Analysis of sialic acids by gas chromatography of the mannosamine derivatives released by the action of N-acetylneuraminate lyase

Abstract

A convenient method for the analysis of sialic acids is proposed, which is based on their dissociation into pyruvate and N-acylmannosamines by the action of N-acetylneuraminate lyase, followed by gas-chromatographic analysis of the latter products as trimethylsilylated diethyl dithioacetals. Conjugated sialic acids should be freed with neuraminidase before being subjected to the action of the lyase, but these sequential enzymic reactions may be performed in one pot. N-Acetyl-, N-glycolyl- and N,O-diacetylneuraminic acids gave the corresponding mannosamines, and the dithioacetal derivatives of these mannosamines were well separated on a column of silicone OV-1. Quantitation of this enzymic and gas chromatographic method indicated that the error and coefficient of variation for free N-acetylneuraminic acid were 1.1% and 2.5%, respectively, for ten determinations at the 100 nmol level. The values for conjugated N-acetylneuraminic acid in N-acetylneuraminlactose were 2.9% and 5.9%, respectively. This method was applied to the analysis of sialic acids in some biological samples, and the results were compared with those obtained by the conventional colorimetric method. Preliminary data on urinary sialic acids indicated that cancerous patients gave significantly higher levels of urinary N-acetylneuraminic acid than normal subjects.