Isolation of disaturated phosphatidylcholine with osmium tetroxide

Abstract

A simple, rapid, inexpensive method for isolating disaturated phosphatidylcholine from adult rat lung has been devised.Total lipids are reacted with osmium tetroxide dissolved in carbon tetrachloride, and the disaturated phosphatidylcholine is isolated on a column of neutral alumina.More than 99% of the fatty acids in the phosphatidylcholine fraction are saturated and 94% of this material migrates as phosphatidylcholine on subsequent thinlayer chromatography. Supplementary key words lung lipids * phosphatidylcholineFor investigators interested in phospholipid metabolism in the lung, there is a need for a rapid method for isolating disaturated phosphatidylcholine.This species of phosphatidylcholine, which has only saturated fatty acids, is found in high concentration in purified surface active material and is mainly re- sponsible for the ability of the latter substance to produce low surface tensions (1-3).Ever since dipalmitoylphosphatidylcholine was first isolated from lung by Thannhauser, Benotti, and Boncoddo (4), many means of isolating disaturated phosphatidylcholine have been devised.These methods include ' Established Investigator of the American Heart Association.* Career Investigator of the American Heart Association.precipitation in cold ethanol (1) or acetone (4, 5), oxidation with ozone (6) or permanganate and periodate (7), formation of mercuric acetate adducts (8lo), and argentation thin-layer chromatography ( 1 1).Species of phosphatidylcholines can also be separated by diglycerides after treatment with phospholipase C (12, 13).Most of these methods require isolation of phosphatidylcholines before the specific separation procedures.Exceptions are the method of Young and Tierney (14), in which mercuric acetate adducts are formed with total phospholipids and the disaturated phosphatidylcholine is isolated by thinlayer chromatography, and the method of Brown (1) that uses solvent fractionation for analysis of material obtained by endobronchial lavage.A rapid, simple, quantitative method for isolating disaturated phosphatidylcholine would be useful for analyzing amniotic fluid to assess fetal maturity, for studying secretion of surface active material by type I1 alveolar cells, and for performing turnover studies in intact lung.We have developed a method which does not require prior isolation of phosphatidylcholine from lipid extracts.Osmium tetroxide is a powerful oxidant that reacts with double bonds to form complexes (15-18).One molecule of osmium tetroxide reacts with each double bond in egg phosphatidylcholine, and the reaction proceeds rapidly to completion (15, 17).Our method is simply to react total lipids with osmium tetroxide in carbon tetrachloride and to isolate the disaturated phosphatidylcholine by column chromatography on neutral alumina. MethodsLipids are prepared by extracting cells or homogenized tissue with chloroform-methanol 2: 1 (v/v),