SAMPLE CLEANUP AND ANALYSIS OF N-ACETYL AND N-GLYCOLYLNEURAMINIC ACIDS IN BLOOD-SERUM AND TISSUE SPECIMEN BY HPLC

A procedure for sample clean-up, isolation of glycoconjugates, and ana lysis of the two major sialic acids, Neu5Ac and Neu5Gc, as per-O-benzo ylated derivatives in biologic samples is described. In blood sera, th e bulk of low molecular weight molecules was efficiently removed by pr ecipitation of sialic acids containing glycoconjugates with saturated ammonium sulphate, pH 10, at 0 degrees C, centrifugation, and gel chro matography on a Sephadex G-25. The tissue specimen was homogenized wit h water at 0 degrees C and following centrifugation the water-soluble sialic acids containing glycoconjugates were recovered in the supernat ant. Sialic acids were liberated from their isolated macromolecules by acidic hydrolysis with 25 mM trifluoroacetic acid at 80 degrees C for 2 h. Constituents with positive charged groups and neutral monosaccha rides were removed by ion-exchange chromatographies on Dowex 50X8 and Dowex 1X8, whereas sialic acids were eluted with 2M formic acid. Follo wing derivatization with benzoic anhydride in the presence of p-dimeth ylaminopyridine, the per-O-benzoylated derivatives were completely sep arated and analyzed an a Supelcosil LC-18 column by isocratic elution with acetonitrile-water (67:33, v/v) and detection at 231 nm. Quantita tion was performed using peak areas obtained from external standards t reated under the same hydrolytic conditions as samples. Application of the method in C57BI mouse lung showed that N-glycolylneuraminic acid is the major sialic acid and that the amounts of sialic acids in rat s erum are in excellent agreement with those previously reported.