Sialic Acid Determination

Using the same approach as above a separation of sialic acids in the first dimension is achieved. The plate is then dried and masked, except for the sialic acids which have migrated, and this area is sprayed with alkaline hydroxylamine (equal volumes of 0.35 M hydroxylamine hydrochloride and 2.5 M ammonium hydroxide in water and methanol [1 1, v/v]). The plate is covered to prevent evaporation and left for 60 min. Chromatography in the second dimension together with hydroxamate standards is in propan-l-ol 10% aqueous ammonium carbonate 5M ammonium hydroxide (6 2 1, v/v/v) (Buscher et al 1974, Schauer 1978). The acylhydroxamates are visualized using a spray of 10% FeCl3 solution. The method is specific but not sensitive, at least 20-30 [xg of 0-acyl sialic acid in any one spot is required to yield a useful result (Schauer 1978). 

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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,... 

Sialic acid determination in cultured fibro- Enzyme analysis, sialidase, /1-galactosidase blasts... 

Cardo PP, Lombardo C, Gatti R (1985) A simple detection of sialic acid storage disorders by urinary free and total sialic acid determinations. Clin Chim Acta 150 129-135 Denny PC, Denny PA, Allerton SE (1983) Determination of sialic acid using 2-thiobarbituric acid in the absence of hazardous sodium arsenite. Clin Chim Acta 131 333-336... 

Ito M, Ikeda K, Suzuki Y, Tanaka K, Saito M (2002) An improved fluorometric high-performance liquid chromatography method for sialic acid determination an internal standard method and its application to sialic acid analysis of human apolipoprotein E. Anal Biochem 300 260-266... 

Rats and chickens maintained in the dark for several days were injected with b- H-glucosamine and divided in 2 groups one remained in the dark while the other was exposed to 1000 lux. Rats were exposed to illumination for 1 h chickens for 2 h. Then animals were killed and the nervous tissue fractionated according to Eichberg et al. (1964). Gangliosides and sialic acid determinations as described previously. (Maccioni et al., 1971 Maccioni A. H. R. et al., 1974). p was calculated by Student s test for correlated data. Values for animals in the dark were taken as 100%. 

The most specific method for sialic acid determination is possible with enzymes. Using acylneuraminate pyruvate-lyase (EC 4.1.3.3), sialic acids are cleaved to acylmannosamines and pyruvate. By coupling this reaction to lactate dehydrogenase the reduction of pyruvate allows monitoring of the pyruvate concentration and drives the lyase reaction, otherwise an equilibrium reaction, to completion (Brunetti et al. 1963). The concentration of sialic acid can be measured by spectrophotometry or fluorimetry of the amount of NADH oxidized on completion of the reaction, or by measurement of the initial rate of NADH oxidation. A less sensitive alternative is determination of N-acylmannosamine using colorimetric methods. Brunetti et al. (1962) utilized the Morgan-Elson reaction with dimethyl-aminobenzaldehyde for this purpose. 

Alternative methods of sialic acid determination have been described, but remain largely unused due to their poor sensitivity or difficulty in application relative to the assays described in these sections. These include the sulpho/phospho/vanillin method, originally used with gangliosides (Saifer and Feldman 1971), and the 1,10-phenanthroline method of Dimitrov (1973), which was subsequently refuted as a valid method by Snyder et al (1974). Of the remaining methods, already detailed at the beginning of section II, only the direct Ehrlich reaction has occasionally been employed where great sensitivity was of secondary importance (Werner and Odin 1952, Onodera et al 1965), or in cases where an additional colorimetric assay for identification of sialic acid was required (Onodera et al 1965, Cabezas 1973, Schauer 1978). 

Luppi, A., and Cavazzini, G., 1966, Sialic acid determination in some Corynebacteri-aceae, Nuovi Ann. Ig. Microbiol. 17 183. 

Suzuki Y, Ito T, Suzuki T, HoUand RE Jr, Chambers TM, Kiso M, Ishida H, Kawaoka Y (2000) Sialic acid species as a determinant of the host range of influenza A viruses. J Virol 74 11825-11831... 

Mild hydrolysis with acid for sialic add determination... 

The linkage of sialic acid to Gal, GlcNAc, or GalNAc may be determined on the microscale by methylation analysis before and after treatment with... 

Enzymic hydrolysis is a useful tool for the identification of carbohydrate linkages, as well as for hydrolysis of the (labile) sialic acids. Neeser developed a method wherein the sialic acids are enzymically hydrolyzed and, simultaneously, enzymically converted into stable 2-amino-2-deoxymannose derivatives. This allows determination of carbohydrate constituents of glycoproteins in a single flask. 

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).

The availability of both the cataboHc aldolase and the uniquely synthetic anabolic synthase made it possible to assemble a novel continuous assay for the determination of the metabolite N-acetylneuraminic acid [46]. A combination of both enzymes, in the presence of an excess of PEP, will start a cycle in which the determinant sialic acid will undergo a steady conversion of cleavage and re-syn-thesis as a futile cycle (Scheme 2.2.5.24). With each progression, however, 1 equiv of pyruvate is liberated simultaneously, which causes time-dependent signal amplification. Pyruvate is quantified spectrophotometrically by a corresponding NADH consumption when the system is coupled to the standard pyruvate dehy-... 

After the mild-hydrolysis step at 70°, the sialic acids liberated are removed from the sample by dialysis or ultrafiltration at 2°, and the macromolecular material is rehydrolyzed, using, however, the stronger acidic conditions of 0.1 M acid. The dialysis time ranges between 6 and 24 h, depending on the volume and viscosity of the hydrolysis mixture. Therefore, the optimum dialysis time should be evaluated by determinations of sialic acid in the eluate, or by addition of a trace of radioactive Nen5Ac. The dialyzates, or filtrates, are combined, and processed as will be described. By using this procedure, the overall yield of purified sialic acids is 70-80%, and the loss of O-acetyl groups107 is 40%. 

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