sections were prepared and examined using an electron microscope after staining with uranyl acetate and lead citrate.
BALF Lipid and Protein Analysis
Ten milliliters of the BALF of each patient were analyzed for protein and lipid contents.” Protein concentration was determined by the method of Bradford.” Lipids were extracted according to Bligh and Dyer, dried under a stream of nitrogen, and analyzed. Phospholipid content was determined by the phosphonis content of each lipid extract, assuming that the mean molecular weight of phospholipid was 650.
One dimensional thin layer chromatography (Silica gel 60 plates) of each extract dissolved in chloroform/methanol (2:1, by vol) was performed using two solvent systems to separate phospholipids (chloroform/methanol/acetic acid/water: 75 + 45 + 12 + 6, by vol) and neutral lipids (petroleum ether/diethyl ether/acetic acid: 80 + 20 + 1, by vol). Spots were visualized under iodine vapors and compared to various control liquid paraffin preparations.
Statistical Methods
All results were expressed as mean ± standard deviation. Statistical comparisons were made using the Students two-tailed t-test. A p value less than 0.05 was considered as significant.