THE REACTION OF OSMIUM TETROXIDE WITH LECITHIN: APPLICATION OF STAINING PROCEDURES

Abstract

To obtain additional insight into the chemical reactions occuring furing fixation of tissue lipids by osmium tetroxide, the reaction between osmium tetroxide and lecithin has been studied. Procedures have been developed whereby quantitative information may be obtained about the nature of this reaction. One series of experiments was designed to arrive at the stoichiometry of the reaction and its time-course. To this end air-dried lecithin spots on cover glasses or on filter paper were exposed to oxmium tetroxide vapor. The weight increase of the lecithin spots was determined, and related to time of exposure to osmium tetroxide and to the quantity of lecithin present. Under the conditions chosen the binding of osmium tetroxide rapidly reaches a limiting value; thus the reaction appears to proceed to a definite end-point. The number of double bonds in the lecithin molecule, as determined by gas chromatography, corresponds with the number of moles of osmium tetroxide bound per mole of lecithin. This supplements earlier spectrophotometric findings, and confirms part of the reaction scheme proposed by Criegee for the interaction between osmium tetroxide and unsaturated compounds generally. The chemical properties of lecithin, and of other polar lipids, are determined not only by the presence of unsaturated linkages but also by the presence of an ampho-ionic group. Earlier research has demonstrated that this group is responsible for the staining properties of lecithin with anionic dyes, for instance, Brilliant Scarlet 3 R (Ponceau 4R or 4RS, Colour Index No. 16255). In this paper evidence is presented to show that the stainability of lecithin spots by Brilliant Searlet 3 R, under specified conditions, decreases markedly with increasing time of exposure to osmium tetroxide. It appears likely that as a result of the reaction with osmium tetroxide the lecithin polar group has changed properties. After studying the staining properties of lecithin towards an anionic dye, in relation to time of exposure to osmium tetroxide, its staining capacity for a representative cationic dye was similarly studied. Evidence is presented demonstrating an increased binding capacity of exposed lecithin spots to Brilliant Green (Colour Index no 42040). After considering a few possible interpretations we suggest that the simplest hypothesis to account for this phenomenon is a partial removal of the positive charge of lecithin, whereby the negatively charged groups are freed for interaction with positive dye molecules.