Studies on the Role of Sialic Acid in Fibrinogen: Determination of the Sialic Acid in Fibrinogen by High Performance Liquid Chromatography

Abstract

In blood coagulation, fibrinogen-bound sialic acid has played an important role in the fibrinogen-fibrin conversion through the action of thrombin. The use of high performance liquid chromatography (HPLC) for determining sialic acid was attempted. Namely, a fibrinogen specimen was thoroughly dialyzed against sodium chloride orphosphate buffer solution, and then hydrolyzed in mild acid or by incubation with neuraminidase. After ultrafiltration to remove high molecular substances, the sialic acid in the filtrate was determined by HPLC, eluted by an isocratic procedure from a strong cation exchange resin column with a 1% phosphoric acid solution as a mobile phase. This analysis showed that each molecule of fibrinogen has 6.46 residues of sialic acid on average. This HPLC method can be directly used for quantitative determination of sialic acid released from fibrinogen without any conversion into derivatives and thus with fewer errors than conventional methods. In addition, the measurement was highly reproducible. Samples containing more than about 0.05 nmol of sialic acid or more than about 3.4μg (0.01 nmol) of fibrinogen can be determined by this method. The HPLC analysis required less than 9 minutes for the elution of the sialic acid. The results of this HPLC analysis suggest that fibrinogen molecules having different numbers of sialic acid residues are present in the blood. And it seems possible to determine sialic acid in other sialo-compounds of high molecular weights.