Automated method for determination of bound N-acetylneuraminic acid in serum.

The usefulness of determining protein-bound sialic acid in serum is obvious,because a significant increase in various serum sialoglycoprotein concentrations (haptoglobin, orosomucoid, a1-antitrypsin, and ceruloplasmin) has been observed in many pathological states (1, 2). We earlier described(3) a fully automated method in which thiobarbitunc acid is used as a reagent.This very sensitive method, specific forfree sialic acid, has an inconvenience: it requires first a mild acid-hydrolysis step, which is time consuming and complicates the manifold. In thispaper,we present a simplerautomated method for the quantitative estimation of bound N-acetylneurazninic acid, which is as sensitive as thethiobarbituric a idassay but which is applicable directly to the serum without a previous hydrolysis step. The method is based on a modification of the Svennerholm resorcinol method (4). Spiro (5) has shown that a mild periodate oxidation of free and bound sialic acid increases the chromogenicity of the sialic acid in theSvennerholm assay. Taking advantage of these observations, Jourdian et al. (6) reported recently a differential method for determining totaland bound sialic acid. With suitable modifications, we were able to automate the periodate resorcinol technique, avoiding the step in which the chromogen is extracted with an organic solvent. The techniquedescribedhere was successfully used routinely to determine glycosidically-bound sialic acid, but also can be applied to free sialic acid.