Bial reaction

For their detection, the papers may be sprayed with reagents commonly used for the detection of keto and aminodeoxy sugars. Those of particular value include the direct Ehrlich reaction, the orcinol-trichloroacetic acid reagent, the chlorine-benzidine reagent for the detection of —NH—CO— groupings, and the Bial reaction as modified by Bohm und Baumeister. The limit of detection with the orcinol-trichloroacetic acid reagent is reported to be 5 ixg. of V-acetylneuraminic acid. For the detection of methoxyneuraminic acid, ninhydrin may also be used. 

Although several colorimetric methods have been developed in the past, only two main procedures are currently routinely applied, namely the orcinol/Fe /HCl assay, known as the Bial reaction, and the periodie acid/thiobarbituric acid assay. For microadaptations of these two different tests, see ref. [11]. 

By careful choice of both the reaction conditions and the phenolic compound used, it may be possible to produce a colour that is characteristic of a particular carbohydrate or related group, so giving some degree of specificity to the method. Thus, Seliwanoff s test uses hydrochloric acid and either resorcinol or 3-indolylacetic acid to measure fructose with minimal interference from glucose. The colour produced by pentoses with orcinol (Bial s reagent) or p-bromoaniline is sufficiently different from that produced by hexoses to permit their quantitation in the presence of hexoses. However, none of the methods based on the formation of furfural or its derivatives can be considered to be entirely specific. 

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%. 

Qualitatively, the presence of the nonulosaminic acids is best indicated by the brilliant-red coloration formed on addition of an acidic solution of p-dimethylaminobenzaldehyde (the so-called direct Ehrlich reaction ), and by the bright-purple coloration which develops upon boiling with Bial s reagent for several minutes at 100°. In addition, the nonulosaminic acids give a blue-violet coloration with Dische s diphenylamine reagent for deoxypentoses and a positive reaction in the tryptophan-perchloric acid test. Since no single one of these color reactions is absolutely specific for a nonulosaminic acid, it is advisable to carry out at least two of these for a qualitative analysis. For quantitative determinations, all four reactions have been employed, using either A/ -acetylneuraminic acid (m. p., 183-185° [a] —32.0°) or methoxyneuraminic acid [m. p., 200° (dec.) [a]o —55.0°] as colorimetric standards. 

The Bial color reaction has been utilized for the detection of nonulosaminic acids in defatted histological sections. This technique has... 

A comparison between certain chemical characteristics of the siaUc, iV-acetylneuraminic, and methoxyneuraminic acids, especially their functional groups and the color reactions afforded with Ehrlich s and Bial s reagents, suggested a close relationship between these nonulosaminic acids and indicated that methoxyneuraminic acid might be the methyl glycoside of a completely deacylated sialic acid. This concept was substantiated with the conversion of A-acetylneuraminic acid into methoxyneuraminic... 

The mechanically fragmented brain or spinal cord was dehydrated with acetone, as in the isolation of cerebrosides.7 Gangliosides are present in the water-soluble fraction they were recognized by color reactions (for example, Bial s orcinol reaction) based on the presence of sialic acid.159 The ganglioside mixture may be purified by partition... 

On the basis of characteristic color-reactions for (acylated) neuraminic acids (for example, Bial s orcinol reaction140,159 and the thio-barbituric acid method181), quantitative methods for the determination of gangliosides have been developed (for example, on thin-layer plates157,161,184,182). 

The orcinol reaction (Al) is simple, and further evidence of identification can be gained by determining the absorption spectrum of the color obtained by the reaction. The reaction is not specific for pentoses. Bial s test (HI) for pentoses in the urine represents the qualitative version of this reaction. 

Such materials may interfere with the determination of hexosamines (see Chapter VIII). With the colors produced by sialic acid as a reference, Werner and Odin 100) have estimated the amounts of sialic acid in several glycoproteins, including serum glycoprotein and ovomucin. They used Bial s orcinol, the direct Ehrlich reaction, diphenylamine, and tryptophan-perchloric acid. 

Measuring the rate of disappearance of ribose-5-phosphate in the first reaction with orcinol reagent (i.e. Bial s test, q.v.). 

Detailed colorimetric assay methods for nucleic acid derivatives will be confined to descriptions of slight modifications of three procedures diich have withstood well the test of time (f) the Mejbaum (76) modification of the Bial orcinol reaction for pentose (2) the Dische (42) diphenylamine method for desoxypentose and (S) the method of Fiske and Subbarow (45) for inorganic orthophosphate. Many other modifications of the original techniques have been advanced but, in our opinion, none offer a complete solution of such difficulties as may be encountered in nudmc acid studies. Discussions of the colorimetric techniques will be foimd in Schlenk s review (89), in the manual edited by Umbreit cf oZ. (61), and in the review by Dische (41). 

This work was continued by Dickens, who also used a fraction from autolyzed yeast, TPN, and as the autoxidizable hydrogen carrier, phenazine or a flavoprotein system. Dickens isolated various products, one of which appeared to be a mucture of a phosphopentonic acid and a phosphoketohexonic acid, and others analyzed as 5- and 4-carbon phosphate esters. The 5-carbon compound gave a reaction in the Bial FeClj-orcinol test which was characteristic of pentose. In general, the enzyme preparations of this period did not yield simple reaction products, nor were the methods available for the separation and characterization of the small amounts of products which were isolated. For instance, although suggestions were obtained of the presence of pentose in the products, this was not unequivocally demonstrated. 

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