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Am. J. Respir. Crit. Care Med., Volume 162, Number 2, August 2000, 617-622

Separation of Alveolar Surfactant into Subtypes
A Comparison of Methods

NICHOLAS J. GROSS, MATTHEW KELLAM, JARED YOUNG, SUBBIAH KRISHNASAMY, and RAJIV DHAND

Medical and Research Services, Hines Veterans Affairs Hospital; Department of Molecular Biochemistry, Stritch School of Medicine, Loyola University; and Department of Medicine, Stritch School of Medicine, Loyola University, Hines, Illinois and Maywood, Illinois

Alveolar surfactant is known to exist in several morphologic forms or subtypes which have been separated from bronchoalveolar lavage fluid (BAL) by two types of methods---differential centrifugation (DC) and equilibrium buoyant density gradient centrifugation (EBDC). DC separates BAL into large aggregates (LA) and small aggregates (SA); EBDC separates BAL into three peaks called ultraheavy (UH), heavy (H), and light (L). We compared these two separation methods by subjecting replicates of the same pools of BALF from groups of mice to DC and EBDC in parallel assays. We found that each method was highly internally consistent, but that the amount of phospholipid in the LA fraction of DC was consistently and substantially less (by 33 to 43%) than that found in the UH + H fractions of EBDC. This appeared to be due to failure of DC to sediment all of the phospholipid that banded as UH or H in EBDC despite adjustments in the time and g-force of DC. In experiments where differentially labeled purified H and L subtypes were subjected to DC over a wide range of g-force and time conditions, cross-contamination of the DC pellet and supernatant with heterologous subtypes was always present (4 to 33% cross-contamination). Addition of extraneous serum proteins to the BAL, as a model of lung damage, resulted in further inconsistencies in DC but not EBDC. Investigators may wish to bear these considerations in mind when planning or interpreting the results of experiments bearing on surfactant subtype analysis.




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