A modified thiobarbituric acid method for the determination of N-acetylneuraminic acid

Abstract

Sialic acid, a family of acylated derivatives of neuraminic acid, is widely distributed in mammals and usually occurs as a terminal component at the non-reducing end of carbohydrate chains of glycoproteins and glycolipids [ 11. The most commonly occuring of these is N-acetylneuraminic acid (NANA). Elevated levels of total serum NANA have been associated with tumour burden, especially in cases of advanced disease [2]. However, other work has shown that the extent of tumour burden may not be reflected in total serum NANA levels [3,4]. Significant increases have also been associated with non-neoplastic disorders, including bacterial infections and rheumatoid arthritis (51. It has been suggested that sialic acid associated with gangliosides, known as lipid-bound sialic acid (LSA), is a more useful and discriminating tumour marker than total NANA levels [6]. However, the specificity of the method most commonly used to serum LSA levels [7] has been questioned [8]. A procedure which claims to extract only gangliosidic sialic acid has been developed [9]. Early methods of NANA quantitation were colourimetric. Warren developed the thiobarbituric acid method [ 101. More recent methods have included fluorometric analysis [ 111, high performance liquid chromatography 1121, and enzymatic assays [ 131. Our aim was to modify and improve the Warren thiobarbituric acid method. In the original method NANA was subjected to periodate oxidation. After terminating this reaction with arsenite the oxidation product (b-formylpyruvate) was further reacted with thiobarbituric acid to form a pink chromophore. The colour was developed by placing the tubes in a boiling water-bath for 25 minutes. This was extracted into cyclohexanone. The purpose of this was to increase the solubility, stability, and colour intensity of the chromophore. The upper organic layer was then removed. To determine NANA levels, optical densities were measured at two wavelengths (549nm & 532nm), and then inserted into a formula devised by Warren. 2-Deoxyribose, a contaminant in cellular material from dead cells, forms an interfering chromophore in the assay and the formula is necessary to correct for this interference.The modified Warren assay was used to determine total serum NANA levels in patients suffering from cancer of the prostate and was as follows.The initial periodate oxidation was unchanged. After addition of the thiobarbituric acid and colour development a solution containing 5% (v/v) hydrochloric acid (HCI) in ethanol was added to each tube. The temperature of the tubes was maintained at 50°C. Optical densities were then measured at 560nm. Serum NANA levels were determined from a standard curve. The elimination of the use of cyclohexanone and the associated tedious and time consuming extraction step was a major improvement. The use of 50°C as the reaction temperature also eliminated cloudiness in samples, a problem often associated with the original method. The new solvent fulfilled all the requirements that had previously necessitated the use of cyclohexa...