Periodate thiobarbituric acid assay

The periodate oxidation of 9-O-acetyl and 4,9-di-O-acetyI derivatives of the methyl ester of 7V-acetyl-)8-D-neuraminic acid methyl glycoside has been studied the 9-O-acetyl group was found to hinder the oxidation, thus accounting for the low molar absorbancy coefficient of such derivatives in the periodate-thiobarbituric acid assay. The reactivity of a seven-carbon analogue of AT-acetylneuraminic acid, namely 5-acetamido-3,5-dideoxy-L-ara //io-heptulosonic acid, in the thiobarbituric acid and resorcinol assays has also been evaluated and the formation of the various chromophores discussed. ... 

Most of the structural and biochemical work related to KDO is based on the estimation of the compound or its derivatives by the periodate-thiobarbituric acid (TBA) assay in its various modifications. Indeed, KDO (see Fig. 3) was discovered9 through the formation of a characteristic, purple, TBA chromophore (Xmax 549 nm) from its 8-phosphate (2), which is the product of the condensation of D-arabinose 5-phosphate with enolpyruvate phosphate, catalyzed by 3-deoxy-8-0-phosphonooctulosonate synthetase (EC 4.1.2.16) (see Scheme 1 and Section V,2). 

Diphenol and Periodic Acid-Thiobarbituric Acid Assays... 

Hammond and Papermaster130 reported an increase of the sensitivity of the periodic acid-thiobarbituric acid assay by a factor of —50 (over the micro-assay just mentioned) when the chromophore extracted into the acidic 1-butanol phase is excited at 550 nm and the emitted light is measured at 570 nm. 

Periodate oxidation of sialic acids had earlier been used for structural determination of O-substituted sialic acids.89 Whereas, for example, one mole of a 4-O-acetylated sialic acid consumes 2 moles of periodate within 10-20 min at 0°, the same amount of the 7-O-acetyl isomer is oxidized by only one mole. As already discussed in connection with the periodic acid-thiobarbituric acid assay, 9-O-substituted sialic acids exhibit a very low rate of oxidation as compared with the unsubstituted sialic acids13 this observation originally led to the erroneous assignment89 of the 9-O-acetyl group to 0-8. All other sialic acids having O-acetyl groups on the side chain are expected to be unaffected by periodate. 

Chondroitin ABC and Chondroitin AC Lyases An improved method described for the micro determination of glycosamino-glycans is based on digestion with chondroitin ABC and AC lyases. The method has, however, been published previously. The determination is based on the colour production of 4-deoxy-a-L-t/fr 4-enepyranosyluronic acid-containing disaccharides produced by the action of chondroitin ABC and AC lyases when the periodate-thiobarbituric acid method is applied to the a,j8-unsaturated disaccharides. Suitable conditions for the quantitative assay were described. 

Sialic acids, released from human lymphocytes by hydrolysis, have been determined in the nanomolar range by a modified periodic acid - thiobarbituric acid assay. ... 

Reports of sialic acids in plants exist in the literature (Mayer et al. 1964, Onodera et al. 1966), but the analytical methods employed were insufficient to exclude other compounds such as 2-keto-3-deoxy acids noted earlier in this chapter. Several investigations produced negative results, although a reaction in the periodic acid/thiobarbituric acid assay was obtained. The compounds in question gave no colour in the direct Ehrlich, orcinol/Fe and resorcinol assays for sialic acids (Gielen 1968, Cabezas 1968, 1973, CABEZAsand Feo 1969, Unger 1981). 

Complex carbohydrates frequently contain adsorbed lipids, and this is especially the case where tissue, cell and membrane preparations are desialylated. For this reason, two extractions of the supernatant or dialysate containing free sialic acids with one volume each of diethyl ether or n-hexane is advisable, prior to ion-exchange chromatography (Schauer et al. 1975, Schauer 1978). If this extraction is not performed, erroneous results due to non-sialic acid compounds may be obtained in the analytical procedures, especially in the periodic acid/thiobarbituric acid assay of sialic acids (Schauer et al. 1975). Furthermore, lipid extraction improves sialic acid resolution on the following column chromatographic steps. 

Those colorimetric methods developed at this time and found to be suitably sensitive and specific are the orcinol and resorcinol methods (see II. 1 below) and the periodic acid/thiobarbituric acid assay (see II. 2). These methods together with a recently introduced assay using methyl-3-benzothiazolinone-2-hydrazone and fluorimetric methods (II. 3 and II. 4, respectively) form the basis of currently used techniques. 

Compounds Diphenol assays Periodic acid/thiobarbituric acid assay ... 

The periodic acid/thiobarbituric acid assay for sialic acid is probably the most widely used for quantitative analysis. This is due to its sensitivity and specificity. 

Fig. 1. Compounds formed during the oxidation step of the periodic acid/thiobarbituric acid assay. The six carbon aldehyde (7) and its hypothetical lactone (II) give rise to the chromogen p-formyl pyruvic acid (III)...

Sialic acids which do not react in the periodic acid/thiobarbituric acid assay are Neu5Ac2en and Neu-p-Me, neither of these sialic acids yield chromogen I (Fig. 1) on periodate oxidation. 

A modification of the periodic acid/thiobarbituric acid method has been described by excitation of the usual chromophore at 550 nm and measurement of the emission at 570 nm (Hammond and Papermaster 1976). The assay is 500-fold more sensitive than the conventional spectrophotometric procedures and can detect lOng sialic acid. Deoxyribose remains a problem with this assay and the precautions detailed for the periodic acid/thiobarbituric acid assay are required if accurate quantitation is to be made (Hammond and Papermaster 1976). 

The periodic acid/thiobarbituric acid assay has been automated by several authors for use with autoanalysers. For instance, Kendall (1968) and Fidgen (1973) employed the conditions of the Aminoff (1961) assay including organic solvent extraction. This assay was also used by Gerbaut er al. (1973), but without extraction, for measurement of sialic acid in serum. Adaptation of the Warren assay to the autoanalyser was reported by Delmotte (1968), who also dispensed with extraction of the chromophore. This method does not correct for 2-deoxyribose contamination (Delmotte 1968) and has subsequently been adapted to analyze both sialic acid and 2-deoxyribose in the same samples from blood and tissues (Engen et al. 1974) by measuring the optical density at 550 and 530 nm. 

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. 

Hyaluronate lyase purified from Streptomyces hyalurolyticus has been used to provide a specific assay for hyaluronic acid, its action resulting in the generation of 8-D-gluco-4-ene-pyranosyluronic acid groups. The assay is based on the long established periodate oxidation of such groups to inter alia j3-formylpyruvic acid which specifically gives a chromophore with thiobarbituric acid. 

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