Analysis of Sialic Acids

Exact analysis of sialic acid is required in biologieal experiments where the biological role of sialic acid is frequently studied with the aid of sialidases, and the amount of sialic acids released is determined. This is also important for periodate oxidation studies on biological systems, where modification of sialic acids by periodate is only assumed, but chemical analysis of this effect by isolation and analysis of the modified sialic acids is seldom performed. These uncertainties in determinations of sialic acid can be overcome by the purification procedures already described. Furthermore, it must be stressed that unequivocal determination of the structure of a sialic acid, especially 

In contrast, the diphenol reactions leading to a blue-purple color are in wide use for sialic acid determinations. Sialic acids are heated either with orcinol and Fe3+ (the Bial reagent23), or with resorcinol and Cu2+ (Refs. 30 and 120) in concentrated hydrochloric acid.107 In 1903, 

Minimum amounts of 2-3 /ig of sialic acids can be determined in, for example, the orcinol-Fe3+ assay, if the volumes of the Bial reagents23 are one-fifth of those originally described.107 By using this method, sialic acids can be accurately determined only if they have been prepurified, as free or glycosidically bound pentoses, hexoses, or alduronic acids interfere with the reaction by giving green chromo-phores.107 In our experience, an approximate determination of the sialic acid in a complex carbohydrate is only possible if its sialic acid content is 5%. 

An increase in sensitivity of 30-50% can be achieved if resorcinol and Cu2+ are used.30 This increase may be by a factor of 3 to 6 if the sialic acid side-chains are oxidized by periodate prior to application of the resorcinol-Cu2+ reaction.127 

More sensitive, and most frequently used, is the periodic acid-thio-barbituric acid assay devised by Warren22 and modified by Amin-off.107 122 In a reaction sequence initiated by periodate oxidation, the 

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Figure 2. Competitive Binding Assay for CT The mixture of sample solution and P-CT-B (l,(XX)-fold diluted. List Biological Laboratories) was preincubated at room temperature for 30 min, and then added to a GMi-coated microtiter plate (50 ng for each well). CT bound to Gmi was determined spectrophotometrically at 405 nm, after adding a solution of 2,2 -azino-bis-p-ethylbenzothioazoline-6-sulfonic acid) as a substrate. A quantitative analysis of sialic acid using TB A method (25) indicate that sialidase-treatment completely eliminated sialic acids of CMP (6). The degree of hydrolysis with pronase-treat CMP was 71 % (6), which was measured by the TNBS method according to the NOVO (NOVO Industry) manual (26).

Although the different methods for the analysis of sialic acids thus far described give some insight into the structure of these compounds, mass spectrometry permits unambiguous determination of the chain length and the number, type, and position of N- or O-acyl substituents in Neu derivatives. Thus, a variety of new and rare sialic acids have been discovered in the past few years, and the presence of known, mainly O-acetylated, sialic ac ids has been established in a variety of biological materials.34-37,62 85,94,95-97-96-10 -107-147-149... 

Combination of pyrolysis and m.s. has been found to enable rapid analysis of sialic acids and other carbohydrates (for ng quantities of oligosaccharides and polysaccharides). Thus, the presence of (2— 8) and (2—>9) linkages in Neisseria meningitidis capsular polysaccharides has been confirmed.154... 

The chemistry and analysis of sialic acids have been reviewed. Picomole quantities of sialic acids have been measured by the fluorescence produced by the periodate-oxidized acid in the thiobarbituric acid reaction. " Contamination of the sialic acids with 2-deoxy-D-eryt/iro-pentose (derived from cellular material) could be detected by a downfield shift of the excitation maximum. Fluorescent derivatives are also produced when free sialic acids react with pyridoxamine, a procedure that compares favourably with the thiobarbituric acid reaction for determining sialic acids. Keto-acids e.g. pyruvic acid) interfere with the determination, but 2-deoxy-D-arabmo-hexose and 2-deoxy-D-c/-ytliro-pentose do not. A nonfading chromophore is produced when DMSO is used instead of n-butanol in the thiobarbituric acid ssay for sialic acids. A new histochemical method for the visualization and identification of unmodified or 0-acylated sialic acids has been reported. ... 

Dionex Corporation (2000) Dionex Technical Note No. 41 Analysis of Sialic Acids Using HPAE-PAD. Dionex Corporation (now part of Thermo Fisher Scientific), Sunnyvale, USA. 

Interference due to protein, especially haemoglobin, results in a reduction of sensitivity, and proteins should therefore be removed prior to analysis of sialic acid. A method using ethanol/chloroform precipitation has been put forward (Rivetz et al. 1980), but such deproteination methods still require additional correction for lipid, and the ion-exchange purification and lipid extraction should be part of the procedure to achieve reproducible and accurate quantitation (LEDEENandYu 1976, Schauer 1978, 1982). 

Analysis of Sialic Acid Linkage Types in Glycoconjugates... 

Both methods, NMR and FAB-MS, require preparation and purification of individual sialylated oligosaccharides, glycopeptides, or gangliosides before analysis, making this approach less attractive if only low amounts of the corresponding materials are available. Therefore, sensitive methods for isolation and analysis of sialic acids and sialoglycoconjugates are required. Several HPLC methods have been developed that fulfill this need. 

FIGURE 4. Analysis of sialic acids from human colon as DMB derivatives by fluorimetric HPLC on RP-18. (a) Sialic acids after direct derivatization with DMB (b) after treatment with influenza C virus (c) after treatment with 0.1 M NaOH (d) after treatment with aldolase. The following sialic acid peaks were identified by comparison with known standards 1, Neu5Ac 2, Neu5,9Ac2 3, higher O-acetylated sialic acids. 

Schauer, R., 1987a, Analysis of sialic acids. Methods Enzymol 138 132-161. 

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