Isolated lamellar bodies from rat lung: correlated ultrastructural and biochemical studies

Abstract

Lamellar bodies isolated from rat lung homogenates were prepared for electron microscopy by two methods: OsO4 fixation followed by dehydration either with acetone or ethanol. Electron microscopy revealed that the acetone-dehydrated fraction contained many well preserved lamellar bodies with intact outer membrane, and the high retention of a material of low electron density between the fine and closely spaced concentric lamellae. This appearance was contrasted by the increased loss of this interlamellar material and coarse and irregularly spaced lamellae in the ethanol-dehydrated fraction. The supernatants from each respective fixation and dehydration were collected for lipid and protein analyses in an attempt to correlate any detected biochemical difference with a correspondingly modified ultrastructural appearance. Thin layer chromatography on the supernatant from the ethanol-dehydrated fraction revealed the extraction of 40 per cent lecithin and 4 per cent phosphatidylglycerol as compared to 1 per cent lecithin and an undeterminably small percentage of phosphatidylglycerol extracted by acetone dehydration. The effect of fixation and dehydration upon the extraction of chemical components of the lamellar bodies was compared to extraction by those of the following treatments: (1) freezing and thawing (a loss of 7 per cent lecithin, undetectably small losses of phosphatidylglycerol and protein); (2) incubation at 37 degrees C. with magnetic stirring (losses of 35 per cent lecithin, 5 per cent phosphatidylglycerol, and 4 percent protein, respectively); (3) sonication (loss of 57 per cent lecithin, 31 per cent phosphatidylglycerol, and 27 per cent protein, respectively). Further lipid analyses of the supernatants from dehydration and those obtained from the mechanical treatments revealed dipalmitoyl lecithin as the most readily extracted lipid. These data indicate that OsO4 fixation followed by acetone dehydration improves the preservation of the lamellar body ultrastructure by retaining an amorphous material between the lamellae, thus preserving the spatial relationship between the lamellae, and further that this improved morphologic preservation correlates well with retention of disaturated lecithin.