Fluorimetric Determination of Unsubstituted and 9(8)-O-Acetylated Sialic Acids in Erythrocyte Membranes

Abstract

A method is described for the quantitative determination of free or glycosidically bound sialic acids, with special reference to erythrocyte membranes. Sialic acids, unsubstituted in their side chains, quantitatively yielded formaldehyde after mild periodate oxidation (1 mM NaIO4, 15 min, 4.degree. C, in the dark). The formaldehyde was determined by the reaction with acetylacetone and ammonium acetate which led to a sensitive fluorogen (F 410/510 nm). Sialic acids O-acetylated at C-9 or C-8 were not oxidized under these conditions. They were determined quantitatively by measuring the increase of fluorogen after O-deacetylation by 0.1 M NaOH. A minimum amount of 100 ng of sialic acids was determined. Total sialic acids (.mu.g/ml packed erythrocytes) and the relative amount of 9(8)-mono-O-acetylated sialic acids were determined by this procedure in erythrocytes from the following mammals: donkey (360; 8%), horse (350; 4%), mouse (300; 60%), rat (280; 40%), cow (200; 0%), cat (190; 25%), man (180; 0%), guinea pig (160; 22%), pig (130; 12%) and rabbit (70; 20%).