Glycosidically-bound sialic acid

Free sialic acids are used by the enzymes of sialic acid metabolism, CMP-sialate synthase and Neu5Ac aldolase. However the majority of sialic acid-metabolising and -recognising proteins interact with glycosidically bound sialic acid. There-... 

In the first assay, the sample is mixed with orcinol, FeCls and concentrated HCl and heated at 96°C. The formed purple to red-violet chromophore is extracted with isoamyl alcohol and its absorbance measured at 572 nm. Because of the use of HCl, the method can be used to quantitate the total amount of both free and glycosidically bound sialic acids. Due to the strongly acidic conditions, ester groups are released. As the assay does not discriminate between bound and free sialic acids, it is widely used to monitor the presence of sialic acids in either form during fractionation of biological material. It should be noted that other monosaccharides, especially pentoses, hexoses and uronic acids interfere with the assay, which is of importance when small amounts of sialic acid are present. 

When both tests are used in combination, a differentiation between total and free sialic acid is possible, allowing the calculation of the amount of glycosidically bound sialic acid. 

Free as well as glycosidically bound sialic acid give rise to highly characteristic H NMR parameters. The H NMR spectra are generally recorded in D2O, and because of the pH dependency of the proton chemical shifts, the spectral data are standardized at pD 6-7. The choice of the pH is also of importance in view of the earlier discussed de-O-acylation, 0-acyl migration, and autohydrolysis. 

The preparation of acylneuraminic acids is desirable to provide characterized standards for analytical methods and as a basis for the development of synthetic and new analytical techniques. Several methods exist for this, involving hydrolysis of material containing glycosidically bound sialic acids by dilute aqueous or methanolic acids or by enzymic means. Problems exist with respect to O-acyl-sialic acid preparation due to their lability during acid hydrolysis and their reduced or non-susceptibility towards the action of sialidases. Standard techniques have been developed over the last 15 years in the authors laboratory and these are discussed in the light of alternative methods and improvements which have appeared in the literature. 

As mentioned above, the periodic acid/thiobarbituric acid methods only function with free sialic acids. This means that prior release of glycosidically bound sialic acids must be undertaken before analysis. Care should be taken in the case of sialic acid glycosides known to break down under the conditions of the assay, e.g. CMP-p-Neu5Ac (Aminoff 1961) and various synthetic ketosides of sialic acid (Meindl and Tupfy 1967, Khorlin et al. 1970). This inevitably leads to erroneous values of free sialic acid and involves other parameters which need consideration. These are discussed below in II. 10. 

A new colorimetric method for the measurement of free and glycosidically bound sialic acid was introduced by Durand et al. (1974) and Massamiri et al. (1978, 1979), based on the reaction of methyl-3-benzothiazolinone-2-hydrazone (MBTH) with the formaldehyde liberated on periodate oxidation of the sialic acid side chain. The molar extinction coefficient for Neu5Ac is 67,000 at 625 nm, and the method is thus similar in sensitivity to the periodic acid/thiobarbituric acid methods. 

Several laboratories have reported automated procedures for the quantitation of sialic acid in biological fluids, column effluents or for miscellaneous samples. The periodic acid/resorcinol assay has been adapted for use in a Technicon autoanalyser for routine assay of serum sialic acid (Rey et al. 1975). The method is based on the assay developed by Jourdian et al. (1971) and does not involve the organic solvent extraction. As with the original assay, the automated procedure can be adapted to measure free or glycosidically bound sialic acid (Reyc/ al. 1975). Modification of this system allows measurement of erythrocyte membrane sialic acid simultaneously with protein (Gerbaut et al. 1978). 

In 1977 we introduced the application of high-resolution iR-NMR spectroscopy as a new method for structure elucidation of carbohydrate chains present in glycoproteins. These studies have shown that sialylated carbohydrate chains can completely be characterized with regard to the sialic acid residues. In this chapter relevant iH- and i C-NMR parameters of free and glycosidically bound sialic acids will be discussed. 

A role for glycosidically bound sialic acid in brain complex carbohydrates in development, learning ability and environmental stimulation has been described (Morgan and Winick 1979, 1980 a, b). The regulation in these events probably exists at hormonal and nervous levels and may involve mediation of Neu5Ac uptake into the cell as discussed in section V. 

Sialate 9-0-acetylesterases (EC 3.1.1.53) have been isolated and characterized from bovine brain, horse and rat liver, and influenza C virus (Schauer et al, 1988b,c, 1989 Butor et al, 1993b). These enzymes specifically release 9-0-acetyl groups from free and glycosidically bound sialic acids 9-0-lactoyl... 

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