A MICROTITER PLATE ASSAY FOR THE DETERMINATION OF URONIC-ACIDS

The amount of uronic acid residues in samples containing glycosaminogl ycans or pectin is an important parameter in the quantitative and stru ctural analysis of these complex carbohydrates. This paper describes a method to determine the content of uronic acids in biological samples , using conventional polystyrene microtiter plates and microtiter plat e-reading equipment with standard interference filters (i.e., 540 or 4 92 nm). This assay is a modification of a commonly used procedure, viz , hydrolysis of uronic acid containing carbohydrate polymers in 80% su lfuric acid containing tetraborate ions at 80 degrees C followed by a coloring step with an m-hydroxydiphenyl reagent at room temperature. T he use of microtiter plates has several practical advantages: (i) less risk of handling hot, concentrated sulfuric acid is present; (ii) an accurate estimate of background absorbance by multiple reading of the plates is possible; and (iii) many samples can be assayed in one serie s without errors due to fading of the final color. The validity of the assay was checked for the quantification of hyaluronic acid in equine synovial fluid samples. We consider this the method of choice when a large number of samples must be analyzed for their content of uronic a cid residues, (C) 1998 Academic Press.