Development and characterization of an HPLC (high-performance liquid chromatography) system for the fractionation of bronchoalveolar lavage fluid

A high-performance liquid chromatography (HPLC) method has been developed for fractionating the protein components of the lung's extracellular lining fluid, as sampled by bronchoalveolar lavage. With this method, 10 ml of rat bronchoalveolar lavage fluid (BALF) in phosphate buffered saline can be quantitatively analyzed rapidly and reproducibly. This volume (25% of the lavage fluid volume from one rat using a standardized lavage technique) is made 0.2% with respect to trifluoroacetic acid (TFA) and pumped through a ..mu..Bondapak C/sub 18/ Radial-PAK HPLC column equilibrated with H/sub 2/O/O.2% TFA. Six fractions are then eluted with a series of acetonitrile gradients and isocratic steps that progress from H/sub 2/O/O.2% TFA to 65% Ch/sub 3/CN/O.2% TFA. Following these steps in the procedure, five additional fractions are eluted with methanol. All eleven fractions are detected by monitoring the column effluents at 206 nm and can be recovered by lyophilization since all the components of the HPLC solvent system are volatile. Nine of the eleven fractions were found to contain protein. Three of the fractions contained proteins common to the blood compartment. Of the largest fraction contained albumin and the next largest contained the immunoglobulins. Six other protein fractions appeared to be derived from the cellsmore » of the lung inasmuch as they were not detected in plasma. 60 refs., 7 figs., 1 tab.« less