Sialic acid colorimetric methods

The sensors discussed so far are based on ligands covalently bound to the polymer backbone. Other methods of detection - often referred to as mix and detect methods - work by simple noncovalent incorporation of the polymer with the ligand of interest. Reichert et al. generated liposomes of polydiacetylene with sialic acid for the same purpose of detection as Charych s surface-bound polymers, but realized that covalent functionalization of the polymer was not necessary [17]. Through simple mixing of the lipid-bound sialic acid with the polymer before sonication and liposome formation, they were able to form a functional colorimetric recognition system (Fig. 8). 

Finally, for the quantitative estimation of the 0-acyl content of sialic-acid-containing samples (Hestrin assay), also a colorimetric assay is routinely used. The method is based on the reaction with hydroxylamine in alkaline medium yielding hydroxamates, which form with Fe(C104)3 red chromophores, the absorbance of which is measured at 520 run [262]. 

Neutral monosaccharides, uronic acids, hexosa-mines, and sialic (neurominic acids) are identified and determined by specific colorimetric reactions. The principle behind the techniques rests on the condensation of the degraded products of the neutral monosaccharides (hexose, pentose and methyl pentose) by sulfuric acid with anthrone, cysteine hydrochloride, orcinol, and phenol reagents. Uronic acids may be determined by colorimetric and manometric procedures. While sialic acids are determined after chemical/enzymatic hydrolysis, gravimetric and Van-Soest detergent based methods are used to determine cellulose, hemicellulose, and fiber. 

Svennerhohn L (1957) Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim Biophys Acta 24 604-611... 

The initial attempts to develop a quantitative assay for sialic acids were colorimetric. The majority of these early methods were unsuitable because of their lack of specificity and/or sensitivity required for sialic acid analysis in biological materials or for enzymic assays. Some of these methods included diphenylamine (Ayala et al. 1951), tryptophane/perchloric acid (Seibert et al. 1948), sulphuric acid/acetic acid (Hess et al. 1957), hydrochloric acid (Folch et al. 1951) and the Ehrlich reagent, dimethyl-aminobenzaldehyde (Werner and Odin 1952). 

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. 

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). 

To date, various assay methods for sialidase activity have been developed. In principle, the hydrolytic reaction catalyzed by sialidase can be assayed by quantitating the released sialic acid or aglycone. The thiobarbituric acid procedure described by Warren (1959) or its modification by Aminoff (1961) is used frequently for the colorimetric determination of released, free sialic acid. 

The earliest methods for detection and assay of sialic acids were colorimetric procedures that capitalized on the more or less specific chromogens derived from these carbohydrates. Over the years a variety of colorimetric agents have been employed orcinol (Klenk and Langer-beins, 1941), diphenylamine (Ayala et aL, 1951), hydrochloric acid... 

The other widely employed colorimetric assay is the thiobarbituric acid procedure which is useful for free but not bound sialic acid. Proposed independently by Warren (1959) and Aminoff (1959), the method is based on periodate oxidation of sialic acid to )8-formylpyruvic acid (Figure 11) which then reacts with two moles of 2-thiobarbituric acid to form a chromophore (XLIX) with absorption maximum at 549 nm. The identity of this chromophore was established through synthesis... 

A scaled-down version of the Warren method was developed that can be used to assay as little as 25 ng of NANA (Hahn et aL, 1974). A number of automated procedures based on periodate-thiobarbituric acid have been described (Kendal, 1968 Delmotte, 1968 Fidgen, 1973 Gerbant et aL, 1973). Additional spectrophotometric techniques have employed the sulfo-phospho-vanillin reaction (Saifer and Feldman, 1971) and 1,10-phenanthroline (Dimitrov, 1973). The direct Ehrlich reaction, utilizing dimethylaminobenzaldehyde, played a key role in the early studies of sialic acid chemistry and analysis (Gottschalk, 1960) and still finds occasional use although its sensitivity is considerably below that of resorcinol and thiobarbituric acid (Werner and Odin, 1952 Onodera et aL, 1965). In comparing this method with others, Onodera et al. (1965) concluded that the possibility of false chromophores requires at least two colorimetric methods for reliable estimate of sialic acid in biological materials. 

Svennerholm, L., 1957 , Quantitative estimation of sialic acids. I. A colorimetric method with orcinol-hydrochloric acid (Dial s) reagent, Arkiv Kemi. 10 577. 

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