FOURIER-TRANSFORM INFRARED SPECTROSCOPIC ANALYSIS OF RABBIT LUNG SURFACTANT - SUBFRACTION-ASSOCIATED PHOSPHOLIPID AND PROTEIN PROFILES

Surfactant obtained from bronchoalveolar lavage (BAL) can be separated into subfractions based on sedimentation characteristics. It has been suggested that the 10 000 x g, 60 000 x g and 100 000 x g subfraction s isolated by this approach represent stages of surfactant extracellul ar processing. These three subfractions have been reported to differ i n their morphology, composition and ability to lower surface tension. We wished to determine if infrared spectroscopy, which may be applied as a non-invasive technique could potentially prove useful for charact erization and quantification of bronchoalveolar lavage (BAL) protein a nd phospholipid, and if this approach could detect differences in inte rmediate surfactant processing stages. Subfractions were collected fro m adult rabbit lungs by BAL and differential centrifugation and analyz ed by Fourier transform infrared (FT-IR) spectroscopy. Biochemical ass ay of phospholipid and protein showed differences between subfractions that correlated well with the phospholipid/protein ratios obtained fr om FT-IR spectra (r = 0.939; r(2) = 0.882). The subfraction sedimentin g at 100 000 x g (P-100) exhibited spectral shifts in the Amide I band , suggesting that the protein secondary structure was different compar ed to other fractions. Spectra obtained after separation of lipids and protein components showed an apparent disordering of protein secondar y structure but little or no effect on the structure or mobility of ph ospholipids. These results support the idea that subfractions represen t various processing stages of surfactant. In addition, they show that results from FT-IR analyses correlate significantly with traditional biochemical assay methods which may prove of clinical use, They also s how that each fraction displays characteristic FT-IR spectra suggestin g that this technique may potentially provide a non-invasive method fo r further detailed analysis of BAL.