The remaining fluid was centrifuged at 4°C for 10 min at 1,500 g, and the clarified supernatant was saved at — 70°C until assayed. Prostaglandin Bt (200 ng; Sigma Chemical Co, St. Louis, Mo) was added to one stored aliquot from each patient to serve as an internal standard for future high performance liquid chromatography (HPLC) analysis.
Transbronchial biopsies were done when not contraindicated, at the discretion of the bronchoscopist. All asymptomatic patients (except normal volunteers) and all patients whose conditions were ultimately diagnosed as NIP had transbronchial biopsies. Processing and staining of biopsy specimens were as previously described. The diagnosis of NIP was established by review of transbronchial biopsy specimens by a pathologist using previously described criteria; data from the histopathologic specimens used in this study have been previously reported.
Bronchoalveolar lavage specimens from patients with diagnoses other than NIP or P carinii pneumonia, or from symptomatic patients with no pathologic diagnosis, were excluded from analysis.
Extraction of Leukotriene B4
Frozen BAL specimens were thawed and extracted on octadecyl-silane cartridges (Sep-pak; Waters Associates, Milford, Mass) as previously described.Briefly, individual cartridges were washed with 20 ml ethanol, then with 5 ml of HPLC grade water. Samples were diluted to 10 percent ethanol, and the pH was adjusted to 3.2 before applying the diluted sample to the cartridge. Cartridges were eluted sequentially with 5 ml of 10 percent HPLC grade ethanol, 5 ml of HPLC grade water, and 10 ml of petroleum ether. Eicosanoids, including LTB4, were eluted from the cartridge with 4 ml of a solution of redistilled methyl formate (Eastman Kodak Co., Rochester, NY), ethanol, and acetic acid in a ratio of90:10:0.02. The eluant was evaporated to dryness under nitrogen. For subsequent HPLC, the dried sample was reconstituted in mobile phase A, as described later. The extraction efficiency of this method for LTB4 was 74.0 ±2.5 percent.