Periodate-resorcinol method

Jourdian, G. W., Dean, L., and Roseman, S., 1971, The sialic acids. XI. A periodate-resorcinol method for the quantitative estimation of free sialic acids and their glycosides, J. Biol. Chem. 246 430. 

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. 

The level of 0-acylation of sialic acids can also be quantitated by difference measurements with the periodic acid/thiobarbituric acid method (Neuberger and Ratcliffe 1972, Skoza and Mohos 1976, Sarris and Palade 1979, see section II. 2), with the periodate/resorcinol (II. 1. b)p)) and the MBTH method (II. 3), and a fluorimetric method (Shukla and Schauer 1981, II. 4). Using such assays care should be taken to run suitable controls for the saponification step and to consider... 

Unique methods based on new principles have been developed within the past 10 years. Threonine (27,28,249) is oxidized by lead tetraacetate or periodic acid to acetaldehyde, which is determined by photometric analysis of its p-hydroxydiphenyl complex or iodometric titration of its combined bisulfite. Serine is oxidized similarly to formaldehyde, which is determined gravimetrically (207) as its dimedon (5,5-dimethyldihydro-resorcinol) derivative or photometric analysis (31) of the complex formed with Eegriwe s reagent (l,8-dihydroxynaphthalene-3,5-disulfonic acid). It appears that the data obtained for threonine and serine in various proteins by these oxidation procedures are reasonably accurate. [Block and Bolling (26) have given data on the threonine and serine content of various proteins. ]... 

The reaction conditions necessary to obtain a good yield of the title compound (a difficult isomer), and to avoid hazards during the nitration of resorcinol, are critical and strict adherence to those specified is essential. The necessary 80% white fuming nitric acid must be completely free from oxides of nitrogen and nitrous acid, and procedures for this are detailed. Then the temperature dining addition of the diacetate must be kept between -10 and 0°C by regulating the rate of addition. The alternative use of 80% sulfuric acid as solvent for the 80% nitric acid (5 equiv.) is preferred as more reliable, but both methods have led to violent exothermic decomposition, accompanied by fume-off, after an induction period. In any event, the explosive 2,4,6-trinitroresorcinol ( styphnic acid ) is produced as a by-product. 

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). 

The sensitivity of the resorcinol/Cu " method can be increased significantly (between 3- to 6-fold), if periodate oxidation is carried out prior to heating with the resorcinol/Cu strong acid reagent (Jourdian et al 1971 Table 1). The chromogen formed with sialic acid glycosides under the influence of periodate is stable to further oxidation at 37 °C and leads to a chromophore with an extinction maximum at 630 nm. The chromogen formed with free sialic acids is destroyed at... 

Several laboratories have reported automated procedures for the quantitation of sialic acid in biological fluids, column effluents or for miscellaneous samples. The periodic acid/resorcinol assay has been adapted for use in a Technicon autoanalyser for routine assay of serum sialic acid (Rey et al. 1975). The method is based on the assay developed by Jourdian et al. (1971) and does not involve the organic solvent extraction. As with the original assay, the automated procedure can be adapted to measure free or glycosidically bound sialic acid (Reyc/ al. 1975). Modification of this system allows measurement of erythrocyte membrane sialic acid simultaneously with protein (Gerbaut et al. 1978). 

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. 

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