Thiobarbituric acid assay

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. 

Powell LD, Hart GW (1986) Quantitation of picomole levels of N-acetyl- and N-glycolyl-neuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay. Anal Biochem 157 179-185... 

Warren L (1959) The thiobarbituric acid assay of sialic acids. J Biol Chem 234 1971-1975... 

The second enzyme in the sequence studied was KD0-8-phosphate synthase. This enzyme was purified to homogeneity (24). This enzyme catalyzes the condensation of D-arabinose-5-phosphate and PEP to yield KD0-8-phosphate and inorganic phosphate. One can assay this irreversible reaction either by measuring the formation of KD0-8-phosphate or the release of P. (2Jj). The latter is the method of choice, since a number of analogues were found to interfer with the thiobarbituric acid assay. The enzyme has an apparent K for PEP of 6 x 10 M and an apparent K for D-arabinose-5-phospftiate of 2 x 10 M. m... 

Lipid peroxidation Rat liver microsomes Fe2+/ascorbate Thiobarbituric acid assay V7... 

The degradation of deoxyribose is determined by a method akin to the conventional thiobarbituric acid assay for aldehydic products (see Chapter 5 for more information). 

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]. 

P. (1993) The thiobarbituric acid assay reflects susceptibility to oxygen-induced lipid peroxidation in vitro rather than levels of lipid hydroperoxides in vivo a methodological approach. Neurochem. Int. 22 255-262. 

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

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. 

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