Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans

BackgroundThe relevance of glycosaminoglycan determination in biological fluids is gradually gaining importance in the literature. Nevertheless, the results obtained by different methods vary widely. We evaluated 1,9-dimethylmethylene blue (DMB) dye-binding assays for quantification of urinary glycosaminoglycans, in comparison to densitometry after agarose gel electrophoresis.MethodsUrinary glycosaminoglycans from different mammalian species were quantified by 3 different DMB dye-binding assays. The results were compared to those obtained by densitometry after agarose gel electrophoresis of glycosaminoglycans isolated from urine samples by ion exchange chromatography.ResultsDensitometry after agarose gel electrophoresis showed glycosaminoglycan urinary concentrations of 1–20 mg/l, and glycosaminoglycan/creatinine ratios of 2–25 × 10− 3, for all the mammalian species here studied. A decrease with age was observed for humans, cats and horses. In comparison, DMB assays gave much higher results — up to 200 mg/l and 500 × 10− 3 glycosaminoglycan/creatinine ratios. These values were greatly reduced after 4-h dialysis, suggesting that low molecular weight compounds do interfere. Furthermore, urinary anions such as sulfate, phosphate and citrate, react with metachromatic dyes, such as Toluidine Blue and DMB.ConclusionDMB assays, although rapid and simple, are not appropriate to quantify urinary glycosaminoglycans in normal mammalians, since other urinary components interfere with the reactions.