Fluorimetric determination of unsubstituted and 9(8)-O-acetylated sialic acids in erythrocyte membranes.

A method is described for all quantitative determination of free or glycosidically bound sialic acids with special reference to erythrocyte membranes. Sialic acids, unsubstituted in their side chains, quantitatively yield formaldehyde after mild periodate oxidation (1 mM NaIO4, 15 min, 4 degrees C, in the dark). The formaldehyde is determined by the reaction with acetylacetone and ammonium acetate which leads to a sensitive fluorogen (F 410/510 nm). Sialic acids O-acetylated at C-9 or C-8 are not oxidized under these conditions. Therefore, they can be 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 can be determined. Total sialic acids (microgram/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 (data in brackets): 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%).